Patent Publication Number: US-2020283524-A1

Title: Novel bispecific polypeptide complexes

Description:
CROSS-REFERENCE 
     This application claims priority to International Patent Application No. PCT/CN2017/103030, filed Sep. 22, 2017, the entire contents of which are incorporated herein by reference. 
    
    
     FIELD OF THE INVENTION 
     The present disclosure generally relates to soluble polypeptide complexes comprising antibody variable regions fused to the TCR constant regions, and bispecific polypeptide complexes comprising the same. 
     BACKGROUND 
     Bispecific antibodies are growing to be the new category of therapeutic antibodies. They can bind two different targets or two different epitopes on a target, creating additive or synergistic effect superior to the effect of individual antibodies. A lot of antibody engineering efforts have been put into designing new bispecific formats, such as DVD-Ig, CrossMab, BiTE etc. (Spiess et al.,  Molecular Immunology,  67(2), pp. 95-106 (2015)). However, these formats may potentially have various limitations in stability, solubility, short half-life, and immunogenicity. 
     Among these bispecific antibody formats, an IgG-like bispecific antibody is a common format: one arm binding to target A and another arm binding to target B. Structurally it is made from half of antibody A and half of antibody B, with the similar size and shape as natural IgG. In order to facilitate downstream development, it is desired that such bispecific molecules can be easily produced like a normal IgG from a single host cell with high expression level and correctly assembled form. Unfortunately, the pairing of cognate light-heavy chains as well as the assembly of two different half antibodies cannot be automatically controlled. All kinds of mispairings in a random manner could result in significant product heterogeneity. 
     By introducing mutations in the Fc region, such as “knobs-into-holes” (Ridgway et al.,  Protein Engineering,  9(7), pp. 617-21(1996); Merchant et al.,  Nature Biotechnology,  16(7), pp. 677-681(1998)), electrostatics (Gunasekaran et al.,  Journal of Biological Chemistry,  285(25), pp. 19637-19646 (2010)) or negative state designs (Kreudenstein et al., mAbs, 5(5), pp. 646-654 (2013); Leaver-Fay et al.,  Structure,  24(4), pp. 641-651 (2016)), the preferred heterodimeric assembly of two different heavy chains has been accomplished. However, the selective pairing of light-heavy chains of each individual antibody remains challenging. The interface between light-heavy chains includes the variable domain (VH-VL) and the constant domain (CH1-CL). Several strategies have been applied into designing orthogonal interfaces to facilitate cognate pairing. Roche swapped the domains of CH1 and CL and created the CrossMab platform (Schaefer et al.,  Proceedings of the National Academy of Sciences of the United States of America,  108(27), pp. 11187-11192 (2011)), MedImmune introduced alternatively disulphide bond (Mazor et al., mAbs, 7(2), pp. 377-389 (2015)), Amgen made further electrostatics in the CH1-CL region (Liu et al.,  Journal of Biological Chemistry,  290(12), pp. 7535-7562 (2015)), and Lilly (Lewis et al.,  Nature Biotechnology,  32(2), pp. 191-198 (2014)) and Genentech (Dillon et al.,  mAbs,  9(2), pp. 213-230 (2017)) introduced mutations in both variable and constant domains. 
     Therefore, there is great need to design bispecific molecules with desirable expression level and affinity to antigens. 
     BRIEF SUMMARY OF THE INVENTION 
     In one aspect, the present disclosure provides a polypeptide complex comprising a first polypeptide comprising, from N-terminus to C-terminus, a first heavy chain variable domain (VH) of a first antibody operably linked to a first T cell receptor (TCR) constant region (C1), and a second polypeptide comprising, from N-terminus to C-terminus, a first light chain variable domain (VL) of the first antibody operably linked to a second TCR constant region (C2), wherein C1 and C2 are capable of forming a dimer comprising at least one non-native interchain bond between C1 and C2, and the non-native interchain bond is capable of stabilizing the dimer, and the first antibody has a first antigenic specificity. 
     In one aspect, the present disclosure provides a bispecific polypeptide complex, comprising a first antigen-binding moiety associated with a second antigen-binding moiety, wherein the first antigen-binding moiety comprising a first polypeptide comprising, from N-terminal to C-terminal, a first heavy chain variable domain (VH) of a first antibody operably linked to a first T cell receptor (TCR) constant region (C1), and a second polypeptide comprising, from N-terminal to C-terminal, a first light chain variable domain (VL) of the first antibody operably linked to a second TCR constant region (C2), wherein C1 and C2 are capable of forming a dimer comprising at least one non-native interchain bond between a first mutated residue comprised in C1 and a second mutated residue comprised in C2, and the non-native interchain bond is capable of stabilizing the dimer, and the first antibody has a first antigenic specificity, a second antigen-binding moiety has a second antigenic specificity which is different from the first antigenic specificity, and the first antigen-binding moiety and the second antigen-binding moiety are less prone to mispair than otherwise would have been if both the first and the second antigen-binding moieties are counterparts of natural Fab. 
     In one aspect, the present disclosure provides herein a bispecific polypeptide complex, comprising a first antigen binding moiety comprising the polypeptide complex provided herein having a first antigenic specificity, associated with a second antigen binding moiety having a second antigenic specificity which is different from the first antigenic specificity, and the first antigen-binding moiety and the second antigen-binding moiety are less prone to mispair than otherwise would have been if both the first and the second antigen-binding moieties are counterparts of natural Fab. 
     In one aspect, the present disclosure provides a bispecific fragment of the bispecific polypeptide complex provided herein. 
     In one aspect, the present disclosure provides herein a conjugate comprising the polypeptide complex provided herein, or the bispecific polypeptide complex provided herein conjugated to a moiety. 
     In one aspect, the present disclosure provides herein an isolated polynucleotide encoding the polypeptide complex provided herein, or the bispecific polypeptide complex provided herein. 
     In one aspect, the present disclosure provides herein an isolated vector comprising the polynucleotide provided herein. 
     In one aspect, the present disclosure provides herein a host cell comprising the isolated polynucleotide provided herein or the isolated vector provided herein. 
     In one aspect, the present disclosure provides herein a method of expressing the polypeptide complex provided herein, or the bispecific polypeptide complex provided herein, comprising culturing the host cell provided herein under the condition at which the polypeptide complex, or the bispecific polypeptide complex is expressed. 
     In one aspect, the present disclosure provides herein a method of producing the polypeptide complex provided herein, comprising a) introducing to a host cell a first polynucleotide encoding a first polypeptide comprising, from N-terminal to C-terminal, a first heavy chain variable domain (VH) of a first antibody operably linked to a first TCR constant domain (C1), and a second polynucleotide encoding a second polypeptide comprising, from N-terminal to C-terminal, a first light chain variable domain (VL) of the first antibody operably linked to a second TCR constant domain (C2), wherein C1 and C2 are capable of forming a dimer comprising at least one non-native interchain bond between a first mutated residue comprised in C1 and a second mutated residue comprised in C2, and the non-native interchain bond is capable of stabilizing the dimer of C1 and C2, and the first antibody has a first antigenic specificity; b) allowing the host cell to express the polypeptide complex. 
     In one aspect, the present disclosure provides herein a method of producing the bispecific polypeptide complex provided herein, comprising a) introducing to a host cell a first polynucleotide encoding a first polypeptide comprising, from N-terminal to C-terminal, a first heavy chain variable domain (VH) of a first antibody operably linked to a first TCR constant region (C1), a second polynucleotide encoding a second polypeptide comprising, from N-terminal to C-terminal, a first light chain variable domain (VL) of the first antibody operably linked to a second TCR constant region (C2), a third polynucleotide encoding a third polypeptide comprising VH of a second antibody, and a fourth polynucleotide encoding a fourth polypeptide comprising VL of the second antibody, wherein C1 and C2 are capable of forming a dimer comprising at least one non-native interchain bond between C2, and the non-native interchain bond is capable of stabilizing the dimer, and the first antibody has a first antigenic specificity and the second antibody has a second antigenic specificity; b) allowing the host cell to express the bispecific polypeptide complex. 
     In certain embodiments, the method of producing the bispecific polypeptide complex provided herein further comprising isolating the polypeptide complex. 
     In one aspect, the present disclosure provides a composition comprising the polypeptide complex provided herein, or the bispecific polypeptide complex provided herein. 
     In one aspect, the present disclosure provides herein a pharmaceutical composition comprising the polypeptide complex provided herein, or the bispecific polypeptide complex provided herein and a pharmaceutically acceptable carrier. 
     In one aspect, the present disclosure provides herein a method of treating a condition in a subject in need thereof, comprising administrating to the subject a therapeutically effective amount of the polypeptide complex provided herein, or the bispecific polypeptide complex provided herein. In certain embodiments, the condition can be alleviated, eliminated, treated, or prevented when the first antigen and the second antigen are both modulated. 
     In certain embodiments, the non-native interchain bond is formed between a first mutated residue comprised in C1 and a second mutated residue comprised in C2. In certain embodiments, at least one of the first and the second mutated residues is a cysteine residue. 
     In certain embodiments, the non-native interchain bond is a disulphide bond. 
     In certain embodiments, the first mutated residue is comprised within a contact interface of C1, and/or the second mutated residue is comprised within a contact interface of C2. 
     In certain embodiments, at least one native cysteine residue is absent or present in C1 and/or C2. In certain embodiments, the native cysteine residue at position C74 of engineered CBeta is absent or present. In certain embodiments, the native C74 is absent in CBeta. 
     In certain embodiments, at least one native N-glycosylation site is absent or present in C1 and/or C2. In certain embodiments, the native N-glycosylation sites are absent in C1 and/or C2. 
     In certain embodiments, the dimer comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more non-native interchain bonds. In certain embodiments, at least one of the non-native interchain bonds is disulphide bond. In certain embodiments, the dimer comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more disulphide bonds. 
     In certain embodiments, a) C1 comprises an engineered CBeta, and C2 comprises an engineered CAlpha; b) C1 comprises an engineered CAlpha, and C2 comprises an engineered CBeta; c) C1 comprises an engineered CBeta, and C2 comprises an engineered CPre-Alpha; d) C1 comprises an engineered CPre-Alpha, and C2 comprises an engineered CBeta; e) C1 comprises an engineered CGamma, and C2 comprises an engineered CDelta; or f) C1 comprises an engineered CDelta, and C2 comprises an engineered CGamma. 
     In certain embodiments, the first VH is operably linked to C1 at a first conjunction domain, and the first VL is operably linked to C2 at a second conjunction domain. In certain embodiments, the first VH associates to C1 at a first conjunction domain via a connector, the first VL associates to C2 at a second conjunction domain via a connector. 
     In certain embodiments, the first and/or the second conjunction domain comprises a proper length (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues) of the C terminal fragment of antibody V/C conjunction, and a proper length (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues) of the N terminal fragment of TCR V/C conjunction. 
     In certain embodiments, the engineered CBeta comprises a mutated cysteine residue within a contact interface selected from the group consisting of amino acid residues 9-35, 52-66, 71-86, and 122-127; and/or the engineered CAlpha comprises a mutated cysteine residue within a contact interface selected from a group consisting of amino acid residues 6-29, 37-67, and 86-95. 
     In certain embodiments, the engineered CBeta comprises a mutated cysteine residue that substitutes for an amino acid residue at a position selected from: S56C, S16C, F13C, V12C, E14C, L62C, D58C, S76C, and R78C, and/or the engineered CAlpha comprises a mutated cysteine residue that substitutes for an amino acid residue at a position selected from: T49C, Y11C, L13C, S16C, V23C, Y44C, T46C, L51C, and S62C. 
     In certain embodiments, the engineered CBeta and the engineered CAlpha comprise a pair of mutated cysteine residues that substitute for a pair of amino acid residues selected from the group consisting of: S16C in CBeta and Y11C in CAlpha, F13C in CBeta and L13C in CAlpha, S16C in CBeta and L13C in CAlpha, V12C in CBeta and S16C in CAlpha, E14C in CBeta and S16C in CAlpha, F13C in CBeta and V23C in CAlpha, L62C in CBeta and Y44C in CAlpha, D58C in CBeta and T46C in CAlpha, S76C in CBeta and T46C in CAlpha, S56C in CBeta and T49C in CAlpha, S56C in CBeta and L51C in CAlpha, S56C in CBeta and S62C in CAlpha, and R78C in CBeta and S62C in CAlpha, and wherein the pair of cysteine residues are capable of forming a non-native interchain disulphide bond. 
     In certain embodiments, at least one native glycosylation site is absent or present in the engineered CBeta and/or in the engineered CAlpha. 
     In certain embodiments, the native glycosylation site in the engineered CBeta is N69, and/or the native glycosylation site(s) in the engineered CAlpha is/are selected from N34, N68, N79, and any combination thereof. 
     In certain embodiments, the engineered CBeta lacks or retains a FG loop encompassing amino acid residues 101-117 of the native CBeta and/or a DE loop encompassing amino acid residues 66-71 of the native CBeta. 
     In certain embodiments, the engineered CAlpha comprises any one of SEQ ID NOs: 43-48, and/or the engineered CBeta comprises any one of SEQ ID NOs: 33-41 and 306. 
     In certain embodiments, C1 comprises the engineered CBeta, and C2 comprises the engineered CAlpha; and wherein the first conjunction domain comprises or is SEQ ID NO: 49 or 50, and/or the second conjunction domain comprises or is SEQ ID NO: 51 or 52. 
     In certain embodiments, the C1 comprises the engineered CAlpha, and the C2 comprises the engineered CBeta; and wherein the first conjunction domain comprises or is SEQ ID NO: 129 or 130, and/or the second conjunction domain comprises or is SEQ ID NO: 49 or 50. 
     In certain embodiments, the engineered CBeta comprises a mutated cysteine residue within a contact interface selected from the group consisting of: amino acid residues 9-35, 52-66, 71-86 and 122-127; and/or the engineered CPre-Alpha comprises a mutated cysteine residue within a contact interface selected from a group consisting of: amino acid residues 7-19, 26-34, 56-75 and 103-106. 
     In certain embodiments, the engineered CBeta comprises a mutated cysteine residue that substitutes for an amino acid residue at a position selected from: S16C, A18C, E19C, F13C, A11C, S56C, and S76C, and/or the engineered CPre-Alpha comprises a mutated cysteine residue that substitutes for an amino acid residue at a position selected from S11C, A13C, I16C, S62C, T65C, and Y59C. 
     In certain embodiments, the engineered CBeta and the engineered CPre-Alpha comprise a pair of mutated cysteine residues that substitute for a pair of amino acid residues selected from the group consisting of: S16C in CBeta and S11C in CPre-Alpha, A18C in CBeta and S11C in CPre-Alpha, E19C in CBeta and S11C in CPre-Alpha, F13C in CBeta and A13C in CPre-Alpha, S16C in CBeta and A13C in CPre-Alpha, A11C in CBeta and I16C in CPre-Alpha, S56C in CBeta and S62C in CPre-Alpha, S56C in CBeta and T65C in CPre-Alpha, and S76C in CBeta, and Y59C in CPre-Alpha, and wherein the pair of mutated cysteine residues are capable of forming a non-native interchain disulphide bond. 
     In certain embodiments, at least one native glycosylation site is absent in the engineered CBeta and/or in the engineered CPre-Alpha. 
     In certain embodiments, the absent or present glycosylation site in the engineered CBeta is N69, and/or the absent glycosylation site in the engineered CPre-Alpha is N50. 
     In certain embodiments, the engineered CBeta lacks or retains a FG loop encompassing the amino acid residues 101-107 of the native CBeta and/or a DE loop at position encompassing the amino acid residues 66-71 of the native CBeta. 
     In certain embodiments, the engineered CPre-Alpha comprises any one of SEQ ID NOs: 82, 83, and 311-318; and/or the engineered CBeta comprises any one of SEQ ID NOs: 84, 33-41, and 319-324. 
     In certain embodiments, C1 comprises the engineered CBeta, and C2 comprises the engineered CPre-Alpha; and wherein the first conjunction domain comprises SEQ ID NO: 49 or 50, and/or the second conjunction domain comprises SEQ ID NO: 81 or 131. 
     In certain embodiments, C1 comprises the engineered CPre-Alpha, and C2 comprises the engineered CBeta; and wherein the first conjunction domain comprises SEQ ID NO: 132 or 133, and/or the second conjunction domain comprises SEQ ID NO: 49 or 50. 
     In certain embodiments, the engineered CDelta comprises a mutated cysteine residue within a contact interface selected from the group consisting of: amino acid residues 8-26, 43-64, and 84-88; and/or the engineered CGamma comprises a mutated cysteine residue within a contact interface selected from a group consisting of: amino acid residues 11-35 and 55-76. 
     In certain embodiments, the engineered CGamma comprises a mutated cysteine residue that substitutes for an amino acid residue at a position selected from: S17C, E20C, F14C, T12C, M62C, Q57C, and A19C, and/or the engineered CDelta comprises a mutated cysteine residue that substitutes for an amino acid residue at a position selected from: F12C, M14C, N16C, D46C, V50C, F87C, and E88C. 
     In certain embodiments, the engineered CGamma and the engineered CDelta comprise a pair of mutated cysteine residues that substitute for a pair of amino acid residues selected from the group consisting of: S17C in CGamma and F12C in CDelta, E20C in CGamma and F12C in CDelta, F14C in CGamma and M14C in CDelta, T12C in CGamma and N16C in CDelta, M62C in CGamma and D46C in CDelta, Q57C in CGamma and V50C in CDelta, A19C in CGamma and F87C in CDelta, and A19C in CGamma and E88C in CDelta, and wherein the introduced pair of cysteine residues are capable of forming an interchain disulphide bond. 
     In certain embodiments, at least one native glycosylation site is absent or present in the engineered CGamma and/or in the engineered CDelta. 
     In certain embodiments, the native glycosylation site in the engineered CGamma is N65, and/or the native glycosylation site(s) in the engineered CDelta is/are one or both of N16 and N79. 
     In certain embodiments, the engineered CGamma comprises SEQ ID NO: 113, 114, 333, 334, 335, 336, 337, 338, 339, or 340, and/or the engineered CDelta comprises SEQ ID NO: 115, 116, 310, 325, 326, 327, 328, 329, 330, 331, or 332. 
     In certain embodiments, C1 comprises the engineered CGamma, and C2 comprises the engineered CDelta; and wherein the first conjunction domain comprises SEQ ID NO: 117 or 118, and/or the second conjunction domain comprises SEQ ID NO: 119 or 120. 
     In certain embodiments, C1 comprises the engineered CDelta, and C2 comprises the engineered CGamma; and wherein the first conjunction domain comprises SEQ ID NO: 123 or 124, and/or the second conjunction domain comprises SEQ ID NO: 125 or 126. 
     In certain embodiments, the first polypeptide further comprises an antibody CH2 domain, and/or an antibody CH3 domain. 
     In certain embodiments, the first antigenic specificity and the second antigenic specificity are directed to two different antigens, or are directed to two different epitopes on one antigen. 
     In certain embodiments, the first antigen-binding moiety binds to CD3. In certain embodiments, the second antigen-binding moiety binds to CD19. In certain embodiments, the first antigen-binding moiety binds to CD19. In certain embodiments, the second antigen-binding moiety binds to CD3. 
     In certain embodiments, the first antigen-binding moiety binds to CTLA-4. In certain embodiments, the second antigen-binding moiety binds to PD-1. In certain embodiments, the first antigen-binding moiety binds to PD-1. In certain embodiments, the second antigen-binding moiety binds to CTLA-4. 
     In certain embodiments, the association is via a connecter, a disulphide bond, a hydrogen bond, electrostatic interaction, a salt bridge, or hydrophobic-hydrophilic interaction, or the combination thereof. 
     In certain embodiments, the second antigen-binding moiety comprises a heavy chain variable domain and a light chain variable domain of a second antibody having the second antigenic specificity. 
     In certain embodiments, the second antigen-binding moiety comprises a Fab. 
     In certain embodiments, the first antigenic specificity and the second antigenic specificity are directed to two different antigens, or are directed to two different epitopes on one antigen. 
     In certain embodiments, one of the first and the second antigenic specificities is directed to a T-cell specific receptor molecule and/or a natural killer cell (NK cell) specific receptor molecule, and the other is directed to a tumor associated antigen. 
     In certain embodiments, one of the first and the second antigenic specificities is directed to CD3, and the other is directed to a tumor associated antigen. 
     In certain embodiments, one of the first and the second antigenic specificities is directed to CD3, and the other is directed to CD19. 
     In certain embodiments, the first antigen-binding moiety further comprises a first dimerization domain, and the second antigen-binding moiety further comprises a second dimerization domain, wherein the first and the second dimerization domains are associated. 
     In certain embodiments, the association is via a connecter, a disulphide bond, a hydrogen bond, electrostatic interaction, a salt bridge, or hydrophobic-hydrophilic interaction, or the combination thereof. 
     In certain embodiments, the first and/or the second dimerization domain comprises at least a portion of an antibody hinge region, optionally derived from IgG1, IgG2 or IgG4. 
     In certain embodiments, the first and/or the second dimerization domain further comprises a dimerization domain. In certain embodiments, the dimerization domain comprises at least a portion of an antibody hinge region, an antibody CH2 domain, and/or an antibody CH3 domain. 
     In certain embodiments, the first dimerization domain is operably linked to the first TCR constant region (C1) at a third conjunction domain. 
     In certain embodiments, a) C1 comprises an engineered CBeta, and the third conjunction domain is comprised in SEQ ID NO: 53 or 54; b) C1 comprises an engineered CAlpha, and the third conjunction domain is comprised in SEQ ID NO: 134, 135, 140, or 141; c) C1 comprises an engineered CPre-Alpha, and the third conjunction domain is comprised in SEQ ID NO: 134, 135, 140, or 141; d) C1 comprises an engineered CGamma, and the third conjunction domain is comprised in SEQ ID NO: 121 or 122; or e) C1 comprises an engineered CDelta, and the third conjunction domain is comprised in SEQ ID NO: 127 or 128. 
     In certain embodiments, the second dimerization domain is operably linked to the heavy chain variable domain of the second antigen-binding moiety. 
     In certain embodiments, the first and the second dimerization domains are different and associate in a way that discourages homodimerization and/or favors heterodimerization. 
     In certain embodiments, the first and the second dimerization domains are capable of associating into heterodimers via knobs-into-holes, hydrophobic interaction, electrostatic interaction, hydrophilic interaction, or increased flexibility. 
     In certain embodiments, the first antigen-binding moiety comprising the first polypeptide comprising VH operably linked to a chimeric constant region, and the second polypeptide comprises VL operably linked to C2, wherein the chimeric constant region and C2 comprises a pair of sequences selected from the group consisting of: SEQ ID NOs: 177/176, 179/178, 184/183, 185/183, 180/176, 181/178, 182/178, 184/186, 185/186, 188/187, 196/187, 190/189, 192/191, 192/193, 195/194, 198/197, 200/199, 202/201, 203/201, 203/204, 205/204, 206/204, 208/207, 208/209, 211/210, 213/212, 213/215, 213/151, 214/212, 214/151, 232/231, 216/215, 218/217, 220/219, 222/221, 224/223, 226/225, 227/223, 229/228, 229/230, 236/235, and 238/237. 
     In certain embodiments, the first antigenicity is directed to CD3, and the first polypeptide and the second polypeptide comprise a pair of sequences selected from the group consisting of: SEQ ID NOs: 2/1, 3/4/, 5/1, 6/3, 7/3, 9/8, 10/8, 9/11, 10/11, 13/12, 15/14, 17/16, 17/18, 20/19, 21/12, 65/64, 67/66, 69/68, 70/68, 70/71, 72/71, 73/71, 75/74, 75/76, 78/77, 86/85, 90/89, 91/92/, 94/93, 96/95, 98/97, 99/95, 101/100, 101/102, 106/105, 108/107, 110/109, 112/111, 137/136, 138/136, 137/139, and 138/139. 
     In certain embodiments, the first antigen-binding moiety and the second antigen-binding moiety comprise a four-sequence combination selected from the group consisting of: SEQ ID NOs: 22/12/24/23, 25/12/26/23, and 25/12/27/23, wherein the first antigen-binding moiety is capable of binding to CD3, and the second antigen-binding moiety is capable of binding to CD19. 
     In certain embodiments, the polypeptide complex provided herein can be made into a Fab, a (Fab) 2 , a bibody, a tribody, a triFabs, tandem linked Fabs, a Fab-Fv, tandem linked V domains, tandem linked scFvs, and among other formats. 
     In another aspect, the present disclosure provides a kit comprising the polypeptide complex provided herein for detection, diagnosis, prognosis, or treatment of a disease or condition. 
     The foregoing and other features and advantages of the invention will become more apparent from the following detailed description of several embodiments which proceeds with reference to the accompanying figures. 
    
    
     
       BRIEF DESCRIPTION OF FIGURES 
         FIG. 1  presents schematic representations of studied antibody formats. Both anti-CD3 antibody T3 and anti-CD19 antibody U4 were developed. The constant region (CL and CH1) of T3 was replaced by the constant domains of TCR to design unique light-heavy chain interface that is orthogonal to regular antibody. The TCR-modified T3 and native U4 in conjunction with “knobs-into-holes” mutations in Fc domain were used to design bispecific antibody formats E17 and F16. 
         FIGS. 2A-2D  present superimposed poses of antibody Fv model and TCR structure providing guidance in fusing antibody Fv and TCR constant region.  FIG. 2A  presents an antibody Fv structure model that was built based on the sequence of an anti-CD3 antibody T3 developed in-house.  FIG. 2B  presents the TCR structure from PDB 4L4T.  FIG. 2C  presents an antibody Fv structural model superimposed on the TCR variable region in different orientations. Rough chimeric proteins were created by removing the TCR variable domain in the superimposed poses, as shown in  FIG. 2D . The overlapped residues in the conjunction area helped design conjunction region. The antibody VL chain and the TCR alpha chain were colored in white. The VH and beta chains were colored in black. 
         FIGS. 3A-3B  show a comparison between TCR constant region and antibody Fab constant region.  FIG. 3A  shows a TCR crystal structure from PDB 4L4T.  FIG. 3B  shows antibody Fab structural model made by the Fv domain of T3 model and the constant domain of antibody from PDB 5DK3. The obvious differences in FG and DE loops between constant domains of TCR and constant domains of antibody Fab were marked by displaying all the residues side chains. 
         FIG. 4  shows SDS-PAGE results of the de-glycosylation mutants of TCR-antibody chimeric antibodies with CAlpha and CBeta chains. Samples were all harvested supernatants from the production of Expi293 expressions. Lanes 1, 3, 5, 7 and 9 are the non-reduced pages of Design_2-QQQQ, Design_2-AAAA, Design_2-QSKE, Design_2-ASKE and Design_2-QQQQQ, respectively. Lanes 2, 4, 6, 8 and 10 are the corresponding reduced pages. 
         FIG. 5  shows dose-dependent FACS bindings of all the de-glycosylated mutants binding to CD3-expressed Jurkat cells. All samples were harvested supernatants of the de-glycosylation mutants expressed in Expi293. The wild type anti-CD3 antibody (T3-IgG1) was used as the positive control. 
         FIGS. 6A-6B  show SDS-PAGE results of the chain mispairing tests of antibody T3 and U4 in IgG1 ( FIG. 6A ) and IgG4 ( FIG. 6B ) isotype. Lanes 1-2 are the pairs of T3 light-U4_heavy and T3_heavy-U4_light, respectively. Lanes 3-4 are the same pair order as lanes 1-2, but with the modified T3 using TCR constant region. Lanes 1-4 in both pictures are the non-reduced samples, and Lanes 5-8 are the corresponding reduced samples. 
         FIGS. 7A-7B  show SDS-PAGE results of purified bispecific antibody, E17-Design_2-QQQQ in ( FIG. 7A ) IgG1 and ( FIG. 7B ) IgG4. The IgG1 isotype was purified by three step purifications: protein A chromatography, Ion-Exchange Chromatography (IEC) and Size Exclusion Chromatography (SEC). The IgG4 was obtained after two-step purifications: protein A chromatography and SEC.  FIGS. 7C-7D  show SEC-HPLC data for the purified samples of IgG1 ( FIG. 7C ) and IgG4 ( FIG. 7D ) to determine the purities of the samples. 
         FIG. 8  shows SDS-PAGE results of Fab fragments of chimeric T3 with a 6×His-tag, purified by Ni Sepharose™ excel chromatography. Lanes 1 and 3 are bands for T3-Fab-Design_2.his1, and Lanes 2 and 4 are bands for T3-Fab-Design_2.his2. 
         FIG. 9  illustrates dose-dependent FACS bindings of the Fab fragment of the TCR-modified chimeric T3. The monovalent form of wild type T3 antibody (T3-Fab-IgG4) was used as a positive control. A regular human IgG4 antibody was used as a negative control. 
         FIGS. 10A-10B  show dose-dependent FACS bindings of the designed bispecific antibody, E17-Design_2-QQQQ, to CD3+ Jurkat cells. The wild type antibody T3 and U4, as well as their monovalent forms, were used as positive controls ( FIG. 10A ) and CD19+ Ramos cells ( FIG. 10B ). Both IgG1 and IgG4 isotypes were tested. A irrelevant human IgG1 or IgG4 antibody was used as a negative control. 
         FIGS. 11A-11B  show comparison of the FACS bindings of two designed bispecific antibodies, E17-Design_2-QQQQ and F16-Design_2-QQQQ, to CD3 on Jurkat cells ( FIG. 11A ) and CD19 expressed on Ramos cells ( FIG. 11B ). The bispecific antibodies in both IgG1 and IgG4 isotypes were tested. A regular human IgG1 or IgG4 antibody was used as a negative control. 
         FIG. 12  illustrates the cytotoxic assay of T-cell directed killing malignant B cell, mediated by the designed bispecific antibodies E17-Design_2-QQQQ in both IgG1 and IgG4. The parental monospecific anti-CD3 (T3-IgG4), anti-CD19 (U4-IgG) antibody and an irrelevant human IgG1 antibody was used as the negative control. 
         FIG. 13  compares the activity of two designed bispecific antibody, E17-Design_2-QQQQ and F16-Design_2-QQQQ in mediating T-cell engaged malignant B cell killing. An irrelevant human IgG antibody was used as the negative control. 
         FIGS. 14A-14B  show deconvoluted mass spectra ofbispecific antibody E17-Design_2-QQQQ in non-reduced ( FIG. 14A ) and reduced ( FIG. 14B ) conditions. Peak at 148180.53 in  FIG. 14A  is the correct molecular weight of the intact WuXiBody. The peak with 22877 Da indicates the light chain found in the reduced mass spectra in  FIG. 14B . The small peak at 149128.45 Da in  FIG. 14A  was deduced to be the O-glycosylation (approximately (947.92 Da more) located on the light chain, as showed in  FIG. 14B . 
         FIGS. 15A-15B  show the role of interchain disulphide bond in antibody expression at alpha/beta interface characterized by SDS-PAGE.  FIG. 15A  shows the antibody containing interchain disulphide bond between CAlpha and CBeta;  FIG. 15B  shows the antibody without interchain disulphide bond between CAlpha and CBeta; Lanes 1 and 3 are the non-reduced PAGE results of Design_2-QQQQ-IgG4 with and without introduced disulphide bond, respectively. Lanes 2 and 4 are the reduced PAGE results of Design_2-QQQQ-IgG4 with and without introduced disulphide bond, respectively. 
         FIG. 16  shows SDS-PAGE of designed disulphide bond at pre-alpha/beta interface. Lane 1 and Lane 2 are “Design_5 Pre_TCR_Conjunction′1_Cys13” and “Design_6_Pre_TCR_Conjunction′1_Cys14”, respectively, treated in non-reduced condition. Lane 4 and lane 5 are “Design_5 Pre_TCR_Conjunction′1_Cys13” and “Design_6_Pre_TCR_Conjunction′1_Cys14”, respectively, treated in reduced condition. 
         FIGS. 17A-17B  show SDS-PAGE of designed disulphide bond at delta/gamma interface. Lane 6 and lane 8 are “Design_2_Cys5 no_Glyco” and “Design_2_hypeCys2no_Glyco”, respectively.  FIG. 17A  is non-reduced SDS-PAGE.  FIG. 17B  is reduced SDS-PAGE. 
         FIG. 18A  shows the sequence of native TCR alpha chain and its counterpart sequence with mutated cysteine residues. TRAC_Human is a natural sequence of alpha chain constant region. 4L4T_Alpha_Crystal is the sequence of a crystal structure (PDB code 4L4T) with S55C mutations that can form inter-chain disulphide bond. The gray region is the constant region used as backbone of chimeric protein in this invention. 
         FIG. 18B  shows the sequence of native TCR beta chain and its counterpart sequence with mutated cysteine residues. TRBC1_Human and TRBC2_Human are natural sequences of beta constant region. 
         FIG. 18C  shows the sequences of native TCR pre-alpha chain. PTCRA_Human is a natural sequence of pre-alpha chain constant region (pre-alpha chain only has no variable region). 30F6_PreAlpha_Crystal is the sequence of a crystal structure (PDB code 30F6). The gray region is the constant region used above to define the numbering. 
         FIG. 18D  shows the sequences of native TCR delta chain. TRA@_Human is the natural sequences of delta constant region. 4LFH_Delta_Crystal is the constant region of a delta chain sequence of a crystal structure (PDB code 4LFH). The gray region is the constant region used above to define the numbering. 
         FIG. 18E  shows the sequences of native TCR gamma chain. TRGC1_Human and TRGC2_Human are natural sequences of gamma constant region. 4LFH_Gamma_Crystal is the constant region of a gamma chain sequence of a crystal structure (PDB code 4LFH). The gray region is the constant region used above to define the numbering. 
         FIGS. 19A-19E  show the sequences and numbering of the TCR constant regions.  FIG. 19A  shows the sequences and numbering of the TCR Alpha constant region.  FIG. 19B  shows the sequences and numbering of the TCR Beta constant region.  FIG. 19C  shows the sequences and numbering of the TCR Pre-Alpha constant region.  FIG. 19D  shows the sequences and numbering of the TCR Delta constant region.  FIG. 19E  shows the sequences and numbering of the TCR Gamma constant region. 
         FIGS. 20A-20D  show the sequences and numbering of the IgG1 and IgG4 knobs-into-holes.  FIG. 20A  shows the sequences and numbering of the IgG1 “knob” mutations.  FIG. 20B  shows the sequences and numbering of the IgG4 “knob” mutations.  FIG. 20C  shows the sequences and numbering of the IgG1 “hole” mutations.  FIG. 20D  shows the sequences and numbering of the IgG4 “hole” mutations. 
         FIGS. 21A-21B  show bindings of E17-Design_2-QQQQ in both IgG4 ( FIG. 21A ) and wild type IgG1 ( FIG. 21B ) formats to human C1Q by ELISA. A human IgG1 antibody was used as control. 
         FIG. 22  shows schematic description of four symmetric WuXiBody formats G19, G19R, G25 and G25R. For formats G19 and G25, two TCR-containing chimeric Fab-like domains were grafted at the C-terminus and N-terminus of a normal antibody, respectively. The rectangles indicate TCR constant domains, and the ovals indicate variable and constant domains of an antibody. The difference between formats G19 and G19R or G25 and G25R is the switched position of normal Fab and chimeric Fab. These formats can accommodate different variable regions from different antibody pairs and usually have a molecular weight around 240-250 kD. 
         FIGS. 23A-23B  show SDS-PAGE ( FIG. 23A ) and SEC-HPLC ( FIG. 23B ) characterizations of two purified bispecific antibodies in G19 format. The lane numbers in SDS-PAGE are consistent with the label numbers in the SEC-HPLC figure. Lanes 1 and 2 are the T1U6 and U6T1 antibody pair, respectively. In T1U6, T1 (anti-CTLA-4) was on the N-terminus of the format, whereas in U6T1 U6 (anti-PD-1) was on the N-terminus of the format. Both bispecific molecules were purified by protein A chromatography, and purities around 90% was achieved. 
         FIGS. 24A-24B  show dose-dependent FACS bindings of purified U6T1 and T1U6 antibodies in G19 format to human PD-1 ( FIG. 24A ) and CTLA-4 ( FIG. 24B ) engineered cells. An IgG4 antibody was used as the negative control. 
         FIGS. 25A-25B  show SDS-PAGE ( FIG. 25A ) and SEC-HPLC ( FIG. 25B ) characterizations of the Protein A-purified bispecific antibodies in different symmetric formats. Lanes 1-3 are the U6T1 antibody pair in G19R, G25, and G25R formats, respectively. PC is a control protein known to have 250 kD molecular weight. All of the three bispecific molecules had more than 90% purity. The lane numbers in SDS-PAGE are consistent with the label numbers in SEC-HPLC figures. 
         FIGS. 26A-26B  show dose-dependent FACS bindings of purified U6T1 bispecific antibodies in G19R, G25, and G25R formats to human PD-1 ( FIG. 26A ) and CTLA-4 ( FIG. 26B ) engineered cells. A benchmark bispecific anti-CTAL-4×PD-1 antibody (BMK1.IgG1) was used as a control, and an IgG4 antibody was used as the negative control. 
         FIGS. 27A-27B  show FACS competition assays of the designed bispecific antibodies in G19R, G25, and G25R formats to block human PD-L1 binding to PD-1 ( FIG. 27A ) and CD80 binding to CTLA-4 ( FIG. 27B ), respectively. A benchmark bispecific anti-CTAL-4×PD-1 antibody (BMK1.IgG1) was used as a control, and an IgG4 antibody was used as the negative control. 
         FIGS. 28A-28B  show SDS-PAGE ( FIG. 28A ) and SEC-HPLC ( FIG. 28B ) characterizations of the Protein A-purified bispecific antibodies in different symmetric formats. Lanes 1-4 are the U6T5 antibody pair in G19, G19R, G25, and G25R formats, respectively. PC is a control protein with 250 kD molecular weight. All the three bispecific molecules had more than 90% purity. The lane numbers in SDS-PAGE are consistent with the label numbers in SEC-HPLC figures. 
         FIGS. 29A-29B  show dose-dependent FACS bindings of purified bispecific antibodies in G19, G19R, G25, and G25R formats to human PD-1 ( FIG. 29A ) and CTLA-4 ( FIG. 29B ) engineered cells. A benchmark bispecific anti-CTAL-4×PD-1 antibody (BMK1.IgG1) was used as a control, and an IgG4 antibody was used as the negative control. 
         FIG. 30  shows ELISA dual binding assay of two molecules U6T5.G25 and U6T1.G25R. A benchmark bispecific anti-CTAL-4×PD-1 antibody (BMK1.IgG1) was used as a control, and an IgG4 antibody was used as the negative control. 
         FIGS. 31A-31B  show FACS competition assays of the designed bispecific antibodies U6T5.G25 and U6T1.G25R to block human PD-L1 binding to PD-1 ( FIG. 31A ), and CD80 binding to CTLA-4 ( FIG. 31B ), respectively. A benchmark bispecific anti-CTAL-4×PD-1 antibody (BMK1.IgG1) was used as a control, and an IgG4 antibody was used as the negative control. 
         FIG. 32  shows schematic description of three symmetric formats G26, G27, and G26R with light-heavy switched chimeric Fab-like domains. 
         FIGS. 33A-33B  show SDS-PAGE ( FIG. 33A ) and SEC-HPLC ( FIG. 33B ) characterizations of the Protein A-purified bispecific antibodies in G27 and G26R formats. Lanes 1-2 are the T4U6 antibody pair in G27 and G26R formats, respectively. Only the one in G26R format achieved 90% purity after purification. The lane numbers in SDS-PAGE are consistent with the label numbers in SEC-HPLC figures. 
         FIGS. 34A-34B  show dose-dependent FACS bindings of purified bispecific T4U6 antibody pair in G26R format to human PD-1 ( FIG. 34A ) and CTLA-4 ( FIG. 34B ) engineered cells. A benchmark bispecific anti-CTAL-4×PD-1 antibody (BMK1.IgG1) was used as a control, and an IgG4 antibody was used as the negative control. 
         FIGS. 35A-35B  show SDS-PAGE ( FIG. 35A ) and SEC-HPLC ( FIG. 35B ) characterizations of the Protein A-purified bispecific U6T4 antibody pair in G26 format. It achieved 90% purity after purification. 
         FIGS. 36A-36D  show dose-dependent ELISA bindings of purified bispecific U6T4 antibody pair in G26 format to human PD-1 ( FIG. 36A ) and CTLA-4 ( FIG. 36B ) engineered cells, as well as dose-dependent FACS bindings of purified bispecific U6T4 antibody pair in G26 format to human PD-1 ( FIG. 36C ) and CTLA-4 ( FIG. 36D ) engineered cells. A benchmark bispecific anti-CTAL-4×PD-1 antibody (BMK1.IgG1) was used as a control, and an irrelevant IgG4 antibody was used as the negative control. 
         FIG. 37  shows flow cytometry histograms of cynomolgus-CD19 transfected cell line WBP701.CHO-K1.cpro1.FL.C9 and CHO-K1 parental cell line. 
         FIG. 38  shows SDS-PAGE of W3438-T3U4.F16-1.uIgG4.SP. M: Protein marker; Lane1: W3438-T3U4.F16-1.uIgG4.SP, non-reduced; Lane3: W3438-T3U4.F16-1.uIgG4.SP, reduced. 
         FIG. 39  shows SEC-HPLC of W3438-T3U4.F16-1.uIgG4. 
         FIG. 40  shows SDS-PAGE of W3438-T3U4.E17-1.uIgG4.SP. M: Protein marker; Lane1: W3438-T3U4.E17-1.uIgG4.SP, non-reduced; Lane2: W3438-T3U4.E17-1.uIgG4.SP, reduced. 
         FIG. 41  shows SEC-HPLC of W3438-T3U4.E17-1.uIgG4.SP. 
         FIGS. 42A-42B  show binding of W3438-T3U4.E17-1.uIgG4.SP to Ramos cells ( FIG. 42A ) and Jurkat cells ( FIG. 42B ) by FACS. 
         FIGS. 43A-43B  show binding of W3438-T3U4.F16-1.uIgG4.SP to Ramos cells ( FIG. 43A ) and Jurkat cells ( FIG. 43B ) by FACS. 
         FIG. 44  shows binding of W3438-T3U4.E17-1.uIgG4.SP to cynomolgus-CD19 expressing cell by FACS. 
         FIG. 45  shows binding of W3438-T3U4.E17-1.uIgG4.SP to cynomolgus CD3 by ELISA. 
         FIGS. 46A-46B  show affinity of W3438-T3U4.E17-1.uIgG4.SP to human CD19 and CD3 as measured by binding to Ramos ( FIG. 46A ) and Jurkat ( FIG. 46B ) cells. 
         FIGS. 47A-47B  show W3438-T3U4.E17-1.uIgG4.SP-mediated CD3+ cells binding to CD19+ cells ( FIG. 47A ). An irrelevant IgG was used as a negative control ( FIG. 47B ). 
         FIGS. 48A-48B  show cytotoxic activity of W3438-T3U4.E17-1.uIgG4.SP mediated T cells killing on Raji cell ( FIG. 48A ) and cytotoxic activity of W3438-T3U4.F16-1.uIgG4.SP mediated T cells killing on Raji cell ( FIG. 48B ). 
         FIGS. 49A-49D  show CD69 and CD25 expression on T cell in the presence or absence of CD19+ target cells. Percentage of CD69+ expression T cell in CD4+ T cell subset ( FIG. 49A ); Percentage of CD69 expression T cell in CD8+ T cell subset ( FIG. 49B ); Percentage of CD25 expression T cell in CD4+ T cell subset ( FIG. 49C ); Percentage of CD25 expression T cell in CD8+ T cell subset ( FIG. 49D ). 
         FIGS. 50A-50D  show IFN-γ and TNF-α cytokine release of T cell in the presence or absence of CD19+ target cells. Release of IFN-γ in CD4+ T cell subset ( FIG. 50A ); Release of TNF-α in CD4+ T cell subset ( FIG. 50B ); Release of IFN-γ in CD8+ T cell subset ( FIG. 50C ); Release of TNF-α in CD8+ T cell subset ( FIG. 50D ). 
         FIGS. 51A-51B  show stability of W3438-T3U4.E17-1.uIgG4.SP in human serum. Binding of W3438-T3U4.E17-1.uIgG4.SP samples incubated in serum to Ramos at indicated days ( FIG. 51A ); Binding of serum incubated W3438-T3U4.E17-1.uIgG4.SP samples to Jurkat at indicated days ( FIG. 51B ). 
         FIG. 52  shows binding of W3438-T3U4.E17-1.uIgG4.SP to C1Q by ELISA. An IgG1 antibody was used as the control. 
         FIG. 53  shows tumor volume trace after administering W3438-T3U4.E17-1.uIgG4.SP at different doses to admixed PBMC humanized mice bearing Raji xenografts tumors. Data points represent group mean, and error bars represent standard error of the mean (SEM). An IgG4 antibody was used as a negative control. 
         FIG. 54  shows pharmacokinetics of W3438-T3U4.E17-1.uIgG4.SP in cynomolgus monkey. The serum samples from two monkeys were detected by ELISA. 
         FIGS. 55A-55B  show the anti-drug antibody (ADA detected by ELISA) in serum samples from monkey #1 ( FIG. 55A ) and monkey #2 ( FIG. 55B ), including both predose and postdose of W3438-T3U4.E17-1.uIgG4.SP. 
         FIGS. 56A-56B  show SDS-PAGE characterizations of W3248-U6T5.G25-1.uIgG4.SP and W3248-U6T1.G25R-1.uIgG4.SP. M: Protein marker. PC: a positive control of a bispecific antibody at around 250 kDa ( FIG. 56A ) and SEC-HPLC characterizations of W3248-U6T1.G25R-1.uIgG4.SP and W3248-U6T5.G25-1.uIgG4.SP ( FIG. 56B ). 
         FIG. 57  shows melting temperatures of W3248-U6T1.G25R-1.uIgG4.SP, W3248-U6T5.G25-1.uIgG4.SP, and a benchmark bispecific anti-CTLA-4×PD-1 antibody WBP324-BMK1.uIgG1.KDL. 
         FIG. 58  shows FACS bindings of W3248-U6T5.G25-1.uIgG4.SP and W3248-U6T1.G25R-1.uIgG4.SP to human PD-1 engineered cells. WBP324-BMK1.uIgG1.KDL, W324-BMK2.uIgG4, and W324-BMK3.uIgG4 are different versions of benchmark bispecific anti-CTLA-4×PD-1 antibodies. WBP305-BMK1.IgG4 is an anti-PD-1 antibody. An IgG4 antibody was used as the negative control. 
         FIG. 59  shows FACS bindings of W3248-U6T5.G25-1.uIgG4.SP and W3248-U6T1.G25R-1.uIgG4.SP to cynomolgus PD-1 engineered cells. WBP3055_1.153.7.hAb and WBP305-BMK1.IgG4 are anti-PD-1 antibodies. An IgG4 antibody was used as the negative control. 
         FIG. 60  shows FACS bindings of W3248-U6T5.G25-1.uIgG4.SP and W3248-U6T1.G25R-1.uIgG4.SP to human CTLA-4 engineered cells. WBP324-BMK1.uIgG1.KDL, W324-BMK2.uIgG4, and W324-BMK3.uIgG4 are different benchmark bispecific anti-CTLA-4×PD-1 antibodies. WBP316-BMK1.IgG4 is an anti-CTLA-4-1 antibody. An IgG4 antibody was used as the negative control. 
         FIG. 61  shows FACS bindings of W3248-U6T5.G25-1.uIgG4.SP and W3248-U6T1.G25R-1.uIgG4.SP to cynomolgus CTLA-4 engineered cells. WBP324-BMK1.uIgG1.KDL is a benchmark bispecific anti-CTLA-4×PD-1 antibody. W3162_1.154.8-z35-IgG1K and WBP316-BMK1.IgG4 are anti-CTLA-4 antibodies. An IgG4 antibody was used as the negative control. 
         FIG. 62  summarizes binding affinities of W3248-U6T5.G25-1.uIgG4.SP and W3248-U6T1.G25R-1.uIgG4.SP to CTLA-4 and PD-1, as measured by SPR. WBP316-BMK1.IgG4 is an anti-CTLA-4-1 antibody. A parent antibody of anti-PD-1 was used as a control. 
         FIG. 63  shows FACS competition assays of W3248-U6T5.G25-1.uIgG4.SP and W3248-U6T1.G25R-1.uIgG4.SP to block human PD-L1 protein binding to PD-1 engineered cells. WBP324-BMK1.uIgG1.KDL is a benchmark bispecific anti-CTLA-4×PD-1 antibody. WBP3055_1.153.7.hAb and WBP305-BMK1.IgG4 are anti-PD-1 antibodies. An IgG4 antibody was used as the negative control. 
         FIG. 64  shows FACS competition assays of W3248-U6T5.G25-1.uIgG4.SP and W3248-U6T1.G25R-1.uIgG4.SP to block human CTLA-4 protein binding to CD80 engineered cells. WBP324-BMK1.uIgG1.KDL is a benchmark bispecific anti-CTLA-4×PD-1 antibody. W3162_1.154.8-z35-IgG1K and WBP316-BMK1.IgG4 are anti-CTLA-4 antibodies. An IgG4 antibody was used as the negative control. 
         FIG. 65  shows FACS competition assays of W3248-U6T5.G25-1.uIgG4.SP and W3248-U6T1.G25R-1.uIgG4.SP to block cynomolgus CTLA-4 protein binding to CD80 engineered cells. WBP324-BMK1.uIgG1.KDL is a benchmark bispecific anti-CTLA-4×PD-1 antibody. W3162_1.154.8-z35-IgG1K and WBP316-BMK1.IgG4 are anti-CTLA-4 antibodies. An IgG4 antibody was used as the negative control. 
         FIG. 66  shows ELISA dual binding assay of W3248-U6T5.G25-1.uIgG4.SP and W3248-U6T1.G25R-1.uIgG4.SP. WBP324-BMK1.uIgG1.KDL is a benchmark bispecific anti-CTLA-4×PD-1 antibody. An IgG4 antibody was used as the negative control. 
         FIG. 67  shows FACS dual binding of W3248-U6T5.G25-1.uIgG4.SP and W3248-U6T1.G25R-1.uIgG4.SP to CTLA-4 and PD-1. An IgG4 antibody was used as the negative control. 
         FIGS. 68A-68B  show stability of W3248-U6T5.G25-1.uIgG4.SP in serum for 14 days, as measured by ELISA dual binding to human CTLA-4 and PD-1 ( FIG. 68A ) and stability of W3248-U6T1.G25R-1.uIgG4.SP in serum for 14 days, as measured by ELISA dual binding to human CTLA-4 and PD-1 ( FIG. 68B ). 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The following description of the disclosure is merely intended to illustrate various embodiments of the disclosure. As such, the specific modifications discussed are not to be construed as limitations on the scope of the disclosure. It will be apparent to one skilled in the art that various equivalents, changes, and modifications may be made without departing from the scope of the disclosure, and it is understood that such equivalent embodiments are to be included herein. All references cited herein, including publications, patents and patent applications are incorporated herein by reference in their entirety. 
     Definitions 
     The articles “a,” “an,” and “the” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “a polypeptide complex” means one polypeptide complex or more than one polypeptide complex. 
     As used herein, the term “about” or “approximately” refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length. In particular embodiments, the terms “about” or “approximately” when preceding a numerical value indicates the value plus or minus a range of 15%, 10%, 5%, or 1%. 
     Throughout this disclosure, unless the context requires otherwise, the words “comprise”, “comprises” and “comprising” will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. By “consisting of” is meant including, and limited to, whatever follows the phrase “consisting of”. Thus, the phrase “consisting of” indicates that the listed elements are required or mandatory, and that no other elements may be present. By “consisting essentially of” is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase “consisting essentially of” indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements. 
     Reference throughout this disclosure to “one embodiment,” “an embodiment,” “a particular embodiment,” “a related embodiment,” “a certain embodiment,” “an additional embodiment,” or “a further embodiment” or combinations thereof means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present disclosure. Thus, the appearances of the foregoing phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. 
     The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues, or an assembly of multiple polymers of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer. The term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an alpha-carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. An alpha-carbon refers to the first carbon atom that attaches to a functional group, such as a carbonyl. A beta-carbon refers to the second carbon atom linked to the alpha-carbon, and the system continues naming the carbons in alphabetical order with Greek letters. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid. The term “protein” typically refers to large polypeptides. The term “peptide” typically refers to short polypeptides. Polypeptide sequences are usually described as the left-hand end of a polypeptide sequence is the amino-terminus (N-terminus); the right-hand end of a polypeptide sequence is the carboxyl-terminus (C-terminus). “Polypeptide complex” as used herein refers to a complex comprising one or more polypeptides that are associated to perform certain functions. In certain embodiments, the polypeptides are immune-related. 
     The term “antibody” as used herein encompasses any immunoglobulin, monoclonal antibody, polyclonal antibody, multispecific antibody, or bispecific (bivalent) antibody that binds to a specific antigen. A native intact antibody comprises two heavy chains and two light chains. Each heavy chain consists of a variable region (“HCVR”) and a first, second, and third constant region (CH1, CH2 and CH3), while each light chain consists of a variable region (“LCVR”) and a constant region (CL). Mammalian heavy chains are classified as α, δ, ε, γ, and μ, and mammalian light chains are classified as λ or κ. The antibody has a “Y” shape, with the stem of the Y consisting of the second and third constant regions of two heavy chains bound together via disulphide bonding. Each arm of the Y includes the variable region and first constant region of a single heavy chain bound to the variable and constant regions of a single light chain. The variable regions of the light and heavy chains are responsible for antigen binding. The variable regions in both chains generally contain three highly variable loops called the complementarity determining regions (CDRs) (light (L) chain CDRs including LCDR1, LCDR2, and LCDR3, heavy (H) chain CDRs including HCDR1, HCDR2, HCDR3). CDR boundaries for antibodies may be defined or identified by the conventions of Kabat, Chothia, or Al-Lazikani (Al-Lazikani, B., Chothia, C., Lesk, A. M., J. Mol. Biol., 273(4), 927 (1997); Chothia, C. et al., J Mol. Biol. December 5; 186(3):651-63 (1985); Chothia, C. and Lesk, A. M., J. Mol. Biol., 196,901 (1987); Chothia, C. et al., Nature. December 21-28; 342(6252):877-83 (1989); Kabat E. A. et al., National Institutes of Health, Bethesda, Md. (1991)). The three CDRs are interposed between flanking stretches known as framework regions (FRs), which are more highly conserved than the CDRs and form a scaffold to support the hypervariable loops. Each HCVR and LCVR comprises four FRs, and the CDRs and FRs are arranged from amino terminus to carboxy terminus in the order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The constant regions of the heavy and light chains are not involved in antigen binding, but exhibit various effector functions. Antibodies are assigned to classes based on the amino acid sequence of the constant region of their heavy chain. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of α, δ, ε, γ, and μ heavy chains, respectively. Several of the major antibody classes are divided into subclasses such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain), or IgA2 (α2 heavy chain). 
     The term “variable domain” with respect to an antibody as used herein refers to an antibody variable region or a fragment thereof comprising one or more CDRs. Although a variable domain may comprise an intact variable region (such as HCVR or LCVR), it is also possible to comprise less than an intact variable region yet still retain the capability of binding to an antigen or forming an antigen-binding site. 
     The term “antigen-binding moiety” as used herein refers to an antibody fragment formed from a portion of an antibody comprising one or more CDRs, or any other antibody fragment that binds to an antigen but does not comprise an intact native antibody structure. Examples of antigen-binding moiety include, without limitation, a variable domain, a variable region, a diabody, a Fab, a Fab′, a F(ab′) 2 , an Fv fragment, a disulphide stabilized Fv fragment (dsFv), a (dsFv) 2 , a bispecific dsFv (dsFv-dsFv′), a disulphide stabilized diabody (ds diabody), a multispecific antibody, a camelized single domain antibody, a nanobody, a domain antibody, and a bivalent domain antibody. An antigen-binding moiety is capable of binding to the same antigen to which the parent antibody binds. In certain embodiments, an antigen-binding moiety may comprise one or more CDRs from a particular human antibody grafted to a framework region from one or more different human antibodies. For more and detailed formats of antigen-binding moiety are described in Spiess et al, 2015 (Supra), and Brinkman et al., mAbs, 9(2), pp. 182-212 (2017), which are incorporated herein by their entirety. 
     “Fab” with regard to an antibody refers to that portion of the antibody consisting of a single light chain (both variable and constant regions) associating to the variable region and first constant region of a single heavy chain by a disulphide bond. In certain embodiments, the constant regions of both the light chain and heavy chain are replaced with TCR constant regions. 
     “Fab′” refers to a Fab fragment that includes a portion of the hinge region. 
     “F(ab′) 2 ” refers to a dimer of Fab′. 
     “Bibody” refers to a fusion protein formed by fusing a scFv to the C-terminus of either the light chain (Fab-L-scFv) or Fd (Fab-H-scFv). 
     “Tribody” refers to a fusion protein formed by fusing a scFv to both light chain and heavy chain (Fab-(scFv) 2 ). 
     A “WuXiBody” is a bispecific antibody comprising soluble chimeric protein with variable domains of an antibody and the constant domains of TCR, wherein the subunits (such as alpha and beta domains) of TCR constant domains are linked by engineered disulfide bond. 
     A “fragment difficult (Fd)” with regard to an antibody refers to the amino-terminal half of the heavy chain fragment that can be combined with the light chain to form Fab. 
     “Fc” with regard to an antibody refers to that portion of the antibody consisting of the second (CH2) and third (CH3) constant regions of a first heavy chain bound to the second and third constant regions of a second heavy chain via disulphide bonding. The Fc portion of the antibody is responsible for various effector functions such as ADCC, and CDC, but does not function in antigen binding. 
     “Hinge region” in terms of an antibody includes the portion of a heavy chain molecule that joins the CH1 domain to the CH2 domain. This hinge region comprises approximately 25 amino acid residues and is flexible, thus allowing the two N-terminus antigen binding regions to move independently. 
     “CH2 domain” as used herein refers to includes the portion of a heavy chain molecule that extends, e.g., from about amino acid 244 to amino acid 360 of an IgG antibody using conventional numbering schemes (amino acids 244 to 360, Kabat numbering system; and amino acids 231-340, EU numbering system; see Kabat, E., et al., U.S. Department of Health and Human Services, (1983)). 
     The “CH3 domain” extends from the CH2 domain to the C-terminus of the IgG molecule and comprises approximately 108 amino acids. Certain immunoglobulin classes, e.g., IgM, further include a CH4 region. 
     “Fv” with regard to an antibody refers to the smallest fragment of the antibody to bear the complete antigen binding site. An Fv fragment consists of the variable domain of a single light chain bound to the variable domain of a single heavy chain. A number of Fv designs have been provided, including dsFvs, in which the association between the two domains is enhanced by an introduced disulphide bond; and scFvs can be formed using a peptide linker to bind the two domains together as a single polypeptide. Fvs constructs containing a variable domain of a heavy or light immunoglobulin chain associated to the variable and constant domain of the corresponding immunoglobulin heavy or light chain have also been produced. Fvs have also been multimerised to form diabodies and triabodies (Maynard et al., Annu Rev Biomed Eng 2 339-376 (2000)). 
     “ScFab” refers to a fusion polypeptide with a Fd linked to a light chain via a polypeptide linker, resulting in the formation of a single chain Fab fragment (scFab). 
     “TriFabs” refers to a trivalent, bispecific fusion protein composed of three units with Fab-functionalities. TriFabs harbor two regular Fabs fused to an asymmetric Fab-like moiety. 
     “Fab-Fab” refers to a fusion protein formed by fusing the Fd chain of a first Fab arm to the N-terminus of the Fd chain of a second Fab arm. 
     “Fab-Fv” refers to a fusion protein formed by fusing a HCVR to the C-terminus of a Fd chain and a LCVR to the C-terminus of a light chain. A “Fab-dsFv” molecule can be formed by introducing an interdomain disulphide bond between the HCVR domain and the LCVR domain. 
     “MAb-Fv” or “IgG-Fv” refers to a fusion protein formed by fusion of HCVR domain to the C-terminus of one Fc chain and the LCVR domain either expressed separately or fused to the C-terminus of the other resulted in a bispecific, trivalent IgG-Fv (mAb-Fv) fusion protein, with the Fv stabilized by an interdomain disulphide bond. 
     “ScFab-Fc-scFv 2 ” and “ScFab-Fc-scFv” refer to a fusion protein formed by fusion of a single-chain Fab with Fc and disulphide-stabilized Fv domains. 
     “Appended IgG” refers to a fusion protein with a Fab arm fused to an IgG to form the format of bispecific (Fab) 2 -Fc. It can form a “IgG-Fab” or a “Fab-IgG”, with a Fab fused to the C-terminus or N-terminus of an IgG molecule with or without a connector. In certain embodiments, the appended IgG can be further modified to a format of IgG-Fab 4  (see, Brinkman et al., 2017, Supra). 
     “DVD-Ig” refers to a dual-variable-domain antibody that is formed by fusion of an additional HCVR domain and LCVR domain of a second specificity to an IgG heavy chain and light chain. “CODV-Ig” refers to a related format where the two HCVR and two LCVR domains are linked in a way that allows crossover pairing of the variable HCVR-LCVR domains, which are arranged either (from N- to C-terminus) in the order HCVRA-HCVRB and LCVRB-LCVRA, or in the order HCVRB-HCVRA and LCVRA-LCVRB. 
     A “CrossMab” refers to a technology of pairing of unmodified light chain with the corresponding unmodified heavy chain and pairing of the modified light chain with the corresponding modified heavy chain, thus resulting an antibody with reduced mispairing in the light chain. 
     A “BiTE” is a bispecific T-cell engager molecule, comprising a first scFv with a first antigen specificity in the LCVR-HCVR orientation linked to a second scFv with a second specificity in the HCVR-LCVR orientation. 
     “Percent (%) sequence identity” with respect to amino acid sequence (or nucleic acid sequence) is defined as the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to the amino acid (or nucleic acid) residues in a reference sequence, after aligning the sequences and, if necessary, introducing gaps, to achieve the maximum number of identical amino acids (or nucleic acids). Conservative substitution of the amino acid residues may or may not be considered as identical residues. Alignment for purposes of determining percent amino acid (or nucleic acid) sequence identity can be achieved, for example, using publicly available tools such as BLASTN, BLASTp (available on the website of U.S. National Center for Biotechnology Information (NCBI), see also, Altschul S. F. et al., J. Mol. Biol., 215:403-410 (1990); Stephen F. et al., Nucleic Acids Res., 25:3389-3402 (1997)), ClustalW2 (available on the website of European Bioinformatics Institute, see also, Higgins D. G. et al., Methods in Enzymology, 266:383-402 (1996); Larkin M. A. et al., Bioinformatics (Oxford, England), 23(21): 2947-8 (2007)), and ALIGN or Megalign (DNASTAR) software. Those skilled in the art may use the default parameters provided by the tool, or may customize the parameters as appropriate for the alignment, such as for example, by selecting a suitable algorithm. 
     An “antigen” or “Ag” as used herein refers to a compound, composition, peptide, polypeptide, protein or substance that can stimulate the production of antibodies or a T cell response in cell culture or in an animal, including compositions (such as one that includes a cancer-specific protein) that are added to a cell culture (such as a hybridoma), or injected or absorbed into an animal. An antigen reacts with the products of specific humoral or cellular immunity (such as an antibody), including those induced by heterologous antigens. 
     An “epitope” or “antigenic determinant” refers to the region of an antigen to which a binding agent (such as an antibody) binds. Epitopes can be formed both from contiguous amino acids (also called linear or sequential epitope) or noncontiguous amino acids juxtaposed by tertiary folding of a protein (also called configurational or conformational epitope). Epitopes formed from contiguous amino acids are typically arranged linearly along the primary amino acid residues on the protein and the small segments of the contiguous amino acids can be digested from an antigen binding with major histocompatibility complex (MHC) molecules or retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5, about 7, or about 8-10 amino acids in a unique spatial conformation. 
     The term “specific binding” or “specifically binds” as used herein refers to a non-random binding reaction between two molecules, such as for example between an antibody and an antigen. In certain embodiments, the polypeptide complex and the bispecific polypeptide complex provided herein specifically bind an antigen with a binding affinity (K D ) of ≤10 −6  M (e.g., ≤5×10 −7  M, ≤2×10 −7  M, ≤10 −7  M, ≤5×10 −8  M, ≤2×10 −8  M, ≤10 −8  M, ≤5×10 −9  M, ≤2×10 −9  M, ≤10 −9  M, or ≤10 −10  M). K D  as used herein refers to the ratio of the dissociation rate to the association rate (k off /k on ), may be determined using surface plasmon resonance methods for example using instrument such as Biacore. 
     The term “operably link” or “operably linked” refers to a juxtaposition, with or without a spacer or linker, of two or more biological sequences of interest in such a way that they are in a relationship permitting them to function in an intended manner. When used with respect to polypeptides, it is intended to mean that the polypeptide sequences are linked in such a way that permits the linked product to have the intended biological function. For example, an antibody variable region may be operably linked to a constant region so as to provide for a stable product with antigen-binding activity. The term may also be used with respect to polynucleotides. For one instance, when a polynucleotide encoding a polypeptide is operably linked to a regulatory sequence (e.g., promoter, enhancer, silencer sequence, etc.), it is intended to mean that the polynucleotide sequences are linked in such a way that permits regulated expression of the polypeptide from the polynucleotide. 
     The term “fusion” or “fused” when used with respect to amino acid sequences (e.g. peptide, polypeptide or protein) refers to combination of two or more amino acid sequences, for example by chemical bonding or recombinant means, into a single amino acid sequence which does not exist naturally. A fusion amino acid sequence may be produced by genetic recombination of two encoding polynucleotide sequences, and can be expressed by a method of introducing a construct containing the recombinant polynucleotides into a host cell. 
     The term “spacer” as used herein refers to an artificial amino acid sequence having 1, 2, 3, 4 or 5 amino acid residues, or a length of between 5 and 15, 20, 30, 50 or more amino acid residues, joined by peptide bonds and are used to link one or more polypeptides. A spacer may or may not have a secondary structure. Spacer sequences are known in the art, see, for example, Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993); Poljak et al. Structure 2:1121-1123 (1994). Any suitable spacers known in the art can be used. For example, a useful spacer in the present disclosure may be rich in glycine and proline residues. Examples include spacers having a single or repeated sequences composed of threonine/serine and glycine, such as TGGGG (SEQ ID NO: 266), GGGGS (SEQ ID NO: 267) or SGGGG (SEQ ID NO: 268) or its tandem repeats (e.g. 2, 3, 4, or more repeats). Alternatively, a spacer may be a long peptide chain containing one or more sequential or tandem repeats of the amino acid sequence of GAPGGGGGAAAAAGGGGG (SEQ ID NO: 269). In certain embodiment, the spacer comprises 1, 2, 3, 4 or more sequential or tandem repeats of SEQ ID NO: 269. 
     The term “antigenic specificity” refers to a particular antigen or an epitope thereof that is selectively recognized by an antigen-binding molecule. 
     The term “substitution” with regard to amino acid residue as used herein refers to naturally occurring or induced replacement of one or more amino acids with another in a peptide, polypeptide or protein. Substitution in a polypeptide may result in diminishment, enhancement, or elimination of the polypeptide&#39;s function. 
     Substitution can also be “conservative substitution” with reference to amino acid sequence refers to replacing an amino acid residue with a different amino acid residue having a side chain with similar physiochemical properties or substitution of those amino acids that are not critical to the activity of the polypeptide. For example, conservative substitutions can be made among amino acid residues with nonpolar side chains (e.g., Met, Ala, Val, Leu, and Ile, Pro, Phe, Trp), among residues with uncharged polar side chains (e.g., Cys, Ser, Thr, Asn, Gly and Gln), among residues with acidic side chains (e.g., Asp, Glu), among amino acids with basic side chains (e.g., His, Lys, and Arg), among amino acids with beta-branched side chains (e.g., Thr, Val and Ile), among amino acids with sulfur-containing side chains (e.g., Cys and Met), or among residues with aromatic side chains (e.g., Trp, Tyr, His and Phe). In certain embodiments, substitutions, deletions or additions can also be considered as “conservative substitution.” The number of amino acids that are inserted or deleted can be in the range of about 1 to 5. Conservative substitution usually does not cause significant change in the protein conformational structure, and therefore could retain the biological activity of a protein. 
     The term “mutation” or “mutated” with regard to amino acid residue as used herein refers to substitution, insertion, or addition of an amino acid residue. 
     As used herein, a “homologue sequence” and “homologous sequence” are used interchangeably and refer to polynucleotide sequences (or its complementary strand) or amino acid sequences that have sequences identity of at least 80% (e.g. at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) to another sequences when optionally aligned. 
     “T cell” as used herein refers to a type of lymphocyte that plays a critical role in the cell-mediated immunity, including helper T cells (e.g. CD4 +  T cells, T helper 1 type T cells, T helper 2 type T cells, T helper 3 type T cells, T helper 17 type T cells), cytotoxic T cells (e.g. CD8 +  T cells), memory T cells (e.g. central memory T cells (TCM cells), effector memory T cells (TEM cells and TEMRA cells) and resident memory T cells (TRM) that are either CD8+ or CD4+), natural killer T (NKT) cells and inhibitory T cells. 
     A native “T cell receptor” or a native “TCR” is a heterodimeric T cell surface protein which is associated with invariant CD3 chains to form a complex capable of mediating signal transduction. TCR belongs to the immunoglobulin superfamily, and is similar to a half antibody with a single heavy chain and a single light chain. Native TCR has an extracellular portion, a transmembrane portion and an intracellular portion. The extracellular domain of a TCR has a membrane-proximal constant region and a membrane-distal variable region. 
     The term “subject” or “individual” or “animal” or “patient” as used herein refers to human or non-human animal, including a mammal or a primate, in need of diagnosis, prognosis, amelioration, prevention and/or treatment of a disease or disorder. Mammalian subjects include humans, domestic animals, farm animals, and zoo, sports, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, swine, cows, bears, and so on. 
     A. Polypeptide Complex 
     Provided herein are novel polypeptide complexes that comprise an antibody heavy chain variable domain operably linked to a first T cell receptor (TCR) constant region, and an antibody light chain variable domain operably linked to a second TCR constant region, wherein the first TCR constant region and the second TCR constant region are associated via at least one non-native interchain bond. The polypeptide complex comprises at least two polypeptide chains, each of which comprises a variable domain derived from an antibody and a constant region derived from a TCR. The two polypeptide chains of the polypeptide complexes comprise a pair of heavy chain variable domain and a light chain variable domain, which are operably linked to a pair of TCR constant regions respectively. Examples of pairs of TCR constant regions include, for example, alpha/beta, pre-alpha/beta, and gamma/delta TCR constant regions. The TCR constant regions in the polypeptide complexes provided herein can be in full length or in a fragment, and can be engineered, as long as the pair of TCR constant regions are capable of associating with each other to form a dimer. 
     It is surprisingly found that the polypeptide complexes provided herein with at least one non-native interchain bond (in particular a non-native disulphlide bond) can be recombinantly expressed and assembled into the desired conformation, which stabilizes the TCR constant region dimer while providing for good antigen-binding activity of the antibody variable regions. Moreover, the polypeptide complexes are found to well tolerate routine antibody engineering, for example, modification of glycosylation sites, and removal of some natural sequences. Furthermore, the polypeptide complexes provided herein can be incorporated into a bispecific format which can be readily expressed and assembled with minimal or substantially no mispairing of the antigen-binding sequences due to the presence of the TCR constant regions in the polypeptide complexes. Additional advantages of the polypeptide complexes and constructs provided herein will become more evident in the following disclosure below. 
     In one aspect, the present disclosure provides polypeptide complexes, comprising a first polypeptide comprising, from N-terminus to C-terminus, a first heavy chain variable domain (VH) of a first antibody operably linked to a first T cell receptor (TCR) constant region (C1), and a second polypeptide comprising, from N-terminus to C-terminus, a first light chain variable domain (VL) of the first antibody operably linked to a second TCR constant region (C2), wherein: C1 and C2 are capable of forming a dimer comprising at least one non-native interchain bond between C1 and C2, and the non-native interchain bond is capable of stabilizing the dimer, and the first antibody has a first antigenic specificity. 
     i. TCR Constant Region 
     The polypeptide complexes provided herein comprise constant regions derived from a TCR. 
     Native TCR consists of two polypeptide chains, and has in general two types: one consists of alpha and beta chains (i.e. alpha/beta TCR), and the other consists of gamma and delta chains (i.e. gamma/delta TCR). These two types are structurally similar but have distinct locations and functions. About 95% human T cells have alpha/beta TCRs, whereas the rest 5% have gamma/delta TCRs. A precursor of alpha chain is also found and named as pre-alpha chain. Each of the two TCR polypeptide chains comprises an immunoglobulin domain and a membrane proximal region. The immunoglobulin region comprises a variable region and a constant region, and is characterized by the presence of an immunoglobulin-type fold. Each TCR polypeptide chain has a cysteine residue (e.g. at C terminal of the constant domain or at N terminal of the membrane proximal region) which together can form a disulphide bond that tethers the two TCR chains together. 
       FIGS. 18A-18E  set forth the amino acid sequences of native TCR constant regions of TCR alpha, pre-alpha, beta, gamma and delta chains. For clarity and consistency, each of the amino acid residues in these sequences are numbered in  FIGS. 19A-19E , and such numbering is used throughout the present disclosure to refer to a particular amino acid residue on a particular TCR constant region. 
     Human TCR alpha chain constant region is known as TRAC, with the NCBI accession number of P01848, or an amino acid sequence of SEQ ID NO: 254. 
     Human TCR beta chain constant region has two different variants, known as TRBC1 and TRBC2 (IMGT nomenclature), with corresponding sequences set forth in SEQ ID NO: 256 and SEQ ID NO: 257, respectively (see also Toyonaga B, et al., PNAs, Vol. 82, pp. 8624-8628, Immunology (1985)). These two beta constant domains are different in the 4 th , 5 th  and 37 th  amino acid residues of exon 1. Specifically, TRBC1 has 4N, 5K and 37F in exon 1, and TRBC2 has 4K, 5N and 37Y in exon 1. 
     Specifically, the native TCR beta chain contains a native cysteine residue at position 74 (see  FIG. 19B ), which is unpaired and therefore does not form a disulphide bond in a native alpha/beta TCR. In certain embodiments, in the polypeptide complexes provided herein, this native cysteine residue is absent or mutated to another residue. This may be useful to avoid incorrect intrachain or interchain pairing. In certain embodiments, the native cysteine residue is substituted for another residue, for example serine or alanine. In certain embodiments, the substitution in certain embodiments can improve the TCR refolding efficiencies in vitro. 
     Human TCR gamma chain constant regions have two variants, known as TRGC1 and TRGC2 (see Lefranc et al., Eur. J. Immunol. 19:989-994 (1989)), with the NCBI accession number of A26659 and P03986, respectively, or amino acid sequences of SEQ ID NO: 263 and SEQ ID NO: 265, respectively. 
     Human TCR delta chain constant region is known as TRDC, with the NCBI accession number of A35591, or an amino acid sequence of SEQ ID NO: 261. 
     The constant region of TCR in the polypeptide complexes provided herein may also be derived from pre-T-cell antigen receptor (pre-TCR). Pre-TCR is expressed by immature thymocytes, which has a pivotal role in early T-cell development. Pre-TCR has a regular beta chain, but a special pre-alpha chain with only constant region available, with sequence and structure distinct from those of regular alpha chain (see Harald von Boehmer, Nat Rev Immunol, July; 5(7):571-7 (2005)). The sequence of human pre-alpha chain constant region (PTCRA) has the NCBI accession number of AAF89556.1, or an amino acid sequence of SEQ ID NO: 259. 
     In the present disclosure, the first and the second TCR constant regions of the polypeptide complexes provided herein are capable of forming a dimer comprising, between the TCR constant regions, at least one non-native interchain bond that is capable of stabilizing the dimer. 
     The term “dimer” as used herein refers to an associated structure formed by two molecules, such as polypeptides or proteins, via covalent or non-covalent interactions. A homodimer or homodimerization is formed by two identical molecules, and a heterodimer or heterodimerization is formed by two different molecules. The dimer formed by the first and the second TCR constant regions is a heterodimer. 
     An interchain bond is formed between one amino acid residue on one TCR constant region and another amino acid residue on the other TCR constant region. In certain embodiments, the non-native interchain bond can be any bond or interaction that is capable of associating two TCR constant regions into a dimer. Examples of suitable non-native interchain bond include, a disulphide bond, a hydrogen bond, electrostatic interaction, a salt bridge, or hydrophobic-hydrophilic interaction, a knobs-into-holes or the combination thereof. 
     A “disulphide bond” refers to a covalent bond with the structure R—S—S—R′. The amino acid cysteine comprises a thiol group that can form a disulphide bond with a second thiol group, for example from another cysteine residue. The disulphide bond can be formed between the thiol groups of two cysteine residues residing respectively on the two polypeptide chains, thereby forming an interchain bridge or interchain bond. 
     Electrostatic interaction is non-covalent interaction and is important in protein folding, stability, flexibility and function, including ionic interactions, hydrogen bonding and halogen bonding. Electrostatic interactions can be formed in a polypeptide, for example, between Lys and Asp, between Lys and Glu, between Glu and Arg, or between Glu, Trp on the first chain and Arg, Val or Thr on the second chain. 
     A salt bridge is close-range electrostatic interactions that mainly arises from the anionic carboxylate of either Asp or Glu and the cationic ammonium from Lys or the guanidinium of Arg, which are spatially proximal pairs of oppositely charged residues in native protein structures. Charged and polar residues in largely hydrophobic interfaces may act as hot spots for binding. Among others, residues with ionizable side chains such as His, Tyr, and Ser can also participate the formation of a salt bridge. 
     A hydrophobic interaction can be formed between one or more Val, Tyr and Ala on the first chain and one or more Val, Leu, and Trp on the second chain, or His and Ala on the first chain and Thr and Phe on the second chain (see Brinkmann, et al., 2017, Supra). 
     A hydrogen bond is formed by electrostatic attraction between two polar groups when a hydrogen atom covalently bound to a highly electronegative atom such as nitrogen, oxygen, or fluorine. A hydrogen bond can be formed in a polypeptide between the backbone oxygens (e.g. chalcogen groups) and amide hydrogens (nitrogen group) of two residues, respectively, such as a nitrogen group in Asn and an oxygen group in His, or an oxygen group in Asn and a nitrogen group in Lys. A hydrogen bond is stronger than a Van der Waals interaction, but weaker than covalent or ionic bonds, and is critical in maintaining the secondary structure and tertiary structure. For example, an alpha helix is formed when the spacing of amino acid residues occurs regularly between positions i and i+4, and a beta sheet is a stretch of peptide chain 3-10 amino acids long formed when two peptides joined by at least two or three backbone hydrogen bonds, forming a twisted, pleated sheet. 
     “Knobs-into-holes” as used herein, refers to an interaction between two polypeptides, where one polypeptide has a protuberance (i.e. “knob”) due to presence of an amino acid residue having a bulky side chain (e.g. tyrosine or tryptophan), and the other polypeptide has a cavity (i.e. “hole”) where a small side chain amino acid residue resides (e.g. alanine or threonine), and the protuberance is positionable in the cavity so as to promote interaction of the two polypeptides to form a heterodimer or a complex. Methods of generating polypeptides with knobs-into-holes are known in the art, e.g., as described in U.S. Pat. No. 5,731,168. 
     In certain embodiments, the TCR constant region dimer comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 non-native interchain bonds. Optionally, at least one of the 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 non-native interchain bonds are disulphide bonds, hydrogen bonds, electrostatic interaction, salt bridge, or hydrophobic-hydrophilic interaction, or any combination thereof. 
     A “non-native” interchain bond as used herein refers to an interchain bond which is not found in a native association of the native counterpart TCR constant regions. For example, a non-native interchain bond can be formed between a mutated amino acid residue and a native amino acid residue, each residing on a respective TCR constant region; or alternatively between two mutated amino acid residues residing respectively on the TCR constant regions. In certain embodiments, the at least one non-native interchain bond is formed between a first mutated residue comprised in the first TCR constant region and a second mutated residue comprised in the second TCR constant region of the polypeptide complex. 
     A “mutated” amino acid residue refers to one which is substituted, inserted or added and is different from its native counterpart residue in a corresponding native TCR constant region. For example, if an amino acid residue at a particular position in the wild-type TCR constant region is referred to as the “native” residue, then its mutated counterpart is any residue that is different from the native residue but resides at the same position on the TCR constant region. A mutated residue can be a different residue which substitutes the native residue at the same position, or which is inserted before the native residue and therefore takes up its original position. 
     In certain embodiments, the mutated residue may be a naturally-occurring amino acid residue. In certain embodiments, at least one of the first and the second non-native amino acid residues is a mutated cysteine residue. In certain embodiments, one or more of the non-native interchain bond is a disulphide bond. In certain embodiments, the non-native disulphide bond can be formed between two mutated cysteine residues comprised in the first and the second TCR constant regions respectively. 
     In certain embodiments, at least one of the first and the second mutated residues is a non-naturally-occurring amino acid residue. A non-naturally-occurring amino acid residue refers to an amino acid residue that is not naturally found in human proteins but can be expressed via a nucleic acid codon that can be incorporated into the encoding polynucleotide. For example, non-naturally occurring amino acid such as  L -3,4-dihydroxyphenylalanine ( L -DOPA) can react and crosslink to natural amino acids such as cysteine, histidine and lysine by periodate induced oxidation. It has been shown that by incorporating  L -DOPA into an antibody, the non-natural amino acid was able to effectively crosslink to residues on the antigen, resulting in a covalently bonded antibody-antigen complex (Xu, J. et al., 2014, Structure-based non-canonical amino acid design to covalently crosslink an antibody-antigen complex. Journal of Structural Biology, 185(2), pp. 215-222.). It is contemplated herein that the mutated amino acid residue in the first and/or the second TCR constant regions may comprise a non-naturally occurring amino acid residue such as  L -DOPA which can crosslink with a natural amino acid residue (or alternatively a non-naturally occurring amino acid residue) to form a non-native interchain covalent bond. 
     In certain embodiments, at least one non-native disulphide bonds is formed between a mutated cysteine residue and a native cysteine residue. In certain embodiments, the non-native disulphide bonds are formed between two mutated cysteine residues. In certain embodiments, at least one of the cysteine residues forming the non-native disulphide bond is a mutated cysteine residue. In certain embodiments, both of the cysteine residues forming the non-native disulphide bond are mutated cysteine residues on the first and the second TCR constant regions respectively. 
     In certain embodiments, the first and/or the second TCR constant regions can be engineered to comprise one or more mutated amino acid residues that is responsible for forming the non-native interchain bond. To introduce such a mutated residue to the TCR constant region, an encoding sequence of a TCR region can be manipulated to for example, substitute a codon encoding a native residue for the codon encoding the mutated residue, or to insert a codon encoding the mutated residue before the codon of the native residue. One or more desired mutated amino acid residues can be introduced to the TCR constant region, for example, one or more amino acid residue (e.g. cysteine residue) that is capable of forming a disulphide bond, that may lead to electrostatic interactions between the two TCR constant regions, that may increase the flexibility of the TCR constant regions, that position at least one of the covalent bond forming amino acids away from the TCR constant domain, such as a hydrogen bond, that may contribute to formation of a salt bridge; hydrophobic amino acid residues capable of leading to hydrophobic interactions; and hydrophilic amino acid residues capable of leading to hydrophilic interactions, and so on. 
     In certain embodiments, the first and/or the second TCR constant regions can be engineered to comprise one or more mutated cysteine residues. For example, a non-cysteine residue can be replaced to a cysteine residue, or a cysteine residue can be inserted in between two originally adjacent native non-cysteine residues. The positions of replacement can be determined such that, after replacement to cysteine residues, a non-native interchain disulphide bond could be formed between the two TCR constant regions. To this end, multiple factors can be considered, including, for example, the cysteine residues forming the disulphide bond may be in sufficiently close proximity, may have suitable alpha-beta bond orientation, the thiol groups of the cysteine residues may be oriented to face each other, the residue to be replaced may have a side chain with relatively similar chemical property to that of cysteine, and/or the replacement would not substantially perturb the tertiary structure of the TCR constant region or the polypeptide complex itself. 
     A skilled person in the art may determine the distance and angle between two amino acid residues to be replaced using suitable methods known in the art, for example without limitation, distance maps by photodetection, computer modelling, NMR spectroscopy or X-ray crystallography. In an illustrative example, for an interested polypeptide (such as a TCR constant region), its protein crystal structure can be obtained from public databases such as PDB database, or alternatively be elucidated using methods such as X-ray crystallography. Suitable computer software can be used to determine distances and angles between amino acid residues based on the protein crystal structure data. In certain embodiments, in the polypeptide complex provided herein, a disulphide bond can be formed between mutated cysteine residues having respective beta carbons sufficiently close, for example, a distance less than 8 angstroms, 7 angstroms, 6 angstroms, 5 angstroms, 4 angstroms, 3 angstroms, 2 angstroms, 1 angstrom, or less when the complex is correctly folded. 
     Further suitable positions for engineering to the first and/or the second TCR constant regions can be taken from the crystal structure data published on the complex between TCR alpha and beta (Boulter, J. M. et al., Protein engineering, 16(9), pp. 707-711 (2003)), or gamma and delta (Allison, T. J. et al., Nature, 411(6839), pp. 820-824 (2001); Uldrich, A. P. et al., Nature Immunology, 14(11), pp. 1137-1145 (2013)). Once the residue to be replaced are determined, a skilled person can readily identify the interested codon to be mutated (for example through sequence alignment using existing software such as ClustalW (European Bioinformatics Institute website (www.ebi.ac.uk/index.html)), and then mutate it to cysteine codon by methods known in the art such as PCR mutagenesis. 
     Formation of the interchain disulphide bond can be determined by suitable methods known in the art. For example, the expressed protein product can be subject to reduced and non-reduced SDS-PAGE respectively, followed by comparison of the resulting bands to identify potential difference which indicates presence of interchain disulphide bond. 
     The non-native interchain bond is capable of stabilizing the polypeptide complex. Such effects in stablization can be embodied in various ways. For example, the presence of the mutated amino acid residue or the non-native interchain bond can enable the polypeptide complex to stably express, and/or to express in a high level, and/or to associate into a stable complex having the desired biological activity (e.g. antigen binding activity), and/or to express and assemble into a high level of desired stable complex having the desired biological activity. The capability of the interchain bond to stabilize the first and the second TCR constant regions can be assessed using proper methods known in the art, such as the molecular weight displayed on SDS-PAGE, or thermostability measured by differential scanning calorimetry (DSC) or differential scanning fluorimetry (DSF). In an illustrative example, formation of a stable polypeptide complex provided herein can be confirmed by SDS-PAGE, if a product shows a molecular weight comparable to the combined molecular weight of the first and the second polypeptides. In certain embodiments, the polypeptide complex provided herein is stable in that its thermal stability is no less than 50%, 60%, 70%, 80%, or 90% of that of a natural Fab. In certain embodiments, the polypeptide complex provided herein is stable in that its thermal stability is comparable to that of a natural Fab. 
     Without wishing to be bound by any theory, it is believed that the non-native interchain bond (such as a disulphide bond) formed between the first and the second TCR constant regions in the polypeptide complexes are capable of stabilizing the heterodimer of TCR constant regions, thereby enhancing the level of correct folding, the structural stability and/or the expression level of the heterodimer and of the polypeptide complexes. Unlike native TCR anchored on the membrane of T cell surface, heterodimers of native TCR extracellular domains are found to be much less stable, despite of its similarity to antibody Fab in 3D structure. As a matter of fact, the instability of native TCR in soluble condition used to be a significant obstacle that prevents elucidation of its crystal structure (see Wang,  Protein Cell,  5(9), pp. 649-652 (2014)). By introducing a pair of Cysteine (Cys) mutations in TCR constant regions and thereby enabling formation of interchain non-native disulphide bond, the polypeptide complexes can be stably expressed while in the meantime the antigen-binding capabilities of the antibody variable region are retained. 
     The TCR constant region comprising a mutated residue is also referred to herein as an “engineered” TCR constant region. In certain embodiments, the first TCR constant region (C1) of the polypeptide complex comprises an engineered TCR Alpha chain (CAlpha), and the second TCR constant region (C2) comprises an engineered TCR Beta chain (CBeta). In certain embodiments, C1 comprises an engineered CBeta, and C2 comprises an engineered CAlpha. In certain embodiments, C1 comprises an engineered TCR Pre-Alpha chain (CPre-Alpha), and C2 comprises an engineered CBeta. In certain embodiments, C1 comprises an engineered CBeta, and C2 comprises an engineered CPre-Alpha. In certain embodiments, C1 comprises an engineered TCR Gamma chain (CGamma), and C2 comprises an engineered TCR Delta chain (CDelta). In certain embodiments, C1 comprises an engineered CDelta, and C2 comprises an engineered CGamma. 
     In certain embodiments, the engineered TCR constant region comprises one or more mutated cysteine residue. In certain embodiments, the one or more mutated residue is comprised within a contact interface of the first and/or the second engineered TCR constant regions. 
     The term “contact interface” as used herein refers to the particular region(s) on the polypeptides where the polypeptides interact/associate with each other. A contact interface comprises one or more amino acid residues that are capable of interacting with the corresponding amino acid residue(s) that comes into contact or association when interaction occurs. The amino acid residues in a contact interface may or may not be in a consecutive sequence. For example, when the interface is three-dimensional, the amino acid residues within the interface may be separated at different positions on the linear sequence. 
     In certain embodiments, the engineered CBeta comprises a mutated cysteine residue within a contact interface selected from the group consisting of: amino acid residues 9-35, 52-66, 71-86, and 122-127. In certain embodiments, the engineered CAlpha comprises a mutated cysteine residue within a contact interface selected from a group consisting of: amino acid residues 6-29, 37-67, and 86-95. Unless specified, the numbering of amino acid residues in the TCR constant region in the present disclosure is as set forth in  FIGS. 19A-19E . 
     In certain embodiments, one or more disulphide bonds can be formed between the engineered CAlpha and the engineered CBeta. The mutated cysteine residue in CBeta can be a substitution selected from the group consisting of: S56C, S16C, F13C, V12C, E14C, F13C, L62C, D58C, S76C, and R78C, and/or the mutated cysteine residues in CAlpha can be a substitution selected from the group consisting of: T49C, Y11C, L13C, S16C, V23C, Y44C, T46C, L51C, and S62C. In certain embodiments, the pair of mutated cysteine residues can be a pair of substitutions selected from the group consisting of: S16C in CBeta and Y11C in CAlpha, F13C in CBeta and L13C in CAlpha, S16C in CBeta and L13C in CAlpha, V12C in CBeta and S16C in CAlpha, E14C in CBeta and S16C in CAlpha, F13C in CBeta and V23C in CAlpha, L62C in CBeta and Y44C in CAlpha, D58C in CBeta and T46C in CAlpha, S76C in CBeta and T46C in CAlpha, S56C in CBeta and T49C in CAlpha, S56C in CBeta and L51C in CAlpha, S56C in CBeta and S62C in CAlpha, and R78C in CBeta and S62C in CAlpha, and wherein the pair of cysteine residues are capable of forming a non-native interchain disulphide bond. 
     As used herein throughout the application, “XnY” with respect to a TCR constant region is intended to mean that the n th  amino acid residue X on the TCR constant region (based on the numbering in  FIGS. 19A-19E  as provided herein) is replaced by amino acid residue Y, where X and Y are respectively the one-letter abbreviation of a particular amino acid residue. It should be noted that the number n is solely based on the numbering provided in  FIGS. 19A-19E , and it could appear different from its actual position. To illustrate, the sequence of CBeta(S56C)(N69Q) shown in SEQ ID NO: 34 is used as an example. While the substitution of S to C occurs at the 48 th  residue in SEQ ID NO:34, the very residue is designated as the 56 th  residue based on the numbering system in  FIGS. 19A-19E , and therefore that substitution of S to C is designated as S56C, but not S48C. Similarly, the substitution of N to Q is also designated as N69Q based on the numbering system in  FIGS. 19A-19E . This designation rule of amino acid residue substitution applies to all TCR constant region in the present disclosure, unless otherwise specified. Similarly, “XnY” when used with respect to an Fc region, is intended to mean that the n th  amino acid residue X on the Fc constant region (based on the numbering in  FIGS. 20A-20D  as provided herein) is replaced by amino acid residue Y. 
     In certain embodiments, the engineered CBeta comprises or is any one of SEQ ID NOs: 33-41, and the engineered CAlpha comprises or is any one of SEQ ID NOs: 43-48. 
     In certain embodiments, one or more non-native disulphide bonds can be formed within the contact interfaces between CPre-Alpha and CBeta. In certain embodiments, the contact interface on CPre-Alpha is selected from substitutions at position amino acid residues 7-19, 26-34, 56-75 and 103-106. In certain embodiments, the contact interface on CBeta is selected from substitutions at position amino acid residues 9-35, 52-66, 71-86 and 122-127. 
     In certain embodiments, one or more disulphide bonds can be formed between the engineered Pre-TCR alpha constant region (CPre-Alpha) and beta chain constant region (CBeta). The mutated cysteine residues in CBeta can be a substitution selected from the group consisting of: S16C, A18C, E19C, F13C, A11C, S56C, and S76C, and/or the mutated cysteine residues in CPre-Alpha can be a substitution selected from the group consisting of: S11C, A13C, I16C, S62C, T65C, and Y59. In certain embodiments, the pair of mutated cysteine residues can be a pair of substitutions selected from the group consisting of: S16C in CBeta and S11C in CPre-Alpha, A18C in CBeta and S11C in CPre-Alpha, E19C in CBeta and S11C in CPre-Alpha, F13C in CBeta and A13C in CPre-Alpha, S16C in CBeta and A13C in CPre-Alpha, A11C in CBeta and I16C in CPre-Alpha, S56C in CBeta and S62C in CPre-Alpha, S56C in CBeta and T65C in CPre-Alpha, and S76C in CBeta, and Y59C in CPre-Alpha, and wherein the pair of mutated cysteine residues are capable of forming a non-native interchain disulphide bond. 
     In certain embodiments, the engineered CBeta comprises or is any one of SEQ ID NOs: 33-41, and the engineered CPre-Alpha comprises or is any one of SEQ ID NOs: 82 and 83. 
     In certain embodiments, one or more non-native disulphide bonds can be formed within the contact interfaces between CGamma and CDelta. In certain embodiments, the contact interface on CGamma is selected from substitutions at position amino acid residues 11-35 and 55-76. In certain embodiments, the contact interface on CDelta is selected from substitutions at position amino acid residues 8-26, 43-64, and 84-88. 
     In certain embodiments, one or more disulphide bonds can be formed between the engineered CGamma and CDelta. The mutated cysteine residue in CGamma can be a substitution selected from the group consisting of: S17C, E20C, F14C, T12C, M62C, Q57C, and A19C, and/or the mutated cysteine residues in CDelta can be a substitution selected from the group consisting of: F12C, M14C, N16C, D46C, V50C, F87C, and E88C. In certain embodiments, the pair of mutated cysteine residues can be a pair of substitutions selected from the group consisting of: S17C in CGamma and F12C in CDelta, E20C in CGamma and F12C in CDelta, F14C in CGamma and M14C in CDelta, T12C in CGamma and N16C in CDelta, M62C in CGamma and D46C in CDelta, Q57C in CGamma and V50C in CDelta, A19C in CGamma and F87C in CDelta, and A19C in CGamma and E88C in CDelta, and wherein the introduced pair of cysteine residues are capable of forming an interchain disulphide bond. 
     In certain embodiments, the engineered CGamma comprises or is any one of SEQ ID NOs: 113 and 114, and the engineered CDelta comprises or is any one of SEQ ID NOs: 115 and 116. 
     In addition to the non-native amino acid residue, the engineered TCR constant region in certain embodiments may further comprise an additional modification to one or more native residues in the wild-type TCR constant region sequence. Examples of such additional modification include, such as modification to a native cysteine residue, modification to a native glycosylation site, and/or modification to a native loop. 
     Certain native TCR constant regions (such as CBeta) comprise a native cysteine residue which, in some embodiments of the present disclosure could be modified (e.g. removed), or alternatively could be kept in some other embodiments. In certain embodiments, a native disulphide bond on the alpha/beta heterodimeric TCR between the TRAC and TRBC1 or TRBC2 constant domain, i.e. between Cys4 of exon 2 of TRAC and Cys2 of exon 2 of TRBC1 or TRBC2, according to IMGT TCR nomenclature, may be present or absent. 
     In certain embodiments, at least one native cysteine residue is absent or present in the engineered CBeta. For example, the native cysteine residue at position C74 of CBeta may be present or absent in the engineered CBeta. In certain embodiments, the engineered CBeta in which the native cysteine residue C74 is absent comprises or is any one of SEQ ID NOs: 32-41. 
     Without wishing to be bound by any theory, but it is believed that the polypeptide complex provided herein is advantageous in that it tolerates both presence and absence of the native cysteine residue on the CBeta. Although it was suggested (see, for example, U.S. Pat. No. 7,666,604) that presence of the native cysteine residues on soluble TCR heterodimers is detrimental to the ligand binding ability of the TCR, the polypeptide complex provided herein can tolerate presence of this native cysteine residue without negatively affecting its antigen-binding activity. Furthermore, the polypeptide complex provided herein in the absence of the native cysteine residue expressed at high level, despite of the contrary teachings by Wu et al. mAbs, 7(2), pp. 364-376 (2005) that native disulphide bond in the TCR heterodimer is good for stabilizing the TCR heterodimer. 
     In certain embodiments, one or more native glycosylation site present in the native TCR constant regions may be modified (e.g. removed) or kept in the polypeptide complex provided in the present disclosure. The term “glycosylation site” as used herein with respect to a polypeptide sequence refers to an amino acid residue with a side chain to which a carbohydrate moiety (e.g. an oligosaccharide structure) can be attached. Glycosylation of polypeptides like antibodies is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue, for example, an asparagine residue in a tripeptide sequence such as asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline. O-linked glycosylation refers to the attachment of one of the sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly to serine or threonine. Removal of native glycosylation sites can be conveniently accomplished by altering the amino acid sequence such that one or more of the above-described tripeptide sequences (for N-linked glycosylation sites) or one or more serine or threonine residues (for O-linked glycosylation sites) are substituted. 
     In certain embodiments, in the polypeptide complex provided herein, at least one native glycosylation site is absent or present in the engineered TCR constant regions, for example, in the first and/or the second TCR constant regions. Without wishing to be bound by any theory, but it is believed that the polypeptide complex provided herein can tolerate removal of all or part of the glycosylation sites without affecting the protein expression and stability, in contrast to existing teachings that presence of N-linked glycosylation sites on TCR constant region, such as CAlpha (i.e. N34, N68, and N79) and CBeta (i.e. N69) are necessary for protein expression and stability (see Wu et al., Mabs, 7:2, 364-376, 2015). 
     In certain embodiments, in the polypeptide complex provided herein, at least one of the N-glycosylation sites in the engineered CAlpha, e.g. N34, N68, N79 and N61 are absent or present. In certain embodiments, the engineered CAlpha sequences absent of a glycosylation site comprises or is any one of SEQ ID NOs: 44-48. In certain embodiments, at least one of the N-glycosylation sites in the engineered CBeta, e.g. N69, is absent or present. The engineered CBeta sequences (TRBC1) absent of glycosylation site comprises or is any one of SEQ ID NOs: 34-36. The engineered CBeta sequences (TRBC2) absent of a glycosylation site comprises or is any one of SEQ ID NOs: 38-40. 
     In certain embodiments, in the polypeptide complex provided herein, at least one of the N-glycosylation sites in the engineered CPre-Alpha, e.g. N50, is absent or present. The engineered CPre-Alpha sequence absent of a glycosylation site comprises or is SEQ ID NO: 83. 
     In certain embodiments, in the polypeptide complex provided herein, at least one of the N-glycosylation sites in the engineered CGamma, e.g. N65, is absent or present. In certain embodiments, the engineered CGamma sequence absent of a glycosylation site comprises or is SEQ ID NO: 114. In certain embodiments, at least one of the N-glycosylation sites in the engineered CDelta, e.g. N16 and N79 is absent or present. The engineered CDelta sequence absent of glycosylation site comprises or is SEQ ID NO: 116. 
     In certain embodiments, one or more native secondary structure present in the native TCR constant regions may be modified (e.g. removed) or kept in the polypeptide complex provided in the present disclosure. In certain embodiments, a native loop (such as FG loop and/or DE loop of native CBeta) is modified (e.g. removed) or kept in the polypeptide complex provided herein. The term “FG loop” and “DE loop” are structures mainly found in the TCR beta chain constant domain. The FG loop encompasses amino acid residues 101-117 of the native CBeta and is an unusually elongated, solvent-exposed structural element that forms one component of an alpha/beta TCR cavity against CD3. The DE loop encompasses amino acid residues 66-71 of the native CBeta. Alignment of sequence of TCR beta chain constant region with that of an immunoglobulin CH1 constant region revealed that the FG loop of TCR beta chain constant region are significantly longer.  FIG. 3  shows the differences of constant regions between T cell beta chain and antibody heavy chain. In certain embodiments, the sequence at FG loop (YGLSENDEWTQDRAKPVT, SEQ ID NO: 79) is absent and/or replaced with YPSN (SEQ ID NO: 80). In certain embodiments, the sequence at native DE loop (QPALNDSR, SEQ ID NO: 88) is absent and/or replaced with QSGR (SEQ ID NO: 87). In certain embodiments, the CBeta sequences absent of native FG loop comprises or is any one of SEQ ID NOs: 37-40. In certain embodiments, the CBeta sequence absent of both native FG loop and native DE loop comprises or is SEQ ID NO: 41. 
     In the polypeptide complex provided herein, the constant regions derived from a TCR are operably linked to the variable regions derived from an antibody. The heavy chain or light chain variable region of an antibody can be operably linked to a TCR constant region, with or without a spacer. 
     In certain embodiments, the first antibody variable domain (VH) is fused to the first TCR constant region (C1) at a first conjunction domain, the first antibody variable domain (VL) is fused to the second TCR constant region (C2) at a second conjunction domain. 
     “Conjunction domain” as used herein refers to a boundary or border region where two amino acid sequences are fused or combined. In certain embodiments, the conjunction domain comprises at least a portion of C terminal fragment from a first fusion partner, fused to at least a portion of N terminal fragment from a second fusion partner, with or without a spacer in between. In such embodiments, the conjunction domain comprises fragments of both fusion partners, and the fusion point resides at the point where the two fragments link to each other, for example, directly or via a spacer. In certain other embodiments, the conjunction domain consists of a fragment of one fusion partner. In such embodiments, the fusion point could be at either end of the conjunction domain. 
     In certain embodiments, the first conjunction domain comprises at least a portion of the C terminal fragment of an antibody V/C conjunction, and at least a portion of the N-terminal fragment of a TCR V/C conjunction. 
     The term “antibody V/C conjunction” as used herein refers to the boundary of antibody variable domain and constant domain, for example, the boundary between heavy chain variable domain and the CH1 domain, or between light chain variable domain and the light chain constant domain. Similarly, the term “TCR V/C conjunction” refers to the boundary of TCR variable domain and constant domain, for example, the boundary between TCR Alpha variable domain and constant domain, or between TCR Beta variable domain and constant domain. 
     If the Fv region of an immunoglobulin is aligned with a TCR immunoglobulin-like domain, the antibody V/C conjunction and the TCR V/C conjunction would also be aligned. An example is given in Table 1 below, where antibody heavy chain V/C conjunction (SEQ ID NO: 270) is aligned to TCR Beta V/C conjunction (SEQ ID NO: 271), and antibody light chain V/C conjunction (SEQ ID NO: 272) is aligned to TCR Beta V/C conjunction (SEQ ID NO: 273). 
     The first and/or the second conjunction domains of the polypeptide complex as provided herein can be selected such that it comprises a proper length (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues) of the C terminal fragment of antibody V/C conjunction, and a proper length (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues) of the N terminal fragment of TCR V/C conjunction. For example, as shown in Table 1, the conjunction domain may be selected to have all sequence from the TCR V/C conjunction (see, e.g. SEQ ID NO: 145), or most sequence (see, e.g. SEQ ID NO: 147), or some sequence (see, e.g. SEQ ID NO: 146) from the TCR V/C conjunction. Still using Table 1 as an example, the conjunction domain may comprise more residues from TCR V/C conjunction than from antibody V/C conjunction (see, e.g. SEQ ID NO:147), or vice versa (see, e.g. SEQ ID NO:146). 
     In certain embodiments, the first and/or the second conjunction domains of the polypeptide complex as provided herein has a total length comparable to that of the antibody V/C conjunction or that of the TCR V/C conjunction. 
     A proper conjunction domain can be determined on a structural basis. For example, the three-dimensional structures of antibody and TCR may be superimposed, and overlappings of the antibody V/C conjunction and the TCR V/C conjunction on the superimposed structure may be determined and considered when determining the length or proportion of sequences from antibody or TCR V/C conjunction. 
     In certain embodiments, the first and/or the second conjunction domain comprises a spacer in between the fragments from antibody V/C conjunction and TCR V/C conjunction. Any suitable sequences or length of spacer sequences can be used, as long as it does not negatively affect the antigen binding or stability of the polypeptide complex. 
     Exemplary sequences of antibody variable/constant domain boundary, and TCR variable/constant domain boundary, and the antibody variable/TCR constant region boundary are provided in the below Tables 1-6. 
     In certain embodiments, C1 comprises an engineered CBeta and C2 comprises an engineered CAlpha. To illustrate, Table 1 shows the exemplary designs for the conjunction domains useful for antibody VH fused to TCR CBeta, or for antibody VL fused to TCR CAlpha. The antibody VH/constant domain boundary is aligned to TCR variable/CBeta boundary, and antibody VL/constant domain boundary is aligned to TCR variable/CAlpha boundary. Exemplary designs of the conjunction domains are also provided in an alignment form (see, e.g., SEQ ID NO: 144, 145, 146, or 147), with the first or the second conjunction domain shown with underline. In such embodiments, the first conjunction domain comprises or is SEQ ID NO: 49 or 50. In such embodiments, the second conjunction domain comprises or is SEQ ID NO: 51 or 52. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 First and second conjunction domain designs for VH-CBeta/VL-CAlpha 
               
            
           
           
               
               
               
               
            
               
                   
                 Conjunction 
                   
                 Conjunction 
               
               
                   
                 (heavy chain) 
                   
                 (light chain) 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 Variable 
                 Constant 
                   
                 Variable 
                 Constant 
               
               
                   
               
               
                 Antibody_VH 
                 ......LVTVSS 
                 AS--TKGPS...... 
                 Antibody_VL 
                 ......KVEIK 
                 RTVAAPSVF...... 
               
               
                 SEQ ID NO: 270 
                   
                   
                 SEQ ID NO: 272 
                   
                   
               
               
                   
               
               
                 TCR_beta 
                 ...... RLTVLE   
                   DLKNVFPPE ...... 
                 TCR_alpha 
                 ...... KLIIK   
                   PDIQNPDPA ...... 
               
               
                 SEQ ID NO: 271 
                   
                   
                 SEQ ID NO: 273 
                   
                   
               
               
                   
               
               
                 H_Conjunction_1 
                 ......LVTV SS   
                   AS KNVFPPE   ...... 
                 L_Conjunction_1 
                 ......KVEI K   
                 RTVAA   PDPA   ...... 
               
               
                 SEQ ID NO: 144 
                   
                   
                 SEQ ID NO: 146 
                   
                   
               
               
                   
               
               
                 H_Conjunction_2 
                 ......LVTV   LE     
                     DLKNVFPPE   ...... 
                 L_Conjunction_2 
                 ......KVEI K   
                     PDIQNPDPA   ...... 
               
               
                 SEQ ID NO: 145 
                   
                   
                 SEQ ID NO: 147 
               
               
                   
               
            
           
         
       
     
     In certain embodiments, C1 comprises an engineered CAlpha and C2 comprises an engineered CBeta. Table 2 shows the exemplary designs for the conjunction domains useful for antibody VH fused to TCR CAlpha, or for antibody VL fused to TCR CBeta. The antibody VH/constant domain boundary is aligned to TCR variable/CAlpha boundary, and antibody VL/constant domain boundary is aligned to TCR variable/CBeta boundary. Exemplary designs of the conjunction domains are also provided in an alignment form (see, e.g., SEQ ID NO: 148, 149, or 150), with the first or the second conjunction domain shown with underline. In such embodiments, the first conjunction domain comprises or is SEQ ID NO: 129 or 130. In such embodiments, the second conjunction domain comprises or is SEQ ID NO: 49 or 50. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 First and second conjunction domain designs for VH-CAlpha/VL-CBeta 
               
            
           
           
               
               
               
               
            
               
                   
                 Conjunction 
                   
                 Conjunction 
               
               
                   
                 (heavy chain) 
                   
                 (light chain) 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 Variable 
                 Constant 
                   
                 Variable 
                 Constant 
               
               
                   
               
               
                 Antibody_H 
                 ......LVTVSS 
                 --ASTKGPS...... 
                 Antibody_L 
                 ......KVEIK-- 
                 RT--- 
               
               
                 SEQ ID NO: 274 
                   
                   
                 SEQ ID NO: 276 
                   
                 VAAPSVF...... 
               
               
                   
               
               
                 TCR_alpha 
                 ...... KLIIK -- 
                   PDIQNPDPA ...... 
                 TCR_beta 
                 ...... RLTVLE   
                 - DLKNVEPPE ...... 
               
               
                 SEQ ID NO: 275 
                   
                   
                 SEQ ID NO: 277 
                   
                   
               
               
                   
               
               
                 H_Conjunction_3 
                 ......LVTV SS   
                   AS IQNPDPA   ...... 
                 L_Conjunction_3 
                 ......KVEI K LE     
                 -   DLKNVEPPE   ...... 
               
               
                 SEQ ID NO: 148 
                   
                   
                 SEQ ID NO: 150 
                   
                   
               
               
                   
               
               
                 H_Conjunction_4 
                 ......LVTV SS   
                     PDIQNPDPA   ...... 
                 L_Conjunction_4 
                 ......KVEI K LE     
                 -   DLKNVEPPE   ...... 
               
               
                 SEQ ID NO: 149 
                   
                   
                 SEQ ID NO: 150 
               
               
                   
               
            
           
         
       
     
     In certain embodiments, C1 comprises an engineered CBeta and C2 comprises an engineered CPre-Alpha. Table 3 shows the exemplary designs for the conjunction domains useful for antibody VH fused to TCR CBeta, or for antibody VL fused to TCR CPre-Alpha. The antibody VH/constant domain boundary is aligned to TCR variable/CBeta boundary, and antibody VL/constant domain boundary is aligned to TCR variable/CPre-Alpha boundary. Exemplary designs of the conjunction domains are also provided in an alignment form (see, e.g., SEQ ID NO: 170, 171, 169, or 156), with the first or the second conjunction domain shown with underline. In such embodiments, the first conjunction domain comprises or is SEQ ID NO: 49 or 50. In such embodiments, the second conjunction domain comprises or is SEQ ID NO: 81 or 131. 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 First and second conjunction domain designs for VH-CBeta/VL-CPre-Alpha 
               
            
           
           
               
               
               
               
            
               
                   
                 Conjunction 
                   
                 Conjunction 
               
               
                   
                 (heavy chain) 
                   
                 (light chain) 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 Variable 
                 Constant 
                   
                 Variable 
                 Constant 
               
               
                   
               
               
                 Antibody_H 
                 ......LVTVSS 
                 AS--TKGPS...... 
                 Antibody_L 
                 ......KVEIK 
                 RTVAAPSVF...... 
               
               
                 SEQ ID NO: 283 
                   
                   
                 SEQ ID NO: 285 
                   
                   
               
               
                   
               
               
                 TCR_beta 
                 ...... RLTVLE   
                   DLKNVFPPE ...... 
                 TCR_alpha 
                 ......KLIIK 
                 PDIQNPDPA...... 
               
               
                 SEQ ID NO: 284 
                   
                   
                 SEQ ID NO: 286 
                   
                   
               
               
                   
               
               
                   
                   
                   
                 Pre-TCR_alpha 
                 ...... GCPAL   
                   PTGVGGTPF ...... 
               
               
                   
                   
                   
                 SEQ ID NO: 287 
                   
                   
               
               
                   
               
               
                 H_Conjunction_A 
                 ......LVTV SS   
                   AS KNVFPPE   ...... 
                 L_Conjunction_A 
                 ......KVEI K   
                   RTVAA GTPF   ...... 
               
               
                 SEQ ID NO: 170 
                   
                   
                 SEQ ID NO: 169 
                   
                   
               
               
                   
               
               
                 H_Conjunction_B 
                 ......LVTV   LE     
                     DLKNVFPPE   ...... 
                 L_Conjunction_B 
                 ......KVEI K   
                     PTGVGGTPF   ...... 
               
               
                 SEQ ID NO: 171 
                   
                   
                 SEQ ID NO: 156 
               
               
                   
               
            
           
         
       
     
     In certain embodiments, C1 comprises an engineered CPre-Alpha and C2 comprises an engineered CBeta. Table 4 shows the exemplary designs for the conjunction domains useful for antibody VH fused to TCR CPre-Alpha, or for antibody VL fused to TCR CBeta. The antibody VH/constant domain boundary is aligned to TCR variable/CPre-Alpha boundary, and antibody VL/constant domain boundary is aligned to TCR variable/CBeta boundary. Exemplary designs of the conjunction domains are also provided in an alignment form (see, e.g., SEQ ID NO: 172, 173, 174, or 175), with the first or the second conjunction domain shown with underline. In such embodiments, the first conjunction domain comprises or is SEQ ID NO: 81, 131, 132, or 133. In such embodiments, the second conjunction domain comprises or is SEQ ID NO: 49 or 50. 
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 First and second conjunction domain designs for VH-CPre-Alpha/VL-CBeta 
               
            
           
           
               
               
               
               
            
               
                   
                 Conjunction 
                   
                 Conjunction 
               
               
                   
                 (heavy chain) 
                   
                 (light chain) 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 Variable 
                 Constant 
                   
                 Variable 
                 Constant 
               
               
                   
               
               
                 Antibody_H 
                 ......LVTVSS 
                 --ASTKGPS...... 
                 Antibody_L 
                 ......KVEIK-- 
                 RTVAAPSVF...... 
               
               
                 SEQ ID NO: 288 
                   
                   
                 SEQ ID NO: 291 
                   
                   
               
               
                   
               
               
                 TCR-alpha 
                 ......KLIIK-- 
                 PDIQNPDPA...... 
                 TCR_beta 
                 ...... RLTVLE   
                 - DLKNVEPPE ...... 
               
               
                 SEQ ID NO: 289 
                   
                   
                 SEQ ID NO: 292 
                   
                   
               
               
                   
               
               
                 Pre-TCR_alpha 
                 ...... GCPAL -- 
                   PTGVGGTPF ...... 
                   
                   
                   
               
               
                 SEQ ID NO: 290 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 H_Conjunction_C 
                 ......LVTV SS   
                   AS GVGGTPF   ...... 
                 L_Conjunction_C 
                 ......KVEI K LE     
                 -   DLKNVEPPE   ...... 
               
               
                 SEQ ID NO: 172 
                   
                   
                 SEQ ID NO: 174 
                   
                   
               
               
                   
               
               
                 H_Conjunction_D 
                 ......LVTV SS   
                     PTGVGGTPF   ...... 
                 L_Conjunction_D 
                 ......KVEI K LE     
                 -   DLKNVEPPE   ...... 
               
               
                 SEQ ID NO: 173 
                   
                   
                 SEQ ID NO: 175 
               
               
                   
               
            
           
         
       
     
     In certain embodiments, C1 comprises an engineered CGamma and C2 comprises an engineered CDelta. Table 5 shows the exemplary designs for the conjunction domains useful for antibody VH fused to TCR CGamma, or for antibody VL fused to TCR CDelta. The antibody VH/constant domain boundary is aligned to TCR variable/CGamma boundary, and antibody VL/constant domain boundary is aligned to TCR variable/CDelta boundary. Exemplary designs of the conjunction domains are also provided in an alignment form (see, e.g., SEQ ID NO: 157, 158, 159, or 160), with the first or the second conjunction domain shown with underline. In such embodiments, the first conjunction domain comprises or is SEQ ID NO: 117 or 118. In such embodiments, the second conjunction domain comprises or is SEQ ID NO: 119 or 120. 
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 First and second conjunction domain designs for VH-CGamma/VL-CDelta 
               
            
           
           
               
               
               
               
            
               
                   
                 Conjunction 
                   
                 Conjunction 
               
               
                   
                 (heavy chain) 
                   
                 (light chain) 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 Variable 
                 Constant 
                   
                 Variable 
                 Constant 
               
               
                   
               
               
                 Antibody_H 
                 ......LVTVSS 
                 AS--TK-GPS...... 
                 Antibody_L 
                 ......KVEIK 
                 ---RTVAAPSVF...... 
               
               
                 SEQ ID NO: 293 
                   
                   
                 SEQ ID NO: 143 
                   
                   
               
               
                   
               
               
                 TCR_gamma 
                 ...... TLVVTD   
                   KQLDADVSPK ...... 
                 TCR_delta 
                 ...... RVTVE   
                   PRSQPHTKPSVF ...... 
               
               
                 SEQ ID NO: 142 
                   
                   
                 SEQ ID NO: 55 
                   
                   
               
               
                   
               
               
                 H_Conjunction_4 
                 ......LVTV SS   
                   AS LDADVSPK   ...... 
                 L_Conjunction_4 
                 ......KVEI K   
                     PRSQPHTKPSVF   ...... 
               
               
                 SEQ ID NO: 157 
                   
                   
                 SEQ ID NO: 159 
                   
                   
               
               
                   
               
               
                 H_Conjunction_5 
                 ......LVTV   TD     
                     KQLDADVSPK   ...... 
                 L_Conjunction_5 
                 ......KVEI   E     
                     PRSQPHTKPSVF   ...... 
               
               
                 SEQ ID NO: 158 
                   
                   
                 SEQ ID NO: 160 
               
               
                   
               
            
           
         
       
     
     In certain embodiments, C1 comprises an engineered CDelta and C2 comprises an engineered CGamma. Table 6 shows the exemplary designs for the conjunction domains useful for antibody VH fused to TCR CDelta, or for antibody VL fused to TCR CGamma. The antibody VH/constant domain boundary is aligned to TCR variable/CDelta boundary, and antibody VL/constant domain boundary is aligned to TCR variable/CGamma boundary. Exemplary designs of the conjunction domains are also provided in an alignment form (see, e.g., SEQ ID NO: 161, 162, 163, or 164), with the first or the second conjunction domain shown with underline. In such embodiments, the first conjunction domain comprises or is SEQ ID NO: 123 or 124. In such embodiments, the second conjunction domain comprises or is SEQ ID NO: 125 or 126. 
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 First and second conjunction domain designs for VH-CDelta/VL-CGamma 
               
            
           
           
               
               
               
               
            
               
                   
                 Conjunction 
                   
                 Conjunction 
               
               
                   
                 (heavy chain) 
                   
                 (light chain) 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 Variable 
                 Constant 
                   
                 Variable 
                 Constant 
               
               
                   
               
               
                 Antibody_H 
                 ......LVTVSS 
                 --ASTKGPS...... 
                 Antibody_L 
                 ......KVEIK- 
                 RTV---AAPSVF...... 
               
               
                 SEQ ID NO: 56 
                   
                   
                 SEQ ID NO: 58 
                   
                   
               
               
                   
               
               
                 TCR_delta 
                 ...... RVTVEP   
                   RSQPHTKPS ...... 
                 TCR_gamma 
                 ...... TLVVTD   
                   KQLDADVSPKPT ...... 
               
               
                 SEQ ID NO: 57 
                   
                   
                 SEQ ID NO: 59 
                   
                   
               
               
                   
               
               
                 H_Conjunction_6 
                 ......LVTV SS   
                     RSQPHTKPS   ...... 
                 L_Conjunction_6 
                 ......KVEI K D     
                     KQLDADVSPKPT   ...... 
               
               
                 SEQ ID NO: 161 
                   
                   
                 SEQ ID NO: 163 
                   
                   
               
               
                   
               
               
                 H_Conjunction_7 
                 ......LVTV   EP     
                     RSQPHTKPS   ...... 
                 L_Conjunction_7 
                 ......KVEI   TD     
                     KQLDADVSPKPT   ...... 
               
               
                 SEQ ID NO: 162 
                   
                   
                 SEQ ID NO: 164 
               
               
                   
               
            
           
         
       
     
     In certain embodiments, the first polypeptide comprises a sequence comprising domains operably linked as in formula (I): VH-HCJ-C1, and the second polypeptide comprises a sequence comprising domains operably linked as in formula (II): VL-LCJ-C2, wherein: 
     VH is a heavy chain variable domain of an antibody; 
     HCJ is a first conjunction domain as defined supra; 
     C1 is a first TCR constant domain as defined supra; 
     VL is a light chain variable domain of an antibody; 
     LCJ is a second conjunction domain as defined supra; 
     C2 is a second TCR constant domain as defined supra. 
     In certain embodiments, C1 is an engineered CAlpha which comprises or is a sequence selected from a group consisting of: SEQ ID NOs: 42-48, and C2 is an engineered CBeta which comprises or is a sequence selected from a group consisting of: SEQ ID NOs: 32-41, the HCJ comprises or is a sequence selected from a group consisting of: SEQ ID NOs: 49 and 50; LCJ comprises or is a sequence selected from a group consisting of: SEQ ID NOs: 51 and 52. 
     In certain embodiments, C1 is an engineered CBeta which comprises or is a sequence selected from a group consisting of: SEQ ID NOs: 32-41, and C2 is an engineered CAlpha which comprises or is a sequence selected from a group consisting of: SEQ ID NOs: 42-48, the HCJ comprises or is a sequence selected from a group consisting of: SEQ ID NOs: 129 and 130; LCJ comprises or is a sequence selected from a group consisting of: SEQ ID NOs: 49 and 50. 
     In certain embodiments, C1 is an engineered CBeta which comprises or is a sequence selected from a group consisting of: SEQ ID NOs: 32-41, 84, 319, 320, 321, 322, 323, and 324, and C2 is an engineered CPre-Alpha which comprises or is a sequence selected from a group consisting of: SEQ ID NOs: 311, 312, 313, 314, 315, 316, 317, and 318, the HCJ comprises or is a sequence selected from a group consisting of: SEQ ID NOs: 49 and 50; LCJ comprises or is a sequence selected from a group consisting of: SEQ ID NOs: 81 and 131. 
     In certain embodiments, C1 is an engineered CPre-Alpha which comprises or is a sequence selected from a group consisting of: SEQ ID NOs: 311, 312, 313, 314, 315, 316, 317, and 318, and C2 is an engineered CBeta which comprises or is a sequence selected from a group consisting of: SEQ ID NOs: 32-41, the HCJ comprises or is a sequence selected from a group consisting of: SEQ ID NOs: 81, 131, 132, and 133; LCJ comprises or is a sequence selected from a group consisting of: SEQ ID NOs: 49 and 50. 
     In certain embodiments, C1 is an engineered CGamma which comprises or is a sequence selected from a group consisting of: SEQ ID NOs: 113, 114, 333, 334, 335, 336, 337, 338, 339, and 340, and C2 is an engineered CDelta which comprises or is a sequence selected from a group consisting of: SEQ ID NOs: 325, 326, 327, 328, 329, 330, 331, and 332, the HCJ comprises or is a sequence selected from a group consisting of: SEQ ID NOs: 117 and 118; LCJ comprises or is a sequence selected from a group consisting of: SEQ ID NOs: 119 and 120. 
     In certain embodiments, C1 is an engineered CDelta which comprises or is a sequence selected from a group consisting of: SEQ ID NOs: 325, 326, 327, 328, 329, 330, 331, and 332, and C2 is an engineered CGamma which comprises or is a sequence selected from a group consisting of: SEQ ID NOs: 113, 114, 333, 334, 335, 336, 337, 338, 339, and 340, the HCJ comprises or is a sequence selected from a group consisting of: SEQ ID NOs: 123 and 124; LCJ comprises or is a sequence selected from a group consisting of: SEQ ID NOs: 125 and 126. 
     U.S. Pat. No. 9,683,052 discloses that certain residues within the contact interface between TCR constant regions can be engineered into an Fc region to facilitate the hetero-dimeric pairing of two heavy chains. Such residues and/or corresponding residues within the contact interface between TCR constant regions disclosed herein can also be engineered into a Fab region, e.g., the CH1 and CL domains, to facilitate the pairing between a light chain and a heavy chain. 
     ii. Antibody Variable Region 
     The polypeptide complex provided herein comprises a first heavy chain variable domain (VH) and a first light chain variable domain (VL) of the first antibody. In a conventional native antibody, a variable region comprises three CDR regions interposed by flanking framework (FR) regions, for example, as set forth in the following formula: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, from N-terminus to C-terminus. The polypeptide complex provided herein can comprise but is not limited to such a conventional antibody variable region. For example, the variable domain may comprise all three or less than three of the CDRs, with all four or less than four of the FRs of the antibody heavy or light chain, as long as the variable domain is capable of specifically binding to an antigen. 
     The first antibody has a first antigenic specificity. In other words, VH and VL form an antigen-binding site which can specifically bind to an antigen or an epitope. The antigenic specificity can be directed to any suitable antigen or epitope, for example, one that is exogenous antigen, endogenous antigen, autoantigen, neoantigen, viral antigen or tumor antigen. An exogenous antigen enters a body by inhalation, ingestion or injection, and can be presented by the antigen-presenting cells (APCs) by endocytosis or phagocytosis and form MHC II complex. An endogenous antigen is generated within normal cells as a result of cell metabolism, intracellular viral or bacterial infection, which can form MHC I complex. An autoantigen (e.g. peptide, DNA or RNA, etc.) is recognized by the immune system of a patient suffering from autoimmune diseases, whereas under normal condition, this antigen should not be the target of the immune system. A neoantigen is entirely absent from the normal body, and is generated because of a certain disease, such as tumor or cancer. In certain embodiments, the antigen is associated with a certain disease (e.g. tumor or cancer, autoimmune diseases, infectious and parasitic diseases, cardiovascular diseases, neuropathies, neuropsychiatric conditions, injuries, inflammations, coagulation disorder). In certain embodiments, the antigen is associated with immune system (e.g. immunological cells such as B cell, T cell, NK cells, macrophages, etc.). 
     In certain embodiments, the first antigenic specificity is directed to an immune-related antigen or an epitope thereof. Examples of an immune-related antigen include a T-cell specific receptor molecule and/or a natural killer cell (NK cell) specific receptor molecule. 
     The T-cell specific receptor molecule allows a T cell to bind to and, if additional signals are present, to be activated by and respond to an epitope/antigen presented by another cell called the antigen-presenting cell or APC. The T-cell specific receptor molecule can be TCR, CD3, CD28, CD134 (also termed OX40), 4-1 BB, CD5, and CD95 (also known as the Fas receptor). 
     Examples of a NK cell specific receptor molecule include CD16, a low affinity Fc receptor and NKG2D, and CD2. 
     In certain embodiments, the first antigenic specificity is directed to a tumor-associated antigen or an epitope thereof. The term “tumor associated antigen” refers to an antigen that is or can be presented on a tumor cell surface and that is located on or within tumor cells. In some embodiments, the tumor associated antigens can be presented only by tumor cells and not by normal, i.e. non-tumor cells. In some other embodiments, the tumor associated antigens can be exclusively expressed on tumor cells or may represent a tumor specific mutation compared to non-tumor cells. In some other embodiments, the tumor associated antigens can be found in both tumor cells and non-tumor cells, but is overexpressed on tumor cells when compared to non-tumor cells or are accessible for antibody binding in tumor cells due to the less compact structure of the tumor tissue compared to non-tumor tissue. In some embodiments the tumor associated antigen is located on the vasculature of a tumor. 
     Illustrative examples of a tumor associated surface antigen are CD10, CD19, CD20, CD22, CD21, CD22, CD25, CD30, CD33, CD34, CD37, CD44v6, CD45, CD133, Fms-like tyrosine kinase 3 (FLT-3, CD135), chondroitin sulfate proteoglycan 4 (CSPG4, melanoma-associated chondroitin sulfate proteoglycan), Epidermal growth factor receptor (EGFR), Her2neu, Her3, IGFR, IL3R, fibroblast activating protein (FAP), CDCP1, Derlin1, Tenascin, frizzled 1-10, the vascular antigens VEGFR2 (KDR/FLK1), VEGFR3 (FLT4, CD309), PDGFR-alpha (CD140a), PDGFR-beta (CD140b) Endoglin, CLEC14, Tem1-8, and Tie2. Further examples may include A33, CAMPATH-1 (CDw52), Carcinoembryonic antigen (CEA), Carboanhydrase IX (MN/CA IX), de2-7 EGFR, EGFRvIII, EpCAM, Ep-CAM, Folate-binding protein, G250, Fms-like tyrosine kinase 3 (FLT-3, CD135), c-Kit (CD117), CSF1R (CD115), HLA-DR, IGFR, IL-2 receptor, IL3R, MCSP (Melanoma-associated cell surface chondroitin sulfate proteoglycane), Muc-1, Prostate-specific membrane antigen (PSMA), Prostate stem cell antigen (PSCA), Prostate specific antigen (PSA), and TAG-72. 
     In certain embodiments, the first antigenic specificity is directed to an antigen or an epitope thereof, selected from the group consisting of: CD3, 4.1BB (CD137), OX40 (CD134), CD16, CD47, CD19, CD20, CD22, CD33, CD38, CD123, CD133, CEA, cdH3, EpCAM, epidermal growth factor receptor (EGFR), EGFRvIII (a mutant form of EGFR), HER2, HER3, dLL3, BCMA, Sialyl-Lea, 5T4, ROR1, melanoma-associated chondroitin sulfate proteoglycan, mesothelin, folate receptor 1, VEGF receptor, EpCAM, HER2/neu, HER3/neu, G250, CEA, MAGE, proteoglycans, VEGF, FGFR, alphaVbeta3-integrin, HLA, HLA-DR, ASC, CD1, CD2, CD4, CD5, CD6, CD7, CD8, CD11, CD13, CD14, CD21, CD23, CD24, CD28, CD30, CD37, CD40, CD41, CD44, CD52, CD64, c-erb-2, CALLA, MHCII, CD44v3, CD44v6, p97, ganglioside GM1, GM2, GM3, GD1a, GD1b, GD2, GD3, GT1b, GT3, GQ1, NY-ESO-1, NFX2, SSX2, SSX4 Trp2, gpl00, tyrosinase, Muc-1, telomerase, survivin, G250, p53, CA125 MUC, Wue antigen, Lewis Y antigen, HSP-27, HSP-70, HSP-72, HSP-90, Pgp, MCSP, EpHA2 and cell surface targets GC182, GT468 or GT512. 
     The antibody variable domains can be derived from a parent antibody. A parent antibody can be any type of antibody, including for example, a fully human antibody, a humanized antibody, or an animal antibody (e.g. mouse, rat, rabbit, sheep, cow, dog, etc.). The parent antibody can be a monoclonal antibody or a polyclonal antibody. 
     In certain embodiments, the parent antibody is a monoclonal antibody. A monoclonal antibody can be produced by various methods known in the art, for example, hybridoma technology, recombinant method, phage display, or any combination thereof. 
     Hybridoma technology involves fusion of antibody-expressing B cells with an immortal B cell line to produce hybridomas, which are further screened for desirable characteristics such as high production level of antibody production, good growth of hybridoma cells, and strong binding or good biological activity of the antibody (see, for example, Harlow et al., (1988) Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.). 
     Recombinant method is another way to produce a parent antibody. Briefly, cells such as lymphocytes secreting antibodies of interest are obtained are identified and single cells are isolated, followed by reverse transcriptase PCR to produce heavy- and light-chain variable region cDNAs. These cDNA sequences of the variable regions can be used to construct the encoding sequence of the polypeptide complex provided herein, and then expressed in a suitable host cell (for reviews, please see, for example, U.S. Pat. No. 5,627,052; PCT Publication No. WO 92/02551; and Babcock et al., (1996) Proc. Natl. Acad. Sci. USA 93:7843-7848). 
     Antibody libraries are still an alternative for obtaining a parent antibody. Briefly, one can screen an antibody library to identify an antibody having the desired binding specificity. Methods for such screening of recombinant antibody libraries are well known in the art and include methods described in, for example, U.S. Pat. No. 5,223,409; PCT Publication Nos. WO 92/18619; WO 91/17271; WO 92/20791; WO 92/15679; WO 93/01288; WO 92/01047; WO 92/09690; and WO 97/29131; Fuchs et al., (1991) Bio/Technology 9:1370-1372; Hay et al., (1992) Hum. Antibod. Hybridomas 3:81-85; Huse et al., (1989) Science 246:1275-1281; McCafferty et al., (1990) Nature 348:552-554; Griffiths et al., (1993) EMBO J. 12:725-734; Hawkins et al., (1992) J. Mol. Biol. 226:889-896; Clackson et al., (1991) Nature 352:624-628; Gram et al., (1992) Proc. Natl. Acad. Sci. USA 89:3576-3580; Garrad et al., (1991) Bio/Technology 9:1373-1377; Hoogenboom et al., (1991) Nucl. Acid Res. 19:4133-4137; and Barbas et al., (1991) Proc. Natl. Acad. Sci. USA 88:7978-7982; and US Patent Application Publication No. 20030186374. 
     Another illustrative method to obtain a parent antibody is phage display (see, e.g., Brinkman et al., (1995) J. Immunol. Methods 182:41-50; Ames et al., (1995) J. Immunol. Methods 184:177-186; Kettleborough et al., (1994) Eur. J. Immunol. 24:952-958; Persic et al., (1997) Gene 187 9-18; and U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743; and 5,969,108). Polynucleotide sequences encoding antibody domains are introduced to phage particles to generate a library of phage particles displaying a variety of functional antibody domains. Fd and M13 are filamentous phage commonly used, and the functional antibody domains displayed on the phage particles can be, for example, Fab, Fv or disulphide stabilized Fv antibody domains, which is recombinantly fused to a phage protein encoded by gene III or gene VIII. The phage library can be screened using an antigen of interest, for example, which is optionally labeled or bound or captured to a solid substrate (e.g. a bead). For a selected phage, its polynucleotide sequences encoding the antibody variable domains are obtained and used in the construction of the polypeptide complex provided herein. Likewise, a library of yeast can be generated displaying antibody variable domains by tethering the antibody domains to the yeast cell wall (see, for example, U.S. Pat. No. 6,699,658), and then screened with a bound antigen to obtain a parent antibody useful for construction of the polypeptide complex provided herein. 
     Furthermore, a parent antibody can also be produced by injecting an antigen of interest to a transgenic non-human animal comprising some, or all, of the human immunoglobulin locus, for example, OmniRat, OmniMouse (see, for example, Osborn M. et al., Journal of Immunology, 2013, 190: 1481-90; Ma B. et al., Journal of Immunological Methods 400-401 (2013) 78-86; Geurts A. et al., Science, 2009, 325:433; U.S. Pat. No. 8,907,157; EP patent 2152880B1; EP patent 2336329B1), HuMab mice (see, for details, Lonberg, N. et al., Nature 368(6474): 856 859 (1994)), Xeno-Mouse (Mendez et al., Nat Genet., 1997, 15:146-156), TransChromo Mouse (Ishida et al., Cloning Stem Cells, 2002, 4:91-102) and VelocImmune Mouse (Murphy et al., Proc Natl Acad Sci USA, 2014, 111:5153-5158), Kymouse (Lee et al., Nat Biotechnol, 2014, 32:356-363), and transgenic rabbit (Flisikowska et al., PLoS One, 2011, 6:e21045). 
     The parent antibodies described herein can be further modified, for example, to graft the CDR sequences to a different framework or scaffold, to substitute one or more amino acid residues in one or more framework regions, to replace one or more residues in one or more CDR regions for affinity maturation, and so on. These can be accomplished by a person skilled in the art using conventional techniques. 
     The parent antibody can also be a therapeutic antibody known in the art, for example those approved by FDA for therapeutic or diagnostic use, or those under clinical trial for treating a condition, or those in research and development. Polynucleotide sequences and protein sequences for the variable regions of known antibodies can be obtained from public databases such as, for example, e.g., www.ncbi.nlm nih gov/entrez-/query.fcgi; www.atcc.org/phage/hdb.html; www.sciquest.com/; www.abcam.com/; www.antibodyresource.com/onlinecomp.html. 
     Examples of therapeutic antibodies include, but are not limited to, rituximab (Rituxan, IDEC/Genentech/Roche) (see for example U.S. Pat. No. 5,736,137), a chimeric anti-CD20 antibody approved to treat Non-Hodgkin&#39;s lymphoma; HuMax-CD20, an anti-CD20 currently being developed by Genmab, an anti-CD20 antibody described in U.S. Pat. No. 5,500,362, AME-133 (Applied Molecular Evolution), hA20 (Immunomedics, Inc.), HumaLYM (Intracel), and PRO70769 (PCT Application No. PCT/US2003/040426), trastuzumab (Herceptin, Genentech) (see for example U.S. Pat. No. 5,677,171), a humanized anti-Her2/neu antibody approved to treat breast cancer; pertuzumab (rhuMab-2C4, Omnitarg), currently being developed by Genentech; an anti-Her2 antibody described in U.S. Pat. No. 4,753,894; cetuximab (Erbitux, Imclone) (U.S. Pat. No. 4,943,533; PCT Publication No. WO 96/40210), a chimeric anti-EGFR antibody in clinical trials for a variety of cancers; ABX-EGF (U.S. Pat. No. 6,235,883), currently being developed by Abgenix-Immunex-Amgen; HuMax-EGFr (U.S. Pat. No. 7,247,301), currently being developed by Genmab; 425, EMD55900, EMD62000, and EMD72000 (Merck KGaA) (U.S. Pat. No. 5,558,864; Murthy et al., (1987) Arch. Biochem. Biophys. 252(2):549-60; Rodeck et al., (1987) J. Cell. Biochem. 35(4):315-20; Kettleborough et al., (1991) Protein Eng. 4(7):773-83); ICR62 (Institute of Cancer Research) (PCT Publication No. WO 95/20045; Modjtahedi et al., (1993) J. Cell Biophys. 22(1-3):129-46; Modjtahedi et al., (1993) Br. J. Cancer 67(2):247-53; Modjtahedi et al., (1996) Br. J. Cancer 73(2):228-35; Modjtahedi et al., (2003) Int. J. Cancer 105(2):273-80); TheraCIM hR3 (YM Biosciences, Canada and Centro de Immunologia Molecular, Cuba (U.S. Pat. Nos. 5,891,996; 6,506,883; Mateo et al., (1997) Immunotechnol. 3(1):71-81); mAb-806 (Ludwig Institute for Cancer Research, Memorial Sloan-Kettering) (Jungbluth et al., (2003) Proc. Natl. Acad. Sci. USA 100(2):639-44); KSB-102 (KS Biomedix); MR1-1 (IVAX, National Cancer Institute) (PCT Publication No. WO 0162931); and SC100 (Scancell) (PCT WO 01/88138); alemtuzumab (Campath, Millenium), a humanized mAb currently approved for treatment of B-cell chronic lymphocytic leukemia; muromonab-CD3 (Orthoclone OKT3), an anti-CD3 antibody developed by Ortho Biotech/Johnson &amp; Johnson, ibritumomab tiuxetan (Zevalin), an anti-CD20 antibody developed by IDEC/Schering AG, gemtuzumab ozogamicin (Mylotarg), an anti-CD33 (p67 protein) antibody developed by Celltech/Wyeth, alefacept (Amevive), an anti-LFA-3 Fc fusion developed by Biogen), abciximab (ReoPro), developed by Centocor/Lilly, basiliximab (Simulect), developed by Novartis, palivizumab (Synagis), developed by Medimmune, infliximab (Remicade), an anti-TNFalpha antibody developed by Centocor, adalimumab (Humira), an anti-TNFalpha antibody developed by Abbott, Humicade, an anti-TNFalpha antibody developed by Celltech, golimumab (CNTO-148), a fully human TNF antibody developed by Centocor, etanercept (Enbrel), an p75 TNF receptor Fc fusion developed by Immunex/Amgen, lenercept, an p55TNF receptor Fc fusion previously developed by Roche, ABX-CBL, an anti-CD147 antibody being developed by Abgenix, ABX-IL8, an anti-IL8 antibody being developed by Abgenix, ABX-MA1, an anti-MUC18 antibody being developed by Abgenix, Pemtumomab (R1549, 90Y-muHMFG1), an anti-MUC1 in development by Antisoma, Therex (R1550), an anti-MUC1 antibody being developed by Antisoma, AngioMab (AS1405), being developed by Antisoma, HuBC-1, being developed by Antisoma, Thioplatin (AS1407) being developed by Antisoma, Antegren (natalizumab), an anti-alpha-4-beta-1 (VLA-4) and alpha-4-beta-7 antibody being developed by Biogen, VLA-1 mAb, an anti-VLA-1 integrin antibody being developed by Biogen, LTBR mAb, an anti-lymphotoxin beta receptor (LTBR) antibody being developed by Biogen, CAT-152, an anti-TGF-.beta.2 antibody being developed by Cambridge Antibody Technology, ABT 874 (J695), an anti-IL-12 p40 antibody being developed by Abbott, CAT-192, an anti-TGF.beta.1 antibody being developed by Cambridge Antibody Technology and Genzyme, CAT-213, an anti-Eotaxin1 antibody being developed by Cambridge Antibody Technology, LymphoStat-B an anti-Blys antibody being developed by Cambridge Antibody Technology and Human Genome Sciences Inc., TRAIL-R1mAb, an anti-TRAIL-R1 antibody being developed by Cambridge Antibody Technology and Human Genome Sciences, Inc., Avastin bevacizumab, rhuMAb-VEGF), an anti-VEGF antibody being developed by Genentech, an anti-HER receptor family antibody being developed by Genentech, Anti-Tissue Factor (ATF), an anti-Tissue Factor antibody being developed by Genentech, Xolair (Omalizumab), an anti-IgE antibody being developed by Genentech, Raptiva (Efalizumab), an anti-CD11a antibody being developed by Genentech and Xoma, MLN-02 Antibody (formerly LDP-02), being developed by Genentech and Millenium Pharmaceuticals, HuMax CD4, an anti-CD4 antibody being developed by Genmab, HuMax-IL15, an anti-IL15 antibody being developed by Genmab and Amgen, HuMax-Inflam, being developed by Genmab and Medarex, HuMax-Cancer, an anti-Heparanase I antibody being developed by Genmab and Medarex and Oxford GcoSciences, HuMax-Lymphoma, being developed by Genmab and Amgen, HuMax-TAC, being developed by Genmab, IDEC-131, and anti-CD40L antibody being developed by IDEC Pharmaceuticals, IDEC-151 (Clenoliximab), an anti-CD4 antibody being developed by IDEC Pharmaceuticals, IDEC-114, an anti-CD80 antibody being developed by IDEC Pharmaceuticals, IDEC-152, an anti-CD23 being developed by IDEC Pharmaceuticals, anti-macrophage migration factor (MIF) antibodies being developed by IDEC Pharmaceuticals, BEC2, an anti-idiotypic antibody being developed by Imclone, IMC-1C11, an anti-KDR antibody being developed by Imclone, DC101, an anti-flk-1 antibody being developed by Imclone, anti-VE cadherin antibodies being developed by Imclone, CEA-Cide (labetuzumab), an anti-carcinoembryonic antigen (CEA) antibody being developed by Immunomedics, LymphoCide (Epratuzumab), an anti-CD22 antibody being developed by Immunomedics, AFP-Cide, being developed by Immunomedics, MyelomaCide, being developed by Immunomedics, LkoCide, being developed by Immunomedics, ProstaCide, being developed by Immunomedics, MDX-010, an anti-CTLA4 antibody being developed by Medarex, MDX-060, an anti-CD30 antibody being developed by Medarex, MDX-070 being developed by Medarex, MDX-018 being developed by Medarex, Osidem (IDM-1), and anti-Her2 antibody being developed by Medarex and Immuno-Designed Molecules, HuMax-CD4, an anti-CD4 antibody being developed by Medarex and Genmab, HuMax-IL15, an anti-IL15 antibody being developed by Medarex and Genmab, CNTO 148, an anti-TNF.alpha. antibody being developed by Medarex and Centocor/J&amp;J, CNTO 1275, an anti-cytokine antibody being developed by Centocor/J&amp;J, MOR101 and MOR102, anti-intercellular adhesion molecule-1 (ICAM-1) (CD54) antibodies being developed by MorphoSys, MOR201, an anti-fibroblast growth factor receptor 3 (FGFR-3) antibody being developed by MorphoSys, Nuvion (visilizumab), an anti-CD3 antibody being developed by Protein Design Labs, HuZAF, an anti-gamma interferon antibody being developed by Protein Design Labs, Anti-.alpha. 5.beta.1 Integrin, being developed by Protein Design Labs, anti-IL-12, being developed by Protein Design Labs, ING-1, an anti-Ep-CAM antibody being developed by Xoma, Xolair (Omalizumab) a humanized anti-IgE antibody developed by Genentech and Novartis, and MLN01, an anti-Beta2 integrin antibody being developed by Xoma. In another embodiment, the therapeutics include KRN330 (Kirin); huA33 antibody (A33, Ludwig Institute for Cancer Research); CNTO 95 (alpha V integrins, Centocor); MEDI-522 (alpha V.beta.3 integrin, Medimmune); volociximab (alpha V.beta.1 integrin, Biogen/PDL); Human mAb 216 (B cell glycosolated epitope, NCI); BiTE MT103 (bispecific CD19 CD3, Medimmune); 4G7 H22 (Bispecific Bcell.times.FcgammaR1, Medarex/Merck KGa); rM28 (Bispecific CD28.times.MAPG, EP Patent No. EP1444268); MDX447 (EMD 82633) (Bispecific CD64 EGFR, Medarex); Catumaxomab (removab) (Bispecific EpCAM anti-CD3, Trion/Fres); Ertumaxomab (bispecific HER2/CD3, Fresenius Biotech); oregovomab (OvaRex) (CA-125, ViRexx); Rencarex (WX G250) (carbonic anhydrase IX, Wilex); CNTO 888 (CCL2, Centocor); TRC105 (CD105 (endoglin), Tracon); BMS-663513 (CD137 agonist, Brystol Myers Squibb); MDX-1342 (CD19, Medarex); Siplizumab (MEDI-507) (CD2, Medimmune); Ofatumumab (Humax-CD20) (CD20, Genmab); Rituximab (Rituxan) (CD20, Genentech); veltuzumab (hA20) (CD20, Immunomedics); Epratuzumab (CD22, Amgen); lumiliximab (IDEC 152) (CD23, Biogen); muromonab-CD3 (CD3, Ortho); HuM291 (CD3 fc receptor, PDL Biopharma); HeFi-1, CD30, NCI); MDX-060 (CD30, Medarex); MDX-1401 (CD30, Medarex); SGN-30 (CD30, Seattle Genentics); SGN-33 (Lintuzumab) (CD33, Seattle Genentics); Zanolimumab (HuMax-CD4) (CD4, Genmab); HCD122 (CD40, Novartis); SGN-40 (CD40, Seattle Genentics); Campathlh (Alemtuzumab) (CD52, Genzyme); MDX-1411 (CD70, Medarex); hLL1 (EPB-1) (CD74.38, Immunomedics); Galiximab (IDEC-144) (CD80, Biogen); MT293 (TRC093/D93) (cleaved collagen, Tracon); HuLuc63 (CS1, PDL Pharma); ipilimumab (MDX-010) (CTLA4, Brystol Myers Squibb); Tremelimumab (Ticilimumab, CP-675,2) (CTLA4, Pfizer); HGS-ETR1 (Mapatumumab) (DR4 TRAIL-R1 agonist, Human Genome Science/Glaxo Smith Kline); AMG-655 (DR5, Amgen); Apomab (DR5, Genentech); CS-1008 (DR5, Daiichi Sankyo); HGS-ETR2 (lexatumumab) (DR5 TRAIL-R2 agonist, HGS); Cetuximab (Erbitux) (EGFR, Imclone); IMC-11F8, (EGFR, Imclone); Nimotuzumab (EGFR, YM Bio); Panitumumab (Vectabix) (EGFR, Amgen); Zalutumumab (HuMaxEGFr) (EGFR, Genmab); CDX-110 (EGFRvIII, AVANT Immunotherapeutics); adecatumumab (MT201) (Epcam, Merck); edrecolomab (Panorex, 17-1A) (Epcam, Glaxo/Centocor); MORAb-003 (folate receptor a, Morphotech); KW-2871 (ganglioside GD3, Kyowa); MORAb-009 (GP-9, Morphotech); CDX-1307 (MDX-1307) (hCGb, Celldex); Trastuzumab (Herceptin) (HER2, Celldex); Pertuzumab (rhuMAb 2C4) (HER2 (DI), Genentech); apolizumab (HLA-DR beta chain, PDL Pharma); AMG-479 (IGF-1R, Amgen); anti-IGF-1R R1507 (IGF1-R, Roche); CP 751871 (IGF-R, Pfizer); IMC-A12 (IGF1-R, Imclone); BIIB022 (IGF-1R, Biogen); Mik-beta-1 (IL-2Rb (CD122), Hoffman LaRoche); CNTO 328 (IL6, Centocor); Anti-KIR (1-7F9) (Killer cell Ig-like Receptor (KIR), Novo); Hu3S193 (Lewis (y), Wyeth, Ludwig Institute of Cancer Research); hCBE-11 (LTBR, Biogen); HuHMFG1 (MUC1, Antisoma/NCI); RAV12 (N-linked carbohydrate epitope, Raven); CAL (parathyroid hormone-related protein (PTH-rP), University of California); CT-011 (PD1, CureTech); MDX-1106 (ono-4538) (PD1, Medarex/Ono); MAb CT-011 (PD1, Curetech); IMC-3G3 (PDGFRa, Imclone); bavituximab (phosphatidylserine, Peregrine); huJ591 (PSMA, Cornell Research Foundation); muJ591 (PSMA, Cornell Research Foundation); GC1008 (TGFb (pan) inhibitor (IgG4), Genzyme); Infliximab (Remicade) (TNF.alpha., Centocor); A27.15 (transferrin receptor, Salk Institute, INSERN WO 2005/111082); E2.3 (transferrin receptor, Salk Institute); Bevacizumab (Avastin) (VEGF, Genentech); HuMV833 (VEGF, Tsukuba Research Lab) PCT Publication No. WO/2000/034337, University of Texas); IMC-18F1 (VEGFR1, Imclone); IMC-1121 (VEGFR2, Imclone). 
     a) Anti-CD3 Binding Moiety 
     In certain embodiments, the first antigen-binding moiety or the second antigen-binding moiety is an anti-CD3 binding moiety derived from an anti-CD3 antibody comprising 1, 2, or 3 heavy chain CDR sequences selected from the group consisting of: SEQ ID NOs: 342-344 and/or 1, 2, or 3 light chain CDR sequences selected from SEQ ID NOs: 345-347. 
     These CDR sequences are derived from the anti-CD3 antibody shown in Table A below. The CDR sequences of the WBP3311_2.306.4 antibody are provided below. 
     
       
         
           
               
               
               
               
               
             
               
                 TABLE A 
               
               
                   
               
               
                 Antibody ID: 
                   
                 CDR1 
                 CDR2 
                 CDR3 
               
               
                   
               
             
            
               
                 WBP3311_2.306.4 
                 VH 
                 SEQ ID NO: 342 
                 SEQ ID NO: 343 
                 SEQ ID NO: 344 
               
               
                   
                   
                 GFAFTDYYIH 
                 WISPGNVNTKY 
                 DGYSLYYFDY 
               
               
                   
                   
                   
                 NENFKG 
                   
               
               
                   
               
               
                 WBP3311_2.306.4 
                 VL 
                 SEQ ID NO: 345 
                 SEQ ID NO: 346 
                 SEQ ID NO: 347 
               
               
                   
                   
                 KSSQSLLNSRTRKN 
                 WASTRQS 
                 TQSHTLRT 
               
               
                   
                   
                 YLA 
               
               
                   
               
            
           
         
       
     
     Heavy and kappa light chain variable region sequences of the WBP3311_2.306.4 antibody are provided below. 
     WBP3311_2.306.4-VH 
     
       
         
           
               
            
               
                 Amino acid sequence (SEQ ID NO: 348): 
               
               
                 QVQLQQSGPELVKPGASVRISCKAS GFAFTDYYIH WVKQRPGQGLEWIG W   
               
               
                   ISPGNVNTKYNENFKG RATLTADLSSSTAYMQLSSLTSEDSAVYFCAR DG   
               
               
                   YSLYYFDY WGQGTTLTVSS 
               
               
                   
               
               
                 Nucleic acid sequence (SEQ ID NO: 349): 
               
               
                 CAGGTCCAGCTGCAGCAGTCTGGACCTGAATTGGTGAAGCCTGGGGCTTC 
               
               
                   
               
               
                 CGTGAGGATATCCTGCAAGGCTTCTGGCTTCGCCTTCACAGACTACTATA 
               
               
                   
               
               
                 TACACTGGGTGAAGCAGAGGCCTGGACAGGGTCTTGAGTGGATTGGATGG 
               
               
                   
               
               
                 ATTTCTCCTGGAAATGTTAATACTAAATACAATGAAAACTTCAAGGGCAG 
               
               
                   
               
               
                 GGCCACACTGACTGCAGACCTATCCTCCAGCACAGCCTACATGCAGCTCA 
               
               
                   
               
               
                 GCAGCCTGACCTCTGAGGACTCTGCGGTCTATTTCTGTGCAAGAGATGGA 
               
               
                   
               
               
                 TATTCCCTGTATTACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGT 
               
               
                   
               
               
                 CTCCTCA 
               
            
           
         
       
     
     WBP3311_2.306.4-VL 
     
       
         
           
               
            
               
                 Amino acid sequence (SEQ ID NO: 350): 
               
               
                 DIVMSQSPSSLTVSAGEKVTMSC KSSQSLLNSRTRKNYLA WYQQKPGQSP 
               
               
                   
               
               
                 KWY WASTRQS GVPDRFTGSGSGTAFTLTISGVQAEDLAVYFCT QSHTLRT   
               
               
                   
               
               
                 FGGGTKLEIK 
               
               
                   
               
               
                 Nucleic acid sequence (SEQ ID NO: 351): 
               
               
                 GACATTGTGATGTCACAGTCTCCATCCTCCCTGACTGTGTCAGCAGGAGA 
               
               
                   
               
               
                 GAAGGTCACTATGAGCTGCAAATCCAGTCAGAGTCTGCTCAACAGTAGAA 
               
               
                   
               
               
                 CCCGAAAGAACTACTTGGCTTGGTACCAGCAGAAGCCAGGGCAGTCTCCT 
               
               
                   
               
               
                 AAACTACTAATCTACTGGGCATCCACTAGGCAATCTGGGGTCCCTGATCG 
               
               
                   
               
               
                 CTTCACAGGCAGTGGATCTGGGACAGCTTTCACTCTCACCATCAGCGGTG 
               
               
                   
               
               
                 TGCAGGCTGAAGACCTGGCAGTTTATTTCTGCACGCAATCTCATACTCTT 
               
               
                   
               
               
                 CGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA 
               
            
           
         
       
     
     CDRs are known to be responsible for antigen binding, however, it has been found that not all of the 6 CDRs are indispensable or unchangeable. In other words, it is possible to replace or change or modify one or more CDRs in the anti-CD3 binding moiety derived from WBP3311_2.306.4, yet substantially retain the specific binding affinity to CD3. 
     In certain embodiments, the anti-CD3 binding moiety provided herein comprises a heavy chain CDR3 sequence of one of the anti-CD3 antibodies WBP3311_2.306.4. In certain embodiments, the anti-CD3 binding moiety provided herein comprises a heavy chain CDR3 comprising SEQ ID NO: 344. Heavy chain CDR3 regions are located at the center of the antigen-binding site, and therefore are believed to make the most contact with antigen and provide the most free energy to the affinity of antibody to antigen. It is also believed that the heavy chain CDR3 is by far the most diverse CDR of the antigen-binding site in terms of length, amino acid composition and conformation by multiple diversification mechanisms (Tonegawa S., Nature. 302:575-81 (1983)). The diversity in the heavy chain CDR3 is sufficient to produce most antibody specificities (Xu J L, Davis M M., Immunity. 13:37-45 (2000)) as well as desirable antigen-binding affinity (Schier R, et al., J Mol Biol. 263:551-67 (1996)). 
     In certain embodiments, the anti-CD3 binding moiety provided herein comprises suitable framework region (FR) sequences, as long as the anti-CD3 binding moiety can specifically bind to CD3. The CDR sequences provided in Table A are obtained from mouse antibodies, but they can be grafted to any suitable FR sequences of any suitable species such as mouse, human, rat, rabbit, among others, using suitable methods known in the art such as recombinant techniques. 
     In certain embodiments, the anti-CD3 binding moiety provided herein is humanized. 
     In certain embodiments, the humanized antigen binding moiety provided herein is composed of substantially all human sequences except for the CDR sequences which are non-human. In some embodiments, the variable region FRs, and constant regions if present, are entirely or substantially from human immunoglobulin sequences. The human FR sequences and human constant region sequences may be derived different human immunoglobulin genes, for example, FR sequences derived from one human antibody and constant region from another human antibody. In some embodiments, the humanized antigen binding moiety comprises human FR1-4. 
     The heavy chain and light chain variable region sequences of the anti-CD3 humanized antibody WBP3311_2.306.4-z1 are provided below. 
     WBP3311_2.306.4-z1-VH 
     
       
         
           
               
            
               
                 Amino acid sequence (SEQ ID NO: 352): 
               
               
                 QVQL V QSG A E VK KPG S SV KV SCKAS GFAFTDYYIH W VR Q A PGQGLEW M G W   
               
               
                   
               
               
                   ISPGNVNTKYNENFKG R V T I TAD K S T STAYM E LSSL R SED T AV Y YCAR DG   
               
               
                   
               
               
                   YSLYYFDY WGQGT LV TVSS 
               
               
                   
               
               
                 Nucleic acid sequence (SEQ ID NO: 353): 
               
               
                 CAGGTGCAGCTTGTGCAGTCTGGGGCAGAAGTGAAGAAGCCTGGGTCTAG 
               
               
                   
               
               
                 TGTCAAGGTGTCATGCAAGGCTAGCGGGTTCGCCTTTACTGACTACTACA 
               
               
                   
               
               
                 TCCACTGGGTGCGGCAGGCTCCCGGACAAGGGTTGGAGTGGATGGGATGG 
               
               
                   
               
               
                 ATCTCCCCAGGCAATGTCAACACAAAGTACAACGAGAACTTCAAAGGCCG 
               
               
                   
               
               
                 CGTCACCATTACCGCCGACAAGAGCACCTCCACAGCCTACATGGAGCTGT 
               
               
                   
               
               
                 CCAGCCTCAGAAGCGAGGACACTGCCGTCTACTACTGTGCCAGGGATGGG 
               
               
                   
               
               
                 TACTCCCTGTATTACTTTGATTACTGGGGCCAGGGCACACTGGTGACAGT 
               
               
                   
               
               
                 GAGCTCC 
               
            
           
         
       
     
     WBP3311_2.306.4-z1-VL 
     
       
         
           
               
            
               
                 Amino acid sequence (SEQ ID NO: 354): 
               
               
                 DIVM T QSP D SL A VS L GE RA T IN C KSSQSLLNSRTRKNYLA WYQQKPGQ P P 
               
               
                   
               
               
                 KLLIY WASTRQS GVPDRF S GSGSGT D FTLTIS SL QAED V AVY Y C TQSHTL   
               
               
                   
               
               
                   RT FGGGTK V EIK 
               
               
                   
               
               
                 Nucleic acid sequence (SEQ ID NO: 355): 
               
               
                 GATATCGTGATGACCCAGAGCCCAGACTCCCTTGCTGTCTCCCTCGGCGA 
               
               
                   
               
               
                 AAGAGCAACCATCAACTGCAAGAGCTCCCAAAGCCTGCTGAACTCCAGGA 
               
               
                   
               
               
                 CCAGGAAGAATTACCTGGCCTGGTATCAGCAGAAGCCCGGCCAGCCTCCT 
               
               
                   
               
               
                 AAGCTGCTCATCTACTGGGCCTCCACCCGGCAGTCTGGGGTGCCCGATCG 
               
               
                   
               
               
                 GTTTAGTGGATCTGGGAGCGGGACAGACTTCACATTGACAATTAGCTCAC 
               
               
                   
               
               
                 TGCAGGCCGAGGACGTGGCCGTCTACTACTGTACTCAGAGCCACACTCTC 
               
               
                   
               
               
                 CGCACATTCGGCGGAGGGACTAAAGTGGAGATTAAG 
               
            
           
         
       
     
     b) Anti-CD19 Antibody 
     In certain embodiments, the first antigen-binding moiety or the second antigen-binding moiety is an anti-CD19 binding moiety derived from an anti-CD19 antibody comprising 1, 2, or 3 heavy chain CDR sequences selected from the group consisting of SEQ ID NOs: 356-359, and/or 1, 2, or 3 kappa light chain CDR sequences selected from the group consisting of: SEQ ID NOs: 360-362. 
     These CDR sequences are derived from the antibodies shown in Table B below. The CDR sequences of these anti-CD19 antibodies are provided below. 
     
       
         
           
               
               
               
               
               
             
               
                 TABLE B 
               
               
                   
               
               
                   
                   
                 CDR1 
                 CDR2 
                 CDR3 
               
               
                   
               
             
            
               
                 WBP7011_4.155.8 
                 VH 
                 SEQ ID NO: 356 
                 SEQ ID NO: 357 
                 SEQ ID NO: 358 
               
               
                   
                   
                 GYAFTSYNMY 
                 YIDPYNGDTTYN 
                 TAYAMDY 
               
               
                   
                   
                   
                 QKFKG 
                   
               
               
                   
               
               
                 W7011-4.155.8-z1- 
                 VH 
                 SEQ ID NO: 356 
                 SEQ ID NO: 359 
                 SEQ ID NO: 358 
               
               
                 P15 
                   
                 GYAFTSYNMY 
                 YIDPYN A DTTYN 
                 TAYAMDY 
               
               
                   
                   
                   
                 QKFKG 
                   
               
               
                   
               
               
                 WBP7011_4.155.8 
                 VL 
                 SEQ ID NO: 360 
                 SEQ ID NO: 361 
                 SEQ ID NO: 362 
               
               
                   
                   
                 SASSTVNYMH 
                 STSNLAS 
                 HQWSSYPYT 
               
               
                   
               
               
                 W7011-4.155.8-z1- 
                 VL 
                 SEQ ID NO: 360 
                 SEQ ID NO: 361 
                 SEQ ID NO: 362 
               
               
                 P15 
                   
                 SASSTVNYMH 
                 STSNLAS 
                 HQWSSYPYT 
               
               
                   
               
            
           
         
       
     
     Heavy and kappa light chain variable region sequences of the WBP7011_4.155.8 antibody are provided below. 
     WBP7011-4.155.8-VH 
     
       
         
           
               
            
               
                 Amino acid sequence (SEQ ID NO: 363): 
               
               
                 EIQLQQSGPELVKPGASVKVSCKAS GYAFTSYNMY WVKQSHGKSLEWIG Y   
               
               
                   
               
               
                   IDPYNGDTTYNQKFKG KATLTVDKSSSTAYMHLNSLTSEDSAVYYCLT TA   
               
               
                   
               
               
                   YAMDY WGQGTSVTVSS 
               
               
                   
               
               
                 Nucleic acid sequence (SEQ ID NO: 364): 
               
               
                 GAGATCCAGCTGCAGCAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTC 
               
               
                   
               
               
                 AGTGAAGGTATCCTGCAAGGCTTCTGGTTATGCATTCACTAGCTACAACA 
               
               
                   
               
               
                 TGTACTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGATAT 
               
               
                   
               
               
                 ATTGATCCTTACAATGGTGATACTACCTACAACCAGAAGTTCAAGGGCAA 
               
               
                   
               
               
                 GGCCACATTGACTGTTGACAAGTCCTCCAGCACAGCCTACATGCATCTCA 
               
               
                   
               
               
                 ACAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTCTCACTACGGCC 
               
               
                   
               
               
                 TATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA 
               
            
           
         
       
     
     WBP7011-4.155.8-VL 
     
       
         
           
               
            
               
                 Amino acid sequence (SEQ ID NO: 365): 
               
               
                 QIVLTQSPAIMSASLGEEITLTC SASSTVNYMH WYQQKSGTSPKLLIY ST   
               
               
                   
               
               
                   SNLAS GVPSRFSGSGSGTFYSLTIRSVEAEDAADYYC HQWSSYPYT FGGG 
               
               
                   
               
               
                 TKLEIK 
               
               
                   
               
               
                 Nucleic acid sequence (SEQ ID NO: 366): 
               
               
                 CAAATTGTTCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCTAGGGGA 
               
               
                   
               
               
                 GGAGATCACCCTAACCTGCAGTGCCAGCTCGACTGTAAATTACATGCACT 
               
               
                   
               
               
                 GGTACCAGCAGAAGTCAGGCACTTCTCCCAAACTCTTGATTTATAGCACA 
               
               
                   
               
               
                 TCCAACCTGGCTTCTGGAGTCCCTTCTCGCTTCAGTGGCAGTGGGTCTGG 
               
               
                   
               
               
                 GACCTTTTATTCTCTCACAATCAGAAGTGTGGAGGCTGAAGATGCTGCCG 
               
               
                   
               
               
                 ATTATTACTGCCATCAGTGGAGTAGTTATCCGTACACGTTCGGAGGGGGG 
               
               
                   
               
               
                 ACCAAGCTGGAAATAAAA 
               
            
           
         
       
     
     In certain embodiments, the anti-CD19 binding moiety disclosed herein comprises a heavy chain CDR3 sequence of the anti-CD19 antibody WBP7011_4.155.8 or W7011-4.155.8-z1-P15. In certain embodiments, the anti-CD19 binding moiety provided herein comprises a heavy chain CDR3 sequence comprising SEQ ID NO: 358. Heavy chain CDR3 regions are located at the center of the antigen-binding site, and therefore are believed to make the most contact with antigen and provide the most free energy to the affinity of antibody to antigen. It is also believed that the heavy chain CDR3 is by far the most diverse CDR of the antigen-binding site in terms of length, amino acid composition and conformation by multiple diversification mechanisms (Tonegawa S., Nature. 302:575-81 (1983)). The diversity in the heavy chain CDR3 is sufficient to produce most antibody specificities (Xu J L, Davis M M. Immunity. 13:37-45 (2000)) as well as desirable antigen-binding affinity (Schier R, etc., J Mol Biol. 263:551-67 (1996)). 
     In certain embodiments, the anti-CD19 antibodies disclosed herein are humanized. The heavy chain and light chain variable region sequences for the anti-CD19 humanized antibody W7011-4.155.8-z1-P15 are provided below. 
     W7011-4.155.8-z1-P15-VH 
     
       
         
           
               
            
               
                 Amino acid sequence (SEQ ID NO: 367): 
               
               
                 QMQLVQSGPEVKKPGTSVKVSCKAS GYAFTSYNMY WVRQARGQRLEWIG Y   
               
               
                   
               
               
                   IDPYNADTTYNQKFKG RVTITRDMSTSTAYMELSSLRSEDTAVYYCLT TA   
               
               
                   
               
               
                   YAMDY WGQGTLVTVSS 
               
               
                   
               
               
                 Nucleic acid sequence (SEQ ID NO: 368): 
               
               
                 CAAATGCAGCTCGTCCAGTCTGGACCTGAAGTGAAGAAGCCCGGGACATC 
               
               
                   
               
               
                 CGTCAAGGTCTCATGTAAGGCTAGCGGGTACGCATTCACTTCCTACAACA 
               
               
                   
               
               
                 TGTACTGGGTGCGCCAGGCCAGAGGACAGAGGTTGGAGTGGATCGGCTAC 
               
               
                   
               
               
                 ATCGACCCATACAACGCCGATACTACCTACAATCAGAAGTTTAAAGGGCG 
               
               
                   
               
               
                 GGTGACCATTACCCGGGATATGTCCACCTCCACCGCCTACATGGAGCTGA 
               
               
                   
               
               
                 GCAGCCTGAGGAGCGAGGACACAGCCGTGTACTACTGCCTGACAACAGCC 
               
               
                   
               
               
                 TATGCCATGGACTATTGGGGCCAGGGCACACTTGTGACTGTGAGCAGT 
               
            
           
         
       
     
     W7011-4.155.8-z1-P15-VL 
     
       
         
           
               
            
               
                 Amino acid sequence (SEQ ID NO: 369): 
               
               
                 DIQLTQSPSFLSASVGDRVTITC SASSTVNYMH WYQQKPGKAPKLLIY ST   
               
               
                   
               
               
                   SNLAS GVPSRFSGSGSGTEFTLTISSLQPEDFATYYC HQWSSYPYT FGQG 
               
               
                   
               
               
                 TKLEIK 
               
               
                   
               
               
                 Nucleic acid sequence (SEQ ID NO: 370): 
               
               
                 GACATCCAGCTCACCCAATCCCCTTCTTTCCTCTCCGCAAGTGTCGGAGA 
               
               
                   
               
               
                 TAGGGTGACTATCACCTGCTCAGCTTCTTCAACCGTGAACTACATGCATT 
               
               
                   
               
               
                 GGTACCAGCAGAAGCCCGGGAAAGCCCCAAAGCTGCTGATCTACAGCACC 
               
               
                   
               
               
                 TCCAATCTGGCCAGTGGAGTGCCAAGCCGGTTTAGCGGGAGCGGCTCCGG 
               
               
                   
               
               
                 CACTGAATTCACTTTGACAATTAGCAGCCTTCAGCCTGAGGACTTTGCCA 
               
               
                   
               
               
                 CATATTACTGTCACCAGTGGTCCAGCTACCCCTACACATTCGGGCAGGGC 
               
               
                   
               
               
                 ACAAAGCTGGAGATTAAG 
               
            
           
         
       
     
     Bispecific Polypeptide Complexes 
     In one aspect, the present disclosure provides herein a bispecific polypeptide complex. The term “bispecific” as used herein means that, there are two antigen-binding moieties, each of which is capable of specifically binding to a different antigen or a different epitope on the same antigen. The bispecific polypeptide complex provided herein comprises a first antigen-binding moiety comprising a first heavy chain variable domain operably linked to a first TCR constant region (C1) and a first light chain variable domain operably linked to a second TCR constant region (C2), wherein C1 and C2 are capable of forming a dimer comprising at least one non-native and stabilizing interchain bond between C1 and C2. The bispecific polypeptide complex provided herein further comprises a second antigen-binding moiety comprising a second antigen-binding site but does not contain a sequence derived from a TCR constant region. 
     In certain embodiments, the present disclosure provides a bispecific polypeptide complex, comprising a first antigen-binding moiety associated with a second antigen-binding moiety, wherein: 
     the first antigen-binding moiety comprising:
         a first polypeptide comprising, from N-terminus to C-terminus, a first heavy chain variable domain (VH) of a first antibody operably linked to a first T cell receptor (TCR) constant region (C1), and   a second polypeptide comprising, from N-terminus to C-terminus, a first light chain variable domain (VL) of the first antibody operably linked to a second TCR constant region (C2),       

     wherein:
         C1 and C2 are capable of forming a dimer comprising at least one non-native interchain bond between a first mutated residue comprised in C1 and a second mutated residue comprised in C2, and the non-native interchain bond is capable of stabilizing the dimer, and   the first antibody has a first antigenic specificity,   a second antigen-binding moiety has a second antigenic specificity which is different from the first antigenic specificity, and   the first antigen-binding moiety and the second antigen-binding moiety are less prone to mispair than otherwise would have been if both the first and the second antigen-binding moieties are counterparts of natural Fab.       

     The bispecific polypeptide complex provided herein is significantly less prone to have mispaired heavy chain and light chain variable domains. Without wishing to be bound by any theory, it is believed that the stabilized TCR constant regions in the first antigen-binding moiety can specifically associate with each other and therefore contribute to the highly specific pairing of the intended VH1 and VL1, while discouraging unwanted mispairings of VH1 or VL1 with other variable regions that do not provide for the intended antigen-binding sites. 
     The bispecific polypeptide complexes in WuXiBody formats have longer in vivo half life and are relatively easier to manufacture when compared to bispecific polypeptide complexes in other formats. 
     In certain embodiments, the second antigen-binding moiety of the bispecific polypeptide complex provided herein comprises a second heavy chain variable domain (VH2) and a second light chain variable domain (VL2) of a second antibody. In certain embodiments, at least one of VH2 and VL2 is operably linked to an antibody constant region, or both VH2 and VL2 are operably linked to antibody heavy chain and light chain constant regions respectively. In certain embodiments, the second antigen-binding moiety further comprises an antibody constant CH1 domain operably linked to VH2, and an antibody light chain constant domain operably linked to VL2. For example, the second antigen-binding moiety comprises a Fab. 
     Where a first, second, third, and fourth variable domains (e.g. VH1, VH2, VL1 and VL2) are expressed in one cell, it is highly desired that VH1 specifically pairs with VL1, and VH2 specifically pairs with VL2, such that the resulting bispecific protein product would have the correct antigen-binding specificities. However, in existing technologies such as hybrid-hybridoma (or quadroma), random pairing of VH1, VH2, VL1 and VL2 occurs and consequently results in generation of up to ten different species, of which only one is the functional bispecific antigen-binding molecule. This not only reduces production yields but also complicates the purification of the target product. 
     The bispecific polypeptide complexes provided herein are exceptional in that the variable domains are less prone to mispair than otherwise would have been if both the first and the second antigen-binding moieties are counterparts of natural Fab. In an illustrative example, the first antigen-binding domain comprises VH1-C1 paired with VL1-C2, and the second antigen-binding domain comprises VH2-CH1 paired with VL2-CL. It has been surprisingly found that C1 and C2 preferentially associates with each other, and are less prone to associate with CL or CH1, thereby formation of unwanted pairs such as C1-CH, C1-CL, C2-CH, and C2-CL are discouraged and significantly reduced. As a result of specific association of C1-C2, VH1 specifically pairs with VL1 and thereby rendering the first antigen binding site, and CH1 specifically pairs with CL, thereby allowing specific pairing of VH2-VL2 which provides for the second antigen binding site. Accordingly, the first antigen binding moiety and the second antigen binding moiety are less prone to mismatch, and mispairings between for example VH1-VL2, VH2-VL1, VH1-VH2, VL1-VL2 would be significantly reduced otherwise could have been if both the first and the second antigen-binding moieties are counterparts of natural Fabs, e.g. in the form of VH1-CH1, VL1-CL, VH2-CH1, and VL2-CL. 
     In certain embodiments, the bispecific polypeptide complex provided herein, when expressed from a cell, would have significantly less mispairing products (e.g., at least 1, 2, 3, 4, 5 or more mispairing products less) and/or significantly higher production yield (e.g., at least 10%, 20%, 30%, 40%, 50%, 60% or more higher yield), than a reference molecule expressed under comparable conditions, wherein the reference molecule is otherwise identical to the bispecific polypeptide complex except having a native CH1 in the place of C1 and a native CL in the place of C2. 
     In certain embodiments, the first and/or the second antigen binding moiety is multivalent, such as bivalent, trivalent, tetravalent. The term “valent” as used herein refers to the presence of a specified number of antigen binding sites in a given molecule. As such, the terms “bivalent”, “tetravalent”, and “hexavalent” denote the presence of two binding site, four binding sites, and six binding sites, respectively, in an antigen-binding molecule. A bivalent molecule can be monospecific if the two binding sites are both for specific binding of the same antigen or the same epitope. Similarly, a trivalent molecule can be bispecific, for example, when two binding sites are monospecific for a first antigen (or epitope) and the third binding site is specific for a second antigen (or epitope). In certain embodiments, the first and/or the second antigen-binding moiety in the bispecific polypeptide complex provided herein can be bivalent, trivalent, or tetravalent, with at least two binding sites specific for the same antigen or epitope. This, in certain embodiments, provides for stronger binding to the antigen or the epitope than a monovalent counterpart. In certain embodiments, in a bivalent antigen-binding moiety, the first valent of binding site and the second valent of binding site are structurally identical (i.e. having the same sequences), or structurally different (i.e. having different sequences albeit with the same specificity). 
     In certain embodiments, the first and/or the second antigen binding moiety is multivalent and comprises two or more antigen binding sites operably linked together, with or without a spacer. 
     In certain embodiments, the first and/or the second antigen binding moiety comprises one or more Fab, Fab′, Fab′-SH, F(ab′) 2 , Fd, Fv, and scFv fragments, and other fragments described in Spiess et al., 2015, supra and Brinkmann et al., 2017, supra, or the combination thereof, which are linked with or without a spacer at the heavy chain and/or the light chain and forms at least one are capable of binding to a second antibody. 
     In certain embodiments, the second antigen binding moiety comprises two or more Fab of the second antibody. The two Fabs can be operably linked to each other, for example the first Fab can be covalently attached to the second Fab via heavy chain, with or without a spacer in between. 
     In certain embodiments, the first antigen-binding moiety further comprises a first dimerization domain, and the second antigen-binding moiety further comprises a second dimerization domain. The term “dimerization domain” as used herein refers to the peptide domain which is capable of associating with each other to form a dimer, or in some examples, enables spontaneous dimerization of two peptides. 
     In certain embodiments, the first dimerization domain can be associated with the second dimerization domain. The association can be via any suitable interaction or linkage or bonding, for example, via a connecter, a disulphide bond, a hydrogen bond, electrostatic interaction, a salt bridge, or hydrophobic-hydrophilic interaction, or the combination thereof. Exemplary dimerization domains include, without limitation, antibody hinge region, an antibody CH2 domain, an antibody CH3 domain, and other suitable protein monomers capable of dimerizing and associating with each other. Hinge region, CH2 and/or CH3 domain can be derived from any antibody isotypes, such as IgG1, IgG2, and IgG4. 
     In certain embodiments, the first and/or the second dimerization domain comprises at least a portion of an antibody hinge region. In certain embodiments, the first and/or the second dimerization domain may further comprise an antibody CH2 domain, and/or an antibody CH3 domain. In certain embodiments, the first and/or the second dimerization domain comprises at least a portion of Hinge-Fc region, i.e. Hinge-CH2-CH3 domain. In certain embodiments, the first dimerization domain can be operably linked to the C terminal of the first TCR constant region. In certain embodiments, the second dimerization domain can be operably linked to the C terminal of the antibody CH1 constant region of the second antigen-binding moiety. 
     In certain embodiments, the first dimerization domain is operably linked to (with or without a spacer in between) the first TCR constant region (C1) at a third conjunction domain. 
     If the Fv region of an immunoglobulin is aligned with a TCR immunoglobulin-like domain, the antibody Hinge N terminal and the TCR Hinge N terminal would also be aligned. An example is given in Table 7 below, where antibody Hinge N terminal (SEQ ID NO: 278 or 279) is aligned to TCR Beta Hinge N terminal (SEQ ID NO: 280). 
     The third conjunction domain of the bispecific polypeptide complex as provided herein can be selected such that it comprises a proper length (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues) of the antibody Hinge N terminal, and a proper length (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues) of the TCR Hinge N terminal. The term “Hinge N terminal” as used herein refers to the most N terminal fragment of the hinge region. For example, the conjunction domain may be selected to have all, or most, or some sequences from the antibody Hinge N terminal or from the TCR Hinge N terminal, or may comprise more residues from antibody Hinge N terminal than from TCR Hinge N terminal, or vice versa. 
     In certain embodiments, the third conjunction domains of the polypeptide complex as provided herein have a total length comparable to that of the antibody Hinge N terminal or that of the TCR Hinge N terminal. 
     Similarly, a proper third conjunction domain can be determined on a structural basis. For example, the three-dimensional structures of antibody and TCR may be superimposed, and overlappings of the antibody Hinge N terminal and the TCR Hinge N terminal on the superimposed structure may be determined and considered when determining the length or proportion of sequences from antibody or TCR Hinge N terminal. 
     In certain embodiments, the third conjunction domain comprises a spacer in between the fragments from antibody Hinge N terminal and TCR Hinge N terminal. Any suitable sequences or length of spacer sequences can be used, as long as it does not negatively affect the antigen binding or stability of the polypeptide complex. 
     Exemplary sequences of antibody Hinge N terminal, TCR Hinge N terminal, and the third conjunction domains are provided in the below Tables 7, 8, 9 and 10. 
     In certain embodiments, C1 comprises an engineered CBeta and the first dimerization domain comprises hinge and Fc of IgG1 or IgG4. Table 7 shows the exemplary designs for the conjunction domains useful for TCR CBeta fused to antibody Hinge. The antibody Hinge N terminal is aligned to TCR Beta Hinge N terminal. Exemplary designs of the conjunction domains are also provided in an alignment form (see, e.g., SEQ ID NO: 152 or 153). In such embodiments, the third conjunction domain is comprised in SEQ ID NO: 53 or 54 (which encompass the third conjunction domain and the Hinge C terminal). 
     
       
         
           
               
             
               
                 TABLE 7 
               
             
            
               
                   
               
               
                 Third conjunction domain designs for VH-CBeta- 
               
               
                 Hinge 
               
            
           
           
               
               
               
            
               
                   
                   
                 SEQ 
               
               
                   
                   
                 ID 
               
               
                   
                 Hinge 
                 NO 
               
               
                   
               
               
                 IgG1_Antibody_H 
                 EPKS- C DKTHTC...... 
                 278 
               
               
                   
               
               
                 IgG4_Antibody_H 
                 ESK----YGPPC...... 
                 279 
               
               
                   
               
               
                 TCR_ beta 
                   WGRADCGFTSVS ...... 
                 280 
               
               
                   
               
               
                 Conjunction′_1 (IgG1) 
                   WGRA - S DKTHTC...... 
                 152 
               
               
                   
               
               
                 Conjunction′_1 (IgG4) 
                   WGR ----YGPPC...... 
                 153 
               
               
                   
               
            
           
         
       
     
     In certain embodiments, C1 comprises an engineered CAlpha or CPre-Alpha and the first dimerization domain comprises hinge and Fc of IgG1 or IgG4. Table 8 shows the exemplary designs for the conjunction domains useful for TCR CAlpha or CPre-Alpha fused to antibody Hinge. The antibody Hinge N terminal is aligned to TCR Alpha or CPre-Alpha Hinge N terminal. In such embodiments, the third conjunction domain is comprised in SEQ ID NO: 134, 135, 140, or 141 (which encompass the third conjunction domain and the Hinge C terminal). 
     
       
         
           
               
             
               
                 TABLE 8 
               
             
            
               
                   
               
               
                 Third conjunction domain designs for VH-CAlpha- 
               
               
                 Hinge 
               
            
           
           
               
               
               
            
               
                   
                   
                 SEQ 
               
               
                   
                   
                 ID 
               
               
                   
                 Hinge 
                 NO 
               
               
                   
               
               
                 IgG1_Antibody_H 
                 EPKS-CDKTHTC...... 
                 281 
               
               
                   
               
               
                 IgG4_Antibody_H 
                 ESK----YGPPC...... 
                 282 
               
               
                   
               
               
                 TCR_alpha or 
                 ------------ 
                   
               
               
                 TCR_pre-alpha 
                   
                   
               
               
                   
               
               
                 Conjunction′_2 (IgG1) 
                 -----SDKTHTC...... 
                 154 
               
               
                   
               
               
                 Conjunction′_2 (IgG4) 
                 -------YGPPC...... 
                 155 
               
               
                   
               
            
           
         
       
     
     In certain embodiments, C1 comprises an engineered CGamma and the first dimerization domain comprises hinge and Fc of IgG1 or IgG4. Table 9 shows the exemplary designs for the conjunction domains useful for TCR CGamma fused to antibody Hinge. The antibody Hinge N terminal is aligned to TCR Gamma Hinge N terminal. Exemplary designs of the conjunction domains are also provided in an alignment form (see, e.g., SEQ ID NO: 165 or 166). In such embodiments, the third conjunction domain is comprised in SEQ ID NO: 121 or 122 (which encompass the third conjunction domain and the Hinge C terminal). 
     
       
         
           
               
             
               
                 TABLE 9 
               
             
            
               
                   
               
               
                 Third conjunction domain designs for VH-CGamma- 
               
               
                 Hinge 
               
            
           
           
               
               
               
            
               
                   
                   
                 SEQ 
               
               
                   
                   
                 ID 
               
               
                   
                 Hinge 
                 NO 
               
               
                   
               
            
           
           
               
               
               
            
               
                 IgG1_Antibody_H 
                 EPKSCDKTHTC...... 
                 60 
               
               
                   
               
               
                 IgG4_Antibody_H 
                 ESK---YGPPC...... 
                 61 
               
               
                   
               
               
                 TCR_gamma 
                   PPIKTDVITMD ...... 
                 62 
               
               
                   
               
               
                 Conjunction′_3 (IgG1) 
                   PPIK SDKTHTC...... 
                 165 
               
               
                   
               
               
                 Conjunction′_3 (IgG4) 
                   PPI ---YGPPC...... 
                 166 
               
               
                   
               
            
           
         
       
     
     In certain embodiments, C1 comprises an engineered CDelta and the first dimerization domain comprises hinge and Fc of IgG1 or IgG4. Table 10 shows the exemplary designs for the conjunction domains useful for TCR CDelta fused to antibody Hinge. The antibody Hinge N terminal is aligned to TCR Delta Hinge N terminal. Exemplary designs of the conjunction domains are also provided in an alignment form. In such embodiments, the third conjunction domain is comprised in SEQ ID NO: 127, or 128 (which encompass the third conjunction domain and the Hinge C terminal). 
     
       
         
           
               
             
               
                 TABLE 10 
               
             
            
               
                   
               
               
                 Third conjunction domain designs for VH-CDelta- 
               
               
                 Hinge-Fc 
               
            
           
           
               
               
               
            
               
                   
                   
                 SEQ 
               
               
                   
                   
                 ID 
               
               
                   
                 Hinge and Fc 
                 NO 
               
               
                   
               
            
           
           
               
               
               
            
               
                 IgG1_Antibody_H 
                 EPKSCDKTHTC...... 
                 63 
               
               
                   
               
               
                 IgG4_Antibody_H 
                 ESK---YGPPC...... 
                 103 
               
               
                   
               
               
                 TCR_delta 
                   FEVKTDSTDHV ...... 
                 104 
               
               
                   
               
               
                 Conjunction′_4 (IgG1) 
                 EPKSSDKTHTC...... 
                 167 
               
               
                   
               
               
                 Conjunction′_4 (IgG4) 
                 ESK---YGPPC...... 
                 168 
               
               
                   
               
            
           
         
       
     
     In certain embodiments, the first dimerization domain is operably linked to the C-terminal of an engineered TCR constant region, and together forms a chimeric constant region. In other words, the chimeric constant region comprises the first dimerization domain operably linked with the engineered TCR constant region. 
     In certain embodiments, the chimeric constant region comprises an engineered CBeta attached to the first hinge-Fc region derived from IgG1, IgG2 or IgG4. Exemplary sequences of such a chimeric constant region are provided in Tables 11, 12, 13 and 14. 
     In certain embodiments, the chimeric constant region comprises an engineered CAlpha attached to the first hinge derived from IgG1, IgG2 or IgG4. Exemplary sequences of such chimeric constant region are provided in Tables 11, 12, and 13. 
     In certain embodiments, the chimeric constant region comprises an engineered CPre-Alpha attached to the first hinge derived from IgG1, IgG2 or IgG4, at the third conjunction domain comprising or being SEQ ID NO: 134, 135, 140 or 141. Exemplary sequences of such a chimeric constant region are provided in Tables 15 and 16. 
     In certain embodiments, the chimeric constant region comprises an engineered CGamma attached to the first hinge derived from IgG1, IgG2 or IgG4. Exemplary sequences of such a chimeric constant region are provided in Tables 17, 18. 
     In certain embodiments, the chimeric constant region comprises an engineered CDelta attached to the first hinge derived from IgG1, IgG2 or IgG4. Exemplary sequences of such a chimeric constant region are provided in Tables 17 and 18. 
     In certain embodiments, the chimeric constant region further comprises a first antibody CH2 domain, and/or a first antibody CH3 domain. For example, the chimeric constant region further comprises a first antibody CH2-CH3 domain attached to the C-terminus of the third conjunction domain. Exemplary sequences of such chimeric constant region are provided in Table 19. 
     In certain embodiments, the first chimeric constant region and the second TCR constant domain comprises a pair of sequences selected from the group consisting of SEQ ID NOs: 177/176, 179/178, 184/183, 185/183, 180/176, 181/178, 182/178, 184/186, 185/186, 188/187, 196/187, 190/189, 192/191, 192/193, 195/194, 198/197, 200/199, 202/201, 203/201, 203/204, 205/204, 206/204, 208/207, 208/209, 211/210, 213/212, 213/151, 214/212, 214/151, 234/233, 232/231, 216/215, 218/217, 220/219, 222/221, 224/223, 226/225, 227/223, 229/228, 229/230, 236/235 and 238/237, as shown in Table 19. 
     These pairs of chimeric constant regions and second TCR constant domains are useful in that they can be manipulated to fuse to a desired antibody variable region, so as to provide for the polypeptide complex as disclosed herein. For example, an antibody heavy chain variable region can be fused to the chimeric constant region (comprising C1), thereby rendering the first polypeptide chain of the polypeptide complex provided herein; and similarly, an antibody light chain variable region can be fused to the second TCR constant domain (comprising C2), thereby rendering the second polypeptide chain of the polypeptide complex provided herein. 
     These pairs of chimeric constant regions and second TCR constant domains can be used as a platform for generating the first antigen-binding moiety of the bispecific polypeptide complexes provided herein. For example, variable regions of a first antibody can be fused at the N-terminus of the platform sequences (e.g. fusing the VH to the chimeric constant domain and the VL to the TCR constant domain, respectively). To produce the bispecific polypeptide complex, the second antigen-binding moiety can be designed and produced, so as to associate into the bispecific polypeptide complex provided herein. 
     In certain embodiments, the second dimerization domain comprises a hinge region. The hinge region may be derived from an antibody, such as IgG1, IgG2, or IgG4. In certain embodiments, the second dimerization domain may optionally further comprise an antibody CH2 domain, and/or an antibody CH3 domain, for example such as a hinge-Fc region. The hinge region may be attached to the antibody heavy chain of the second antigen binding site (e.g. Fab). 
     In the bispecific polypeptide complex, the first and the second dimerization domain are capable of associating into a dimer. In certain embodiments, the first and the second dimerization domains are different and associate in a way that discourages homodimerization and/or favors heterodimerization. For example, the first and the second dimerization domains can be selected so that they are not identical and that they preferentially form heterodimers between each other rather than to form homodimers within themselves. In certain embodiments, the first and the second dimerization domains are capable of associating into heterodimers via formation of knob-into-hole, hydrophobic interaction, electrostatic interaction, hydrophilic interaction, or increased flexibility. 
     In certain embodiments, the first and the second dimerization domains comprise CH2 and/or CH3 domains which are respectively mutated to be capable of forming a knobs-into-holes. A knob can be obtained by replacement of a small amino acid residue with a larger one in the first CH2/CH3 polypeptide, and a hole can be obtained by replacement of a large residue with a smaller one. For details of the mutation sites for knobs into holes please see Ridgway et al., 1996, supra, Spiess et al., 2015, supra and Brinkmann et al., 2017, supra. 
     In certain embodiments, the first and the second dimerization domains comprise a first CH3 domain of the IgG1 isotype containing S139C and T151W substitution (SEQ ID NO: 295, knob) and a second CH3 domain of the IgG1 isotype containing Y134C, T151S, L153A and Y192V substitution (SEQ ID NO: 296, hole). In another embodiments, the first and the second dimerization domains comprise a first CH3 domain of the IgG4 isotype containing S136C and T148W substitution (SEQ ID NO: 298, knob) and a second CH3 domain of the IgG4 isotype containing Y131C, T148S, L150A and Y189V substitution (SEQ ID NO: 299, hole). The sequences and numberings of wild type Fc IgG1 (SEQ ID NO: 294) and Fc IgG4 (SEQ ID NO: 297) are shown in  FIGS. 20A-20D . As noted above, XnY when referring to Fc region (e.g. CH3 domain of the Fc region), the numbering of the amino acid residue is based on the numbering shown in  FIGS. 20A-20D . 
     In certain embodiments, the first and the second dimerization domains further comprise a first hinge region and a second hinge region. For example, charge pairs of substitution can be introduced into the hinge region of IgG1 and IgG2 to promote heterodimerization. For details see Brinkmann et al., 2017, supra. 
     Bispecific Format 
     The bispecific polypeptide complex provided herein can be in any suitable bispecific format known in the art. In certain embodiments, the bispecific polypeptide complex is based on a reference bispecific antibody format. “Based on” as used herein with respect to a bispecific format means that the bispecific polypeptide complex provided herein takes the same bispecific format of a reference bispecific antibody, except that one of the antigen-binding moiety has been modified to comprise a VH operably linked to C1 and a VL operably linked to C2 wherein C1 and C2 are associated with at least one non-native interchain bond, as defined above. Examples of reference bispecific antibody formats known in the art include, without limitation, (i) a bispecific antibody with symmetric Fc, (ii) a bispecific antibody with asymmetric Fc, (iii) a regular antibody appended with an additional antigen-binding moiety, (iv) a bispecific antibody fragment, (v) a regular antibody fragment appended with an additional antigen-binding moiety, (vi) a bispecific antibody appended with human albumin or human albumin-binding peptide. 
     BsIgG is monovalent for each antigen and can be produced by co-expression of the two light and two heavy chains in a single host cell. An appending IgG is engineered to form bispecific IgG by appending either the amino or carboxy termini of either light or heavy chains with additional antigen-binding units. The additional antigen-binding units can be single domain antibodies (unpaired VL or VH), such as DVD-Ig, paired antibody variable domains (e.g. Fv or scFv) or engineered protein scaffolds. Any of the antigen-binding units in BsIgG, in particular paired VH-CH1/VL-CL, can be modified to replace the CH1 to C1 and CL to C2 as disclosed herein, to render the bispecific polypeptide complex as provided herein. 
     Bispecific antibody fragments are antigen-binding fragments that are derived from an antibody but lack some or all of the antibody constant domains. Examples of such a bispecific antibody fragment include, for example, such as single domain antibody, Fv, Fab and diabody etc. To render the bispecific polypeptide complex as provided herein, an antigen-binding site (e.g. particular paired VH-CH1/VL-CL) in a bispecific antibody fragment, can be modified to comprise the polypeptide complex as disclosed herein (e.g. VH-C1/CL-C2). 
     In certain embodiments, the bispecific polypeptide complex as provided herein is based on the format of a “whole” antibody, such as whole IgG or IgG-like molecules, and small recombinant formats, such as tandem single chain variable fragment molecules (taFvs), diabodies (Dbs), single chain diabodies (scDbs) and various other derivatives of these (cf. bispecific antibody formats as described by Byrne H. et al. (2013) Trends Biotech, 31 (11): 621-632. Examples of bispecific antibody is based on a format which include, but is not limited to, quadroma, chemically coupled Fab (fragment antigen binding), and BiTE (bispecific T cell engager). 
     In certain embodiments, the bispecific polypeptide complex as provided herein is based on a bispecific format selected from Triomabs; hybrid hybridoma (quadroma); Multispecific anticalin platform (Pieris); Diabodies; Single chain diabodies; Tandem single chain Fv fragments; TandAbs, Trispecific Abs (Affimed); Darts (dual affinity retargeting; Macrogenics); Bispecific Xmabs (Xencor); Bispecific T cell engagers (Bites; Amgen; 55 kDa); Triplebodies; Tribody (Fab-scFv) Fusion Protein (CreativeBiolabs) multifunctional recombinant antibody derivates; Duobody platform (Genmab); Dock and lock platform; Knob into hole (KIH) platform; Humanized bispecific IgG antibody (REGN1979) (Regeneron); Mab 2  bispecific antibodies (F-Star); DVD-Ig (dual variable domain immunoglobulin) (Abbvie); kappa-lambda bodies; TBTI (tetravalent bispecific tandem Ig); and CrossMab. 
     In certain embodiments, the bispecific polypeptide complex as provided herein is based on a bispecific format selected from bispecific IgG-like antibodies (BsIgG) comprising CrossMab; DAF (two-in-one); DAF (four-in-one); DutaMab; DT-IgG; Knobs-in-holes common LC; Knobs-in-holes assembly; Charge pair; Fab-arm exchange; SEEDbody; Triomab; LUZ-Y; Fcab; kappa-lamda-body; and Orthogonal Fab. For detailed description of the bispecific antibody formats please see Spiess C., Zhai Q. and Carter P. J. (2015) Molecular Immunology 67: 95-106, which is incorporated herein by reference in its entirety. 
     In certain embodiments, the bispecific polypeptide complex as provided herein is based on a bispecific format selected from IgG-appended antibodies with an additional antigen-binding moiety comprising DVD-IgG; IgG(H)-scFv; scFv-(H)IgG; IgG(L)-scFv; scFV-(L)IgG; IgG(L,H)-Fv; IgG(H)-V; V(H)-IgG; IgG(L)-V; V(L)-IgG; KIH IgG-scFab; 2scFv-IgG; IgG-2scFv; scFv4-Ig; scFv4-Ig; Zybody; and DVI-IgG (four-in-one) (see Id.). 
     In certain embodiments, the bispecific polypeptide complex as provided herein is based on a format selected from bispecific antibody fragments comprising Nanobody; Nanobody-HAS; BiTE; Diabody; DART; TandAb; scDiabody; sc-Diabody-CH3; Diabody-CH3; Triple Body; Miniantibody; Minibody; TriBi minibody; scFv-CH3 KIH; Fab-scFv; scFv-CH-CL-scFv; F(ab′)2; F(ab′)2-scFv2; scFv-KIH; Fab-scFv-Fc; Tetravalent HCAb; scDiabody-Fc; Diabody-Fc; Tandem scFv-Fc; and Intrabody (see Id.). 
     In certain embodiments, the bispecific polypeptide complex as provided herein is based on a bispecific format such as Dock and Lock; ImmTAC; HSAbody; scDiabody-HAS; and Tandem scFv-Toxin (see Id.). 
     In certain embodiments, the bispecific polypeptide complex as provided herein is based on a format selected from bispecific antibody conjugates comprising IgG-IgG; Cov-X-Body; and scFv1-PEG-scFv2 (see Id.). 
     In certain embodiments, the first antigen-binding moiety and the second binding moiety can be associated into an Ig-like structure. An Ig-like structure is like a natural antibody having a Y shaped construct, with two arms for antigen-binding and one stem for association and stabilization. The resemblance to natural antibody can provide for various advantages such as good in vivo pharmakinetics, desired immunological response and stability etc. It has been found that the Ig-like structure comprising the first antigen-binding moiety provided herein associated with the second antigen-binding moiety provided herein has thermal stability which is comparable to that of an Ig (e.g. an IgG). In certain embodiments, the Ig-like structure provided herein is at least 70%, 80%, 90%, 95% or 100% of that of a natural IgG. 
     In certain embodiments, the bispecific polypeptide complex comprises four polypeptide chains: i) VH1-C1-Hinge-CH2-CH3; ii) VL1-C2; iii) VH2-CH1-Hinge-CH2-CH3, and iv) VL2-CL, wherein the C1 and C2 are capable of forming a dimer comprising at least one non-native interchain bond, and the two hinge regions and/or the two CH3 domains are capable of forming one or more interchain bond that can facilitate dimerization. 
     Antigenic Specificities of the Bispecific Complex 
     The bispecific complex provided herein have two antigenic specificities. The first and the second antigen-binding moieties are directed to the first and the second antigenic specificities respectively. 
     The first and the second antigenic specificities may be identical, in other words, the first and the second antigen-binding moieties binds to the same antigen molecule, or to the same epitope of the same antigen molecule. 
     Alternatively, the first and the second antigenic specificities may be distinct. For example, the first and the second antigen-binding moieties can bind to different antigens. Such a bispecific polypeptide complex could be useful in, for example, bringing the two different antigens into close proximity and thereby promoting their interactions (e.g. bringing immunological cells in close proximity to a tumor antigen or a pathogen antigen and hence promoting recognition or elimination of such an antigen by the immune system). For another example, the first and the second antigen-binding moieties can bind to different (and optionally non-overlapping) epitopes of one antigen. This may be helpful in enhancing the recognition of or binding to a target antigen, in particular one which is susceptible to mutation (e.g. a viral antigen). 
     In some embodiments, one of the antigenic specificity of the bispecific complex provided herein is directed to a T-cell specific receptor molecule and/or a natural killer cell (NK cell) specific receptor molecule. In some embodiments, one of the first and second antigen-binding moiety is capable of specifically binding to CD3, TCR, CD28, CD16, NKG2D, Ox40, 4-1BB, CD2, CD5 or CD95, and the other is capable of specifically binding to a tumor associated antigen. 
     In certain embodiments, one of the antigenic specificity of the bispecific complex provided herein is directed to CD3. In certain embodiments, the first antigen-binding moiety of the bispecific complex is capable of specifically binding to CD3. In certain embodiments, the second antigen-binding moiety of the bispecific complex is capable of specifically binding to CD3. 
     In certain embodiments, the antigen-binding moiety of the bispecific complex comprises a VH1 and a VL1 both derived from an anti-CD3 antibody. In certain embodiments, the polypeptide complex or the bispecific polypeptide complex provided herein, wherein the first polypeptide and the second polypeptide comprise a pair of sequences selected from the group consisting of SEQ ID NOs: 2/1, 3/4/, 5/1, 6/3, 7/3, 9/8, 10/8, 9/11, 10/11, 13/12, 15/14, 17/16, 17/18, 20/19, 21/12, 28/3, 29/3, 30/12, 31/12, 65/64, 67/66, 69/68, 70/68, 70/71, 72/71, 73/71, 75/74, 75/76, 78/77, 86/85, 90/89, 91/92/, 94/93, 96/95, 98/97, 99/95, 101/100, 101/102, 106/105, 108/107, 110/109, 112/111, 137/136, 138/136, 137/139 and 138/139, wherein the variable regions of anti-CD3 antibody (T3) are fused to the TCR constant region as shown in Table 20. 
     In some embodiments, one of the antigenic specificity of the bispecific complex provided herein is directed to a T-cell specific receptor molecule and/or a natural killer cell (NK cell) specific receptor molecule, and the other antigenic specificity is directed to a tumor associated surface antigen. In certain embodiments, the first antigen-binding moiety of the bispecific complex is capable of specifically binding to T-cell specific receptor molecule and/or a natural killer cell (NK cell) specific receptor molecule (such as CD3), and the second antigen-binding moiety is capable of specifically binding to a tumor associated antigen (such as CD19), or vice versa. 
     In certain embodiments, the bispecific polypeptide complex comprises a four-sequence combination selected from the group consisting of: SEQ ID NOs: 22/12/24/23 (E17, IgG1), 25/12/26/23 (E17, IgG4), and 25/12/27/23 (F16), as shown in Example 8 and Table 20, wherein the first antigen binding moiety binds to CD3, and the second antigen binding moiety binds to CD19. The design of E17 is a bispecific, bivalent antibody, and the design of F16 is a bispecific and trivalent antigen-binding complex, with two repeats of anti-CD19 antibody Fab. 
     In certain embodiments, the bispecific polypeptide complex comprises a first antigen binding moiety that binds to CTLA-4, and a second antigen binding moiety that binds to PD-1, or vice versa. 
     In certain embodiments, the bispecific polypeptide complex comprises four polypeptide chains comprising: i) VH1 operably linked to a first chimeric constant region; ii) VL1 operably linked to a second chimeric constant region; iii) VH2 operably linked to conventional antibody heavy chain constant region, and iv) VL2 operably linked to conventional antibody light chain constant region. In certain embodiments, the first chimeric constant region can comprise C1-Hinge-CH2-CH3, each as defined supra. In certain embodiments, the second chimeric constant region can comprise C2, as defined supra. In certain embodiments, the conventional antibody heavy chain constant region can comprise CH1-Hinge-CH2-CH3, each as defined supra. In certain embodiments, the conventional antibody light chain constant region can comprise CL, as defined supra. 
     The following construct names are used interchangeably in this disclosure: E17-Design_2-QQQQ and W3438-T3U4.E17-1.uIgG4.SP; F16-Design-2-QQQQ and W3438-T3U4.F16-1.uIgG4.SP; U6T5.G25.IgG4 and W3248-U6T5.G25-1.uIgG4.SP; and U6T1.G25R.IgG4 and W3248-U6T1.G25R-1.uIgG4.SP. 
     Method of Preparation 
     The present disclosure provides isolated nucleic acids or polynucleotides that encode the polypeptide complex, and the bispecific polypeptide complex provided herein. 
     The term “nucleic acid” or “polynucleotide” as used herein refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses polynucleotides containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular polynucleotide sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (see Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). 
     The nucleic acids or polynucleotides encoding the polypeptide complex and the bispecific polypeptide complex provided herein can be constructed using recombinant techniques. To this end, DNA encoding an antigen-binding moiety of a parent antibody (such as CDR or variable region) can be isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody). Likewise, DNA encoding a TCR constant region can also be obtained. As an example, the polynucleotide sequence encoding the variable domain (VH) and the polynucleotide sequence encoding the first TCR constant region (C1) are obtained and operably linked to allow transcription and expression in a host cell to produce the first polypeptide. Similarly, polynucleotide sequence encoding VL are operably linked to polynucleotide sequence encoding C1, so as to allow expression of the second polypeptide in the host cell. If needed, encoding polynucleotide sequences for one or more spacers are also operably linked to the other encoding sequences to allow expression of the desired product. 
     The encoding polynucleotide sequences can be further operably linked to one or more regulatory sequences, optionally in an expression vector, such that the expression or production of the first and the second polypeptides is feasible and under proper control. 
     The encoding polynucleotide sequence(s) can be inserted into a vector for further cloning (amplification of the DNA) or for expression, using recombinant techniques known in the art. In another embodiment, the polypeptide complex and the bispecific polypeptide complex provided herein may be produced by homologous recombination known in the art. Many vectors are available. The vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter (e.g. SV40, CMV, EF-1α), and a transcription termination sequence. 
     The term “vector” as used herein refers to a vehicle into which a polynucleotide encoding a protein may be operably inserted so as to bring about the expression of that protein. Typically, the construct also includes appropriate regulatory sequences. For example, the polynucleotide molecule can include regulatory sequences located in the 5′-flanking region of the nucleotide sequence encoding the guide RNA and/or the nucleotide sequence encoding a site-directed modifying polypeptide, operably linked to the coding sequences in a manner capable of expressing the desired transcript/gene in a host cell. A vector may be used to transform, transduce, or transfect a host cell so as to bring about expression of the genetic element it carries within the host cell. Examples of vectors include plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), or P1-derived artificial chromosome (PAC), bacteriophages such as lambda phage or M13 phage, and animal viruses. Categories of animal viruses used as vectors include retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpesvirus (e.g., herpes simplex virus), poxvirus, baculovirus, papillomavirus, and papovavirus (e.g., SV40). A vector may contain a variety of elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selectable elements, and reporter genes. In addition, the vector may contain an origin of replication. A vector may also include materials to aid in its entry into the cell, including but not limited to a viral particle, a liposome, or a protein coating. 
     In some embodiments, the vector system includes mammalian, bacterial, yeast systems, etc., and comprises plasmids such as, but not limited to, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pCMV, pEGFP, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS420, pLexA, pACT2.2 etc., and other laboratorial and commercially available vectors. Suitable vectors may include, plasmid, or viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses). 
     Vectors comprising the polynucleotide sequence(s) provided herein can be introduced to a host cell for cloning or gene expression. The phrase “host cell” as used herein refers to a cell into which an exogenous polynucleotide and/or a vector has been introduced. 
     Suitable host cells for cloning or expressing the DNA in the vectors herein are the prokaryote, yeast, or higher eukaryote cells described above. Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as  Escherichia , e.g.,  E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella , e.g.,  Salmonella typhimurium, Serratia , e.g.,  Serratia marcescans , and  Shigella , as well as Bacilli such as  B. subtilis  and  B. licheniformis, Pseudomonas  such as  P. aeruginosa , and  Streptomyces.    
     In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for the vectors encoding the polypeptide complex and the bispecific polypeptide complex.  Saccharomyces cerevisiae , or common baker&#39;s yeast, is the most commonly used among lower eukaryotic host microorganisms. However, a number of other genera, species, and strains are commonly available and useful herein, such as  Schizosaccharomyces pombe; Kluyveromyces  hosts such as, e.g.,  K. lactis, K. fragilis  (ATCC 12,424),  K. bulgaricus  (ATCC 16,045),  K. wickeramii  (ATCC 24,178),  K. waltii  (ATCC 56,500),  K. drosophilarum  (ATCC 36,906),  K. thermotolerans , and  K. marxianus; yarrowia  (EP 402,226);  Pichia pastoris  (EP 183,070);  Candida; Trichoderma reesia  (EP 244,234);  Neurospora crassa; Schwanniomyces  such as  Schwanniomyces occidentalis ; and filamentous fungi such as, e.g.,  Neurospora, Penicillium, Tolypocladium , and  Aspergillus  hosts such as  A. nidulans  and  A. niger.    
     Suitable host cells for the expression of glycosylated polypeptide complex, the bispecific polypeptide complex provided herein are derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts such as  Spodoptera frugiperda  (caterpillar),  Aedes aegypti  (mosquito),  Aedes albopictus  (mosquito),  Drosophila melanogaster  (fruiffly), and  Bombyx mori  have been identified. A variety of viral strains for transfection are publicly available, e.g., the L-1 variant of  Autographa californica  NPV and the Bm-5 strain of  Bombyx mori  NPV, and such viruses may be used as the virus herein according to the present invention, particularly for transfection of  Spodoptera frugiperda  cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be utilized as hosts. 
     However, interest has been greatest in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)), such as Expi293; baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2). 
     Host cells are transformed with the above-described expression or cloning vectors can be cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the cloning vectors. 
     For production of the polypeptide complex and the bispecific polypeptide complex provided herein, the host cells transformed with the expression vector may be cultured in a variety of media. Commercially available media such as Ham&#39;s F10 (Sigma), Minimal Essential Medium (MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM), Sigma) are suitable for culturing the host cells. In addition, any of the media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S. Pat. Re. 30,985 may be used as culture media for the host cells. Any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCIN™ drug), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art. The culture conditions, such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan. 
     In one aspect, the present disclosure provides a method of expressing the polypeptide complex and the bispecific polypeptide complex provided herein, comprising culturing the host cell provided herein under the condition at which the polypeptide complex, or the bispecific polypeptide complex is expressed. 
     In certain embodiments, the present disclosure provides a method of producing the polypeptide complex provided herein, comprising a) introducing to a host cell: a first polynucleotide encoding a first polypeptide comprising, from N-terminus to C-terminus, a first heavy chain variable domain (VH) of a first antibody operably linked to a first TCR constant region (C1), and a second polynucleotide encoding a second polypeptide comprising, from N-terminus to C-terminus, a first light chain variable domain (VL) of the first antibody operably linked to a second TCR constant region (C2), wherein: C1 and C2 are capable of forming a dimer comprising at least one non-native interchain bond between C1 and C2, and the non-native interchain bond is capable of stabilizing the dimer of C1 and C2, and the first antibody has a first antigenic specificity; b) allowing the host cell to express the polypeptide complex. 
     In certain embodiments, the present disclosure provides a method of producing the bispecific polypeptide complex provided herein, comprising a) introducing to a host cell: a first polynucleotide encoding a first polypeptide comprising, from N-terminus to C-terminus, a first heavy chain variable domain (VH) of a first antibody operably linked to a first TCR constant region (C1), a second polynucleotide encoding a second polypeptide comprising, from N-terminus to C-terminus, a first light chain variable domain (VL) of the first antibody operably linked to a second TCR constant region (C2), and one or more additional polynucleotides encoding a second antigen-binding moiety, wherein: C1 and C2 are capable of forming a dimer comprising at least one non-native interchain bond between a first mutated residue comprised in C1 and a second mutated residue comprised in C2, and the non-native interchain bond is capable of stabilizing the dimer of C1 and C2, the first antigen-binding moiety and the second antigen-binding moiety have reduced mispairing than otherwise would have been if the first antigen-binding moiety was a natural Fab counterpart, and the first antibody has a first antigenic specificity and the second antibody has a second antigenic specificity, b) allowing the host cell to express the bispecific polypeptide complex. 
     In certain embodiments, the method further comprises isolating the polypeptide complex. 
     When using recombinant techniques, the polypeptide complex, the bispecific polypeptide complex provided herein can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the product is produced intracellularly, as a first step, the particulate debris, either host cells or lysed fragments, is removed, for example, by centrifugation or ultrafiltration. Carter et al., Bio/Technology 10:163-167 (1992) describe a procedure for isolating antibodies which are secreted to the periplasmic space of  E. coli . Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min. Cell debris can be removed by centrifugation. Where the product is secreted into the medium, supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants. 
     The polypeptide complex and the bispecific polypeptide complex provided herein prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography, ammonium sulfate precipitation, salting out, and affinity chromatography, with affinity chromatography being the preferred purification technique. 
     Where the polypeptide complex or the bispecific polypeptide complex provided herein comprises immunoglobulin Fc domain, then protein A can be used as an affinity ligand, depending on the species and isotype of the Fc domain that is present in the polypeptide complex. Protein A can be used for purification of polypeptide complexes based on human γ1, γ2, or γ4 heavy chains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G is recommended for all mouse isotypes and for human γ3 (Guss et al., EMBO J. 5:1567 1575 (1986)). The matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. 
     Where the polypeptide complex or the bispecific polypeptide complex provided herein comprises a CH3 domain, the Bakerbond ABX resin (J. T. Baker, Phillipsburg, N.J.) is useful for purification. Other techniques for protein purification such as fractionation on an ion-exchange column, ethanol precipitation, Reverse Phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE™ chromatography on an anion or cation exchange resin (such as a polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also available depending on the antibody to be recovered. 
     Following any preliminary purification step(s), the mixture comprising the polypeptide complex of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5-4.5, preferably performed at low salt concentrations (e.g., from about 0-0.25M salt). 
     In certain embodiments, the bispecific polypeptide complex provided herein can be readily purified with high yields using conventional methods. One of the advantages of the bispecific polypeptide complex is the significantly reduced mispairing between heavy chain and light chain variable domain sequences. This reduces production of unwanted byproducts and make it possible to obtain high purity product in high yields using relatively simple purification processes. 
     Derivatives 
     In certain embodiments, the polypeptide complex or the bispecific polypeptide complex can be used as the base of conjugation with desired conjugates. 
     It is contemplated that a variety of conjugates may be linked to the polypeptide complex or the bispecific polypeptide complex provided herein (see, for example, “Conjugate Vaccines”, Contributions to Microbiology and Immunology, J. M. Cruse and R. E. Lewis, Jr. (eds.), Carger Press, New York, (1989)). These conjugates may be linked to the polypeptide complex or the bispecific polypeptide complex by covalent binding, affinity binding, intercalation, coordinate binding, complexation, association, blending, or addition, among other methods. 
     In certain embodiments, the polypeptide complex or the bispecific polypeptide complex provided herein may be engineered to contain specific sites outside the epitope binding portion that may be utilized for binding to one or more conjugates. For example, such a site may include one or more reactive amino acid residues, such as for example cysteine or histidine residues, to facilitate covalent linkage to a conjugate. 
     In certain embodiments, the polypeptide complex or the bispecific polypeptide complex may be linked to a conjugate indirectly, or indirectly for example through another conjugate or through a linker. 
     For example, the polypeptide complex or the bispecific polypeptide complex having a reactive residue such as cysteine may be linked to a thiol-reactive agent in which the reactive group is, for example, a maleimide, an iodoacetamide, a pyridyl disulphide, or other thiol-reactive conjugation partner (Haugland, 2003, Molecular Probes Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes, Inc.; Brinkley, 1992, Bioconjugate Chem. 3:2; Garman, 1997, Non-Radioactive Labelling: A Practical Approach, Academic Press, London; Means (1990) Bioconjugate Chem. 1:2; Hermanson, G. in Bioconjugate Techniques (1996) Academic Press, San Diego, pp. 40-55, 643-671). 
     For another example, the polypeptide complex or the bispecific polypeptide complex may be conjugated to biotin, then indirectly conjugated to a second conjugate that is conjugated to avidin. For still another example, the polypeptide complex or the bispecific polypeptide complex may be linked to a linker which further links to the conjugate. Examples of linkers include bifunctional coupling agents such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suherate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and his-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). Particularly preferred coupling agents include N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) (Carlsson et al., Biochem. J. 173:723-737 (1978)) and N-succinimidyl-4-(2-pyridylthio)pentanoate (SPP) to provide for a disulphide linkage. 
     The conjugate can be a detectable label, a pharmacokinetic modifying moiety, a purification moiety, or a cytotoxic moiety. Examples of detectable label may include a fluorescent labels (e.g. fluorescein, rhodamine, dansyl, phycoerythrin, or Texas Red), enzyme-substrate labels (e.g. horseradish peroxidase, alkaline phosphatase, luceriferases, glucoamylase, lysozyme, saccharide oxidases or β-D-galactosidase), radioisotopes (e.g.  123 I,  124 I,  125 I,  131 I,  35 S,  3 H,  111 In,  112 In,  14 C,  64 Cu,  67 Cu,  86 Y,  88 Y,  90 Y,  177 Lu,  211 At,  186 Re,  188 Re,  153 Sm,  212 Bi, and  32 P, other lanthanides, luminescent labels), chromophoric moiety, digoxigenin, biotin/avidin, a DNA molecule or gold for detection. In certain embodiments, the conjugate can be a pharmacokinetic modifying moiety such as PEG which helps increase half-life of the antibody. Other suitable polymers include, such as, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, copolymers of ethylene glycol/propylene glycol, and the like. In certain embodiments, the conjugate can be a purification moiety such as a magnetic bead. A “cytotoxic moiety” can be any agent that is detrimental to cells or that can damage or kill cells. Examples of cytotoxic moiety include, without limitation, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin and analogs thereof, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine). 
     Methods for the conjugation of conjugates to proteins such as antibodies, immunoglobulins or fragments thereof are found, for example, in U.S. Pat. Nos. 5,208,020; 6,441,163; WO2005037992; WO2005081711; and WO2006/034488, which are incorporated herein by reference to the entirety. 
     Pharmaceutical Composition 
     The present disclosure also provides a pharmaceutical composition comprising the polypeptide complex or the bispecific polypeptide complex provided herein and a pharmaceutically acceptable carrier. 
     The term “pharmaceutically acceptable” indicates that the designated carrier, vehicle, diluent, excipient(s), and/or salt is generally chemically and/or physically compatible with the other ingredients comprising the formulation, and physiologically compatible with the recipient thereof. 
     A “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is bioactivity acceptable and nontoxic to a subject. Pharmaceutical acceptable carriers for use in the pharmaceutical compositions disclosed herein may include, for example, pharmaceutically acceptable liquid, gel, or solid carriers, aqueous vehicles, nonaqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending/dispending agents, sequestering or chelating agents, diluents, adjuvants, excipients, or non-toxic auxiliary substances, other components known in the art, or various combinations thereof. 
     Suitable components may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavorings, thickeners, coloring agents, emulsifiers or stabilizers such as sugars and cyclodextrins. Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, thioglycerol, thioglycolic acid, thiosorbitol, butylated hydroxanisol, butylated hydroxytoluene, and/or propyl gallate. As disclosed herein, inclusion of one or more antioxidants such as methionine in a pharmaceutical composition provided herein decreases oxidation of the polypeptide complex or the bispecific polypeptide complex. This reduction in oxidation prevents or reduces loss of binding affinity, thereby improving protein stability and maximizing shelf-life. Therefore, in certain embodiments, compositions are provided that comprise the polypeptide complex or the bispecific polypeptide complex disclosed herein and one or more antioxidants such as methionine. 
     To further illustrate, pharmaceutical acceptable carriers may include, for example, aqueous vehicles such as sodium chloride injection, Ringer&#39;s injection, isotonic dextrose injection, sterile water injection, or dextrose and lactated Ringer&#39;s injection, nonaqueous vehicles such as fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, or peanut oil, antimicrobial agents at bacteriostatic or fungistatic concentrations, isotonic agents such as sodium chloride or dextrose, buffers such as phosphate or citrate buffers, antioxidants such as sodium bisulfate, local anesthetics such as procaine hydrochloride, suspending and dispersing agents such as sodium carboxymethylcelluose, hydroxypropyl methylcellulose, or polyvinylpyrrolidone, emulsifying agents such as Polysorbate 80 (TWEEN-80), sequestering or chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid), ethyl alcohol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid, or lactic acid. Antimicrobial agents utilized as carriers may be added to pharmaceutical compositions in multiple-dose containers that include phenols or cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride and benzethonium chloride. Suitable excipients may include, for example, water, saline, dextrose, glycerol, or ethanol. Suitable non-toxic auxiliary substances may include, for example, wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers, or agents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, or cyclodextrin. 
     The pharmaceutical compositions can be a liquid solution, suspension, emulsion, pill, capsule, tablet, sustained release formulation, or powder. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinyl pyrollidone, sodium saccharine, cellulose, magnesium carbonate, etc. 
     In certain embodiments, the pharmaceutical compositions are formulated into an injectable composition. The injectable pharmaceutical compositions may be prepared in any conventional form, such as for example liquid solution, suspension, emulsion, or solid forms suitable for generating liquid solution, suspension, or emulsion. Preparations for injection may include sterile and/or non-pyretic solutions ready for injection, sterile dry soluble products, such as lyophilized powders, ready to be combined with a solvent just prior to use, including hypodermic tablets, sterile suspensions ready for injection, sterile dry insoluble products ready to be combined with a vehicle just prior to use, and sterile and/or non-pyretic emulsions. The solutions may be either aqueous or nonaqueous. 
     In certain embodiments, unit-dose parenteral preparations are packaged in an ampoule, a vial or a syringe with a needle. All preparations for parenteral administration should be sterile and not pyretic, as is known and practiced in the art. 
     In certain embodiments, a sterile, lyophilized powder is prepared by dissolving the polypeptide complex or the bispecific polypeptide complex as disclosed herein in a suitable solvent. The solvent may contain an excipient which improves the stability or other pharmacological components of the powder or reconstituted solution, prepared from the powder. Excipients that may be used include, but are not limited to, water, dextrose, sorbital, fructose, corn syrup, xylitol, glycerin, glucose, sucrose or other suitable agent. The solvent may contain a buffer, such as citrate, sodium or potassium phosphate or other such buffer known to those of skill in the art at, in one embodiment, about neutral pH. Subsequent sterile filtration of the solution followed by lyophilization under standard conditions known to those of skill in the art provides a desirable formulation. In one embodiment, the resulting solution will be apportioned into vials for lyophilization. Each vial can contain a single dosage or multiple dosages of the polypeptide complex, the bispecific polypeptide complex provided herein or composition thereof. Overfilling vials with a small amount above that needed for a dose or set of doses (e.g., about 10%) is acceptable so as to facilitate accurate sample withdrawal and accurate dosing. The lyophilized powder can be stored under appropriate conditions, such as at about 4° C. to room temperature. 
     Reconstitution of a lyophilized powder with water for injection provides a formulation for use in parenteral administration. In one embodiment, for reconstitution the sterile and/or non-pyretic water or other liquid suitable carrier is added to lyophilized powder. The precise amount depends upon the selected therapy being given, and can be empirically determined. 
     Method of Treatment 
     Therapeutic methods are also provided, comprising: administering a therapeutically effective amount of the polypeptide complex or the bispecific polypeptide complex provided herein to a subject in need thereof, thereby treating or preventing a condition or a disorder. In certain embodiments, the subject has been identified as having a disorder or condition likely to respond to the polypeptide complex or the bispecific polypeptide complex provided herein. 
     “Treating” or “treatment” of a condition as used herein includes preventing or alleviating a condition, slowing the onset or rate of development of a condition, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with a condition, reducing or ending symptoms associated with a condition, generating a complete or partial regression of a condition, curing a condition, or some combination thereof. 
     The therapeutically effective amount of the polypeptide complex and the bispecific polypeptide complex provided herein will depend on various factors known in the art, such as for example body weight, age, past medical history, present medications, state of health of the subject and potential for cross-reaction, allergies, sensitivities and adverse side-effects, as well as the administration route and extent of disease development. Dosages may be proportionally reduced or increased by one of ordinary skill in the art (e.g., physician or veterinarian) as indicated by these and other circumstances or requirements. 
     In certain embodiments, the polypeptide complex or the bispecific polypeptide complex provided herein may be administered at a therapeutically effective dosage of about 0.01 mg/kg to about 100 mg/kg (e.g., about 0.01 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 2 mg/kg, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35 mg/kg, about 40 mg/kg, about 45 mg/kg, about 50 mg/kg, about 55 mg/kg, about 60 mg/kg, about 65 mg/kg, about 70 mg/kg, about 75 mg/kg, about 80 mg/kg, about 85 mg/kg, about 90 mg/kg, about 95 mg/kg, or about 100 mg/kg). In certain of these embodiments, the polypeptide complex or the bispecific polypeptide complex provided herein is administered at a dosage of about 50 mg/kg or less, and in certain of these embodiments the dosage is 10 mg/kg or less, 5 mg/kg or less, 1 mg/kg or less, 0.5 mg/kg or less, or 0.1 mg/kg or less. In certain embodiments, the administration dosage may change over the course of treatment. For example, in certain embodiments the initial administration dosage may be higher than subsequent administration dosages. In certain embodiments, the administration dosage may vary over the course of treatment depending on the reaction of the subject. 
     Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single dose may be administered, or several divided doses may be administered over time. 
     The polypeptide complex or the bispecific polypeptide complex provided herein may be administered by any route known in the art, such as for example parenteral (e.g., subcutaneous, intraperitoneal, intravenous, including intravenous infusion, intramuscular, or intradermal injection) or non-parenteral (e.g., oral, intranasal, intraocular, sublingual, rectal, or topical) routes. 
     In certain embodiments, the condition or disorder treated by the polypeptide complex or the bispecific polypeptide complex provided herein is cancer or a cancerous condition, autoimmune diseases, infectious and parasitic diseases, cardiovascular diseases, neuropathies, neuropsychiatric conditions, injuries, inflammations, or coagulation disorder. 
     “Cancer” or “cancerous condition” as used herein refers to any medical condition mediated by neoplastic or malignant cell growth, proliferation, or metastasis, and includes both solid cancers and non-solid cancers such as leukemia. “Tumor” as used herein refers to a solid mass of neoplastic and/or malignant cells. 
     With regard to cancer, “treating” or “treatment” may refer to inhibiting or slowing neoplastic or malignant cell growth, proliferation, or metastasis, preventing or delaying the development of neoplastic or malignant cell growth, proliferation, or metastasis, or some combination thereof. With regard to a tumor, “treating” or “treatment” includes eradicating all or part of a tumor, inhibiting or slowing tumor growth and metastasis, preventing or delaying the development of a tumor, or some combination thereof. 
     For example, with regard to the use of the polypeptide complex or bispecific polypeptide complex disclosed herein to treat cancer, a therapeutically effective amount is the dosage or concentration of the polypeptide complex capable of eradicating all or part of a tumor, inhibiting or slowing tumor growth, inhibiting growth or proliferation of cells mediating a cancerous condition, inhibiting tumor cell metastasis, ameliorating any symptom or marker associated with a tumor or cancerous condition, preventing or delaying the development of a tumor or cancerous condition, or some combination thereof. 
     In certain embodiments, the conditions and disorders include tumors and cancers, for example, non-small cell lung cancer, small cell lung cancer, renal cell cancer, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric carcinoma, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic carcinoma, leukemia, lymphomas, myelomas, mycoses fungoids, merkel cell cancer, and other hematologic malignancies, such as classical Hodgkin lymphoma (CHL), primary mediastinal large B-cell lymphoma, T-cell/histiocyte-rich B-cell lymphoma, EBV-positive and -negative PTLD, and EBV-associated diffuse large B-cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK/T-cell lymphoma, nasopharyngeal carcinoma, and HHV8-associated primary effusion lymphoma, Hodgkin&#39;s lymphoma, neoplasm of the central nervous system (CNS), such as primary CNS lymphoma, spinal axis tumor, brain stem glioma. 
     In certain embodiments, the conditions and disorders include a CD19-related disease or condition, such as, B cell lymphoma, optionally Hodgkin lymphoma or non-Hodgkin lymphoma, wherein the non-Hodgkin lymphoma comprises: Diffuse large B-cell lymphoma (DLBCL), Follicular lymphoma, Marginal zone B-cell lymphoma (MZL), Mucosa-Associated Lymphatic Tissue lymphoma (MALT), Small lymphocytic lymphoma (chronic lymphocytic leukemia, CLL), or Mantle cell lymphoma (MCL), Acute Lymphoblastic Leukemia (ALL), or Waldenstrom&#39;s Macroglobulinemia (WM). 
     In certain embodiments, the conditions and disorders include hyperproliferative conditions or infectious diseases that can be treated via regulation of immune responses by CTLA-4 and/or PD-1. Examples of hyperproliferative conditions include, but are not limited to, solid tumors, hematological cancers, soft tissue tumors, and metastatic lesions. 
     The polypeptide complex or the bispecific polypeptide complex may be administered alone or in combination with one or more additional therapeutic means or agents. 
     In certain embodiments, when used for treating cancer or tumor or prolierative disease, the polypeptide complex or the bispecific polypeptide complex provided herein may be administered in combination with chemotherapy, radiation therapy, surgery for the treatment of cancer (e.g., tumorectomy), one or more anti-emetics or other treatments for complications arising from chemotherapy, or any other therapeutic agent for use in the treatment of cancer or any medical disorder that related. “Administered in combination” as used herein includes administration simultaneously as part of the same pharmaceutical composition, simultaneously as separate compositions, or at different timings as separate compositions. A composition administered prior to or after another agent is considered to be administered “in combination” with that agent as the phrase is used herein, even if the composition and the second agent are administered via different routes. Where possible, additional therapeutic agents administered in combination with the polypeptide complex or the bispecific polypeptide complex provided herein are administered according to the schedule listed in the product information sheet of the additional therapeutic agent, or according to the Physicians&#39; Desk Reference (Physicians&#39; Desk Reference, 70th Ed (2016)) or protocols well known in the art. 
     In certain embodiments, the therapeutic agents can induce or boost immune response against cancer. For example, a tumor vaccine can be used to induce immune response to certain tumor or cancer. Cytokine therapy can also be used to enhance tumor antigen presentation to the immune system. Examples of cytokine therapy include, without limitation, interferons such as interferon-α, -β, and -γ, colony stimulating factors such as macrophage-CSF, granulocyte macrophage CSF, and granulocyte-CSF, interleukins such IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, and IL-12, tumor necrosis factors such as TNF-α and TNF-β. Agents that inactivate immunosuppressive targets can also be used, for example, TGF-beta inhibitors, IL-10 inhibitors, and Fas ligand inhibitors. Another group of agents include those that activate immune responsiveness to tumor or cancer cells, for example, those enhance T cell activation (e.g. agonist of T cell costimulatory molecules such as CTLA-4, ICOS and OX-40), and those enhance dendritic cell function and antigen presentation. 
     Kits 
     The present disclosure further provides kits comprising the polypeptide complex or the bispecific polypeptide complex provided herein. In some embodiments, the kits are useful for detecting the presence or level of, or capturing or enriching one or more target of interest in a biological sample. The biological sample can comprise a cell or a tissue. 
     In some embodiments, the kit comprises the polypeptide complex or the bispecific polypeptide complex provided herein which is conjugated with a detectable label. In certain other embodiments, the kit comprises an unlabeled polypeptide complex or the bispecific polypeptide complex provided herein, and further comprises a secondary labeled antibody which is capable of binding to the unlabeled polypeptide complex or the bispecific polypeptide complex provided herein. The kit may further comprise an instruction of use, and a package that separates each of the components in the kit. 
     In certain embodiments, the polypeptide complex or the bispecific polypeptide complex provided herein are associated with a substrate or a device. Useful substrate or device can be, for example, magnetic beads, microtiter plate, or test strip. Such can be useful for a binding assay (such as ELISA), an immunographic assay, capturing or enriching of a target molecule in a biological sample. 
     The following examples are provided to better illustrate the claimed invention and are not to be interpreted as limiting the scope of the invention. All specific compositions, materials, and methods described below, in whole or in part, fall within the scope of the present invention. These specific compositions, materials, and methods are not intended to limit the invention, but merely to illustrate specific embodiments falling within the scope of the invention. One skilled in the art may develop equivalent compositions, materials, and methods without the exercise of inventive capacity and without departing from the scope of the invention. It will be understood that many variations can be made in the procedures herein described while still remaining within the bounds of the present invention. It is the intention of the inventors that such variations are included within the scope of the invention. 
     
       
         
           
               
             
               
                 TABLE 11 
               
             
            
               
                   
               
               
                 Designs and names of chimeric constant regions (CBeta/CAlpha) 
               
            
           
           
               
               
               
            
               
                   
                   
                 SEQ ID NOs: 
               
               
                   
                 Template designs based 
                 (Heavy Chain (HC)/Light 
               
               
                   
                 on Table 16 
                 Chain (LC))-IgG1 
               
               
                   
                   
               
               
                   
                 Design_1 
                 177/176 
               
               
                   
                 Design_2 
                 179/178 
               
               
                   
                 Design_3 
                 184/183 
               
               
                   
                 Design_4 
                 185/183 
               
               
                   
                 Design_5 
                 180/176 
               
               
                   
                 Design_6 
                 181/178 
               
               
                   
                 Design_6a 
                 182/178 
               
               
                   
                 Design_7 
                 184/186 
               
               
                   
                 Design_8 
                 185/186 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 12 
               
             
            
               
                   
               
               
                 Domains and SEQ ID NOS of chimeric constant regions (CBeta/CAlpha) 
               
            
           
           
               
               
            
               
                 Chimeric 
                 Domains from N-terminal to C-terminal and their SEQ ID NOs 
               
            
           
           
               
               
               
               
               
            
               
                 constant region 
                 First or Second 
                   
                 Third Conjunction 
                   
               
               
                 name and chain 
                 Conjunction 
                 TCR Constant 
                 domain + 
                 Dimerization 
               
               
                 SEQ ID NOs: 
                 domain (CJ) 
                 Domain (C1 or C2) 
                 Hinge (CJ′) 
                 Domain (D) 
               
               
                   
               
               
                 Design_1 HC 
                 HCJ1 
                 Cbeta(S56C) 
                 CJ′1G1 
                 FcG1 
               
               
                 SEQ ID NO: 177 
                 SEQ ID NO: 49 
                 SEQ ID NO: 33 
                 SEQ ID NO: 53 
                 SEQ ID NO: 302 
               
               
                 Design_1 LC 
                 LCJ1 
                 CAlpha(T49C) 
               
               
                 SEQ ID NO: 176 
                 SEQ ID NO: 51 
                 SEQ ID NO: 43 
               
               
                 Design_2 HC 
                 HCJ2 
                 Cbeta(S56C) 
                 CJ′1G1 
                 FcG1 
               
               
                 SEQ ID NO: 179 
                 SEQ ID NO: 50 
                 SEQ ID NO: 33 
                 SEQ ID NO: 53 
                 SEQ ID NO: 302 
               
               
                 Design_2 LC 
                 LCJ2 
                 CAlpha(T49C) 
               
               
                 SEQ ID NO: 178 
                 SEQ ID NO: 52 
                 SEQ ID NO: 43 
               
               
                 Design_3 HC 
                 HCJ3 
                 CAlpha(T49C) 
                 CJ′2G1 
                 FcG1 
               
               
                 SEQ ID NO: 184 
                 SEQ ID NO: 129 
                 SEQ ID NO: 43 
                 SEQ ID NO: 134 
                 SEQ ID NO: 302 
               
               
                 Design_3 LC 
                 LCJ3 
                 Cbeta(S56C) 
               
               
                 SEQ ID NO: 183 
                 SEQ ID NO: 308 
                 SEQ ID NO: 33 + 
               
               
                   
                   
                 NO: 306 
               
               
                 Design_4 HC 
                 HCJ4 
                 CAlpha(T49C) 
                 CJ′2G1 
                 FcG1 
               
               
                 SEQ ID NO: 185 
                 SEQ ID NO: 130 
                 SEQ ID NO: 43 
                 SEQ ID NO: 134 
                 SEQ ID NO: 302 
               
               
                 Design_4 LC 
                 LCJ3 
                 Cbeta(S56C) 
               
               
                 SEQ ID NO: 183 
                 SEQ ID NO: 308 
                 SEQ ID NO: 33 + 
               
               
                   
                   
                 NO: 306 
               
               
                 Design_5 HC 
                 HCJ1 
                 Cbeta(S56C) (FG-) 
                 CJ′1G1 
                 FcG1 
               
               
                 SEQ ID NO: 180 
                 SEQ ID NO: 49 
                 SEQ ID NO: 37 
                 SEQ ID NO: 53 
                 SEQ ID NO: 302 
               
               
                 Design_5 LC 
                 LCJ1 
                 CAlpha(T49C) 
               
               
                 SEQ ID NO: 176 
                 SEQ ID NO: 51 
                 SEQ ID NO: 43 
               
               
                 Design_6 HC 
                 HCJ1 
                 CBeta(S56C)(FG-) 
                 CJ′1G1 
                 FcG1 
               
               
                 SEQ ID NO: 181 
                 SEQ ID NO: 50 
                 SEQ ID NO: 37 
                 SEQ ID NO: 53 
                 SEQ ID NO: 302 
               
               
                 Design_6 LC 
                 LCJ2 
                 CAlpha(T49C) 
               
               
                 SEQ ID NO: 178 
                 SEQ ID NO: 52 
                 SEQ ID NO: 43 
               
               
                 Design_6a HC 
                 HCJ2 
                 CBeta(S56C)(DE- 
                 CJ′1G1 
                 FcG1 
               
               
                   
                   
                 FG-) 
               
               
                 SEQ ID NO: 182 
                 SEQ ID NO: 50 
                 SEQ ID NO: 41 
                 SEQ ID NO: 53 
                 SEQ ID NO: 302 
               
               
                 Design_6a LC 
                 LCJ2 
                 CAlpha(T49C) 
               
               
                 SEQ ID NO: 178 
                 SEQ ID NO: 52 
                 SEQ ID NO: 43 
               
               
                 Design_7 HC 
                 HCJ3 
                 CAlpha(T49C) 
                 CJ′2G1 
                 FcG1 
               
               
                 SEQ ID NO: 184 
                 SEQ ID NO: 129 
                 SEQ ID NO: 43 
                 SEQ ID NO: 134 
                 SEQ ID NO: 302 
               
               
                 Design_7 LC 
                 LCJ3 
                 CBeta(S56C)(FG-) 
               
               
                 SEQ ID NO: 186 
                 SEQ ID NO: 308 
                 SEQ ID NO: 37 + 
               
               
                   
                   
                 NO: 306 
               
               
                 Design_8 HC 
                 HCJ4 
                 CAlpha(T49C) 
                 CJ′2G1 
                 FcG1 
               
               
                 SEQ ID NO: 185 
                 SEQ ID NO: 130 
                 SEQ ID NO: 43 
                 SEQ ID NO: 134 
                 SEQ ID NO: 302 
               
               
                 Design_8 LC 
                 LCJ3 
                 CBeta(S56C)(FG-) 
               
               
                 SEQ ID NO: 186 
                 SEQ ID NO: 308 
                 SEQ ID NO: 37 + 
               
               
                   
                   
                 NO: 306 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 13 
               
             
            
               
                   
               
               
                 Designs and names of Design_2 (CBeta/CAlpha) 
               
               
                 without glycosylation sites 
               
            
           
           
               
               
               
            
               
                   
                   
                 SEQ ID NO: (HC-CBeta/LC-CAlpha) 
               
               
                   
                 Sample 
                 HC/LC 
               
               
                   
                   
               
               
                   
                 Design_2-QQQQ 
                 188/187 (IgG1) 
               
               
                   
                   
                 196/187 (IgG4) 
               
               
                   
                 Design_2-AAAA 
                 190/189 (IgG1) 
               
               
                   
                 Design_2-QSKE 
                 192/191 (IgG1) 
               
               
                   
                 Design_2-ASKE 
                 192/193 (IgG1) 
               
               
                   
                 Design_2-QQQQQ 
                 195/194 (IgG1) 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 14 
               
             
            
               
                   
               
               
                 Domains and SEQ ID NOs of Design_2 (CBeta/CAlpha) without glycosylation sites 
               
            
           
           
               
               
            
               
                   
                 Domains from N-terminal to C-terminal and their SEQ ID NOs 
               
            
           
           
               
               
               
               
               
            
               
                   
                 First or Second 
                   
                 Third Conjunction 
                   
               
               
                 Complex name and 
                 Conjunction 
                 TCR Constant 
                 domain + 
                 Dimerization 
               
               
                 chain SEQ ID NOs: 
                 domain (CJ) 
                 Domain (C1 or C2) 
                 Hinge (CJ′) 
                 Domain (D) 
               
               
                   
               
               
                 Design_2-QQQQ 
                 HCJ2 
                 CBeta(S56C) 
                 CJ′1G1 
                 FcG1 
               
               
                 (IgG1) HC 
                   
                 (N69Q) 
               
               
                 SEQ ID NO: 188 
                 SEQ ID NO: 50 
                 SEQ ID NO: 34 
                 SEQ ID NO: 53 
                 SEQ ID NO: 302 
               
               
                 Design_2-QQQQ 
                 LCJ2 
                 CAlpha(T49C) 
               
               
                 (IgG1) LC 
                   
                 (N34Q + N68Q + 
               
               
                   
                   
                 N79Q) 
               
               
                 SEQ ID NO: 187 
                 SEQ ID NO: 52 
                 SEQ ID NO: 44 
               
               
                 Design_2-QQQQ 
                 HCJ2 
                 CBeta(S56C) 
                 CJ′1G4 
                 FcG4 
               
               
                 (IgG4) HC 
                   
                 (N69Q) 
               
               
                 SEQ ID NO: 196 
                 SEQ ID NO: 50 
                 SEQ ID NO: 34 
                 SEQ ID NO: 54 
                 SEQ ID NO: 303 
               
               
                 Design_2-QQQQ 
                 LCJ2 
                 CAlpha(T49C) 
               
               
                 (IgG4) LC 
                   
                 (N34Q + N68Q + 
               
               
                   
                   
                 N79Q) 
               
               
                 SEQ ID NO: 187 
                 SEQ ID NO: 52 
                 SEQ ID NO: 44 
               
               
                 Design_2-AAAA 
                 HCJ2 
                 CBeta(S56C) 
                 CJ′1G1 
                 FcG1 
               
               
                 (IgG1) HC 
                   
                 (N69A) 
               
               
                 SEQ ID NO: 190 
                 SEQ ID NO: 50 
                 SEQ ID NO: 35 
                 SEQ ID NO: 53 
                 SEQ ID NO: 302 
               
               
                 Design_2-AAAA 
                 LCJ2 
                 CAlpha(T49C) 
               
               
                 (IgG1) LC 
                   
                 (N34A + N68A + 
               
               
                   
                   
                 N79A) 
               
               
                 SEQ ID NO: 189 
                 SEQ ID NO: 52 
                 SEQ ID NO: 45 
               
               
                 Design_2-QSKE 
                 HCJ2 
                 CBeta(S56C) 
                 CJ′1G1 
                 FcG1 
               
               
                 (IgG1) HC 
                   
                 (N69E) 
               
               
                 SEQ ID NO: 192 
                 SEQ ID NO: 50 
                 SEQ ID NO: 36 
                 SEQ ID NO: 53 
                 SEQ ID NO: 302 
               
               
                 Design_2-QSKE 
                 LCJ2 
                 CAlpha(T49C) 
               
               
                 (IgG1) LC 
                   
                 (N34Q + N68S + 
               
               
                   
                   
                 N79K) 
               
               
                 SEQ ID NO: 191 
                 SEQ ID NO: 52 
                 SEQ ID NO: 46 
               
               
                 Design_2-ASKE 
                 HCJ2 
                 CBeta(S56C) 
                 CJ′1G1 
                 FcG1 
               
               
                 (IgG1) HC 
                   
                 (N69E) 
               
               
                 SEQ ID NO: 192 
                 SEQ ID NO: 50 
                 SEQ ID NO: 36 
                 SEQ ID NO: 53 
                 SEQ ID NO: 302 
               
               
                 Design_2-ASKE 
                 LCJ2 
                 CAlpha(T49C) 
               
               
                 (IgG1) LC 
                   
                 (N34A + N68S + 
               
               
                   
                   
                 N79K) 
               
               
                 SEQ ID NO: 191 
                 SEQ ID NO: 52 
                 SEQ ID NO: 47 
               
               
                 Design_2-QQQQQ 
                 HCJ2 
                 CBeta(S56C) 
                 CJ′1G1 
                 FcG1 
               
               
                 (IgG1) HC 
                   
                 (N69Q) 
               
               
                 SEQ ID NO: 195 
                 SEQ ID NO: 50 
                 SEQ ID NO: 34 
                 SEQ ID NO: 53 
                 SEQ ID NO: 302 
               
               
                 Design_2-QQQQQ 
                 LCJ2 
                 CAlpha(T49C) 
               
               
                 (IgG1) LC 
                   
                 (N34Q + N68Q + 
               
               
                   
                   
                 N79Q + N61Q) 
               
               
                 SEQ ID NO: 194 
                 SEQ ID NO: 52 
                 SEQ ID NO: 48 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 15 
               
             
            
               
                   
               
               
                 Designs and names of chimeric constant regions (CBeta/Cpre-Alpha) 
               
            
           
           
               
               
               
            
               
                 Templates based 
                   
                   
               
               
                 on Table 16 (IgG1) 
                 Sequence file 
                 SEQ ID NOs: 
               
               
                   
               
               
                 PreTCR_Design_B 
                 Design_1_Pre_TCR_Conjunction′1 
                 198/197 
               
               
                   
                 Design_2_Pre_TCR_Conjunction′_1_Cys10 
                 200/199 
               
               
                   
                 Design_3_Pre_TCR_Conjunction′_1_Cys11 
                 202/201 
               
               
                   
                 Design_4_Pre_TCR_Conjunction′_1_Cys12 
                 203/201 
               
               
                   
                 Design_5_Pre_TCR_Conjunction′_1_Cys13 
                 203/204 
               
               
                   
                 Design_6_Pre_TCR_Conjunction′_1_Cys14 
                 205/204 
               
               
                   
                 Design_7_Pre_TCR_Conjunction′_1_Cys15 
                 206/204 
               
               
                   
                 Design_8_Pre_TCR_Conjunction′_1_Cys1_4L4T_1 
                 208/207 
               
               
                   
                 Design_9_Pre_TCR_Conjunction′_1_Cys2_4L4T_2 
                 208/209 
               
               
                   
                 Design_10_Pre_TCR_Conjunction′_1_Cys4 
                 211/210 
               
               
                 PreTCR_Design_C 
                 PreTCR_Design_5_crossed_1 
                 213/212 
               
               
                   
                 PreTCR_Design_6_crossed_1 
                 213/215 
               
               
                 PreTCR_Design_D 
                 PreTCR_Design_5_crossed_2 
                 214/212 
               
               
                   
                 PreTCR_Design_6_crossed_2 
                 214/215 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 16 
               
             
            
               
                   
               
               
                 Domains and SEQ ID NOs of chimeric constant regions (CBeta/Cpre-Alpha) 
               
            
           
           
               
               
            
               
                   
                 Domains from N-terminal to C-terminal and their SEQ ID NOs 
               
            
           
           
               
               
               
               
               
            
               
                   
                 First or Second 
                   
                 Third Conjunction 
                   
               
               
                 Complex name and 
                 Conjunction 
                 TCR Constant 
                 domain + 
                 Dimerization 
               
               
                 chain SEQ ID NOs: 
                 domain (CJ) 
                 Domain (C1 or C2) 
                 Hinge (CJ′) 
                 Domain (D) 
               
               
                   
               
               
                 Design_1_Pre_TCR_Conjunction′1 HC 
                 HCJB 
                 CBeta(N69Q) 
                 CJ′1G1 
                 FcG1 
               
               
                 SEQ ID NO: 198 
                 SEQ ID NO: 50 
                 SEQ ID NO: 84 
                 SEQ ID NO: 53 
                 SEQ ID NO: 302 
               
               
                 Design_1_Pre_TCR_Conjunction′1 LC 
                 LCJB 
                 CPreAlpha(N50Q) 
               
               
                 SEQ ID NO: 197 
                 SEQ ID NO: 309 
                 SEQ ID NO: 83 
               
               
                 Design_2_Pre_TCR_Conjunction′_1_Cys10 HC 
                 HCJB 
                 Cbeta(S76C)(N69Q) 
                 CJ′1G1 
                 FcG1 
               
               
                 SEQ ID NO: 200 
                 SEQ ID NO: 50 
                 SEQ ID NO: 319 
                 SEQ ID NO: 53 
                 SEQ ID NO: 302 
               
               
                 Design_2_Pre_TCR_Conjunction′_1_Cys10 LC 
                 LCJB 
                 CPreAlpha 
               
               
                   
                   
                 (Y59C)(N50Q) 
               
               
                 SEQ ID NO: 199 
                 SEQ ID NO: 309 
                 SEQ ID NO: 311 
               
               
                 Design_3_Pre_TCR_Conjunction′_1_Cys11 HC 
                 HCJB 
                 Cbeta(F13C)(N69Q) 
                 CJ′1G1 
                 FcG1 
               
               
                 SEQ ID NO: 202 
                 SEQ ID NO: 50 
                 SEQ ID NO: 320 
                 SEQ ID NO: 53 
                 SEQ ID NO: 302 
               
               
                 Design_3_Pre_TCR_Conjunction′_1_Cys11 LC 
                 LCJB 
                 CPreAlpha 
               
               
                   
                   
                 (A13C)(N50Q) 
               
               
                 SEQ ID NO: 201 
                 SEQ ID NO: 309 
                 SEQ ID NO: 312 
               
               
                 Design_4_Pre_TCR_Conjunction′_1_Cys12 HC 
                 HCJB 
                 Cbeta(S16C)(N69Q) 
                 CJ′1G1 
                 FcG1 
               
               
                 SEQ ID NO: 203 
                 SEQ ID NO: 50 
                 SEQ ID NO: 321 
                 SEQ ID NO: 53 
                 SEQ ID NO: 302 
               
               
                 Design_4_Pre_TCR_Conjunction′_1_Cys12 LC 
                 LCJB 
                 CPreAlpha 
               
               
                   
                   
                 (A13C)(N50Q) 
               
               
                 SEQ ID NO: 201 
                 SEQ ID NO: 309 
                 SEQ ID NO: 312 
               
               
                 Design_5_Pre_TCR_Conjunction′_1_Cys13 HC 
                 HCJB 
                 Cbeta(S16C)(N69Q) 
                 CJ′1G1 
                 FcG1 
               
               
                 SEQ ID NO: 203 
                 SEQ ID NO: 50 
                 SEQ ID NO: 321 
                 SEQ ID NO: 53 
                 SEQ ID NO: 302 
               
               
                 Design_5_Pre_TCR_Conjunction′_1_Cys13 LC 
                 LCJB 
                 CPreAlpha 
               
               
                   
                   
                 (S11C)(N50Q) 
               
               
                 SEQ ID NO: 204 
                 SEQ ID NO: 309 
                 SEQ ID NO: 313 
               
               
                 Design_6_Pre_TCR_Conjunction′_1_Cys14 HC 
                 HCJB 
                 Cbeta(A18C)(N69Q) 
                 CJ′1G1 
                 FcG1 
               
               
                 SEQ ID NO: 205 
                 SEQ ID NO: 50 
                 SEQ ID NO: 322 
                 SEQ ID NO: 53 
                 SEQ ID NO: 302 
               
               
                 Design_6_Pre_TCR_Conjunction′_1_Cys14 LC 
                 LCJB 
                 CPreAlpha 
               
               
                   
                   
                 (S11C)(N50Q) 
               
               
                 SEQ ID NO: 204 
                 SEQ ID NO: 309 
                 SEQ ID NO: 313 
               
               
                 Design_7_Pre_TCR_Conjunction′_1_Cys15 HC 
                 HCJB 
                 Cbeta(E19C)(N69Q) 
                 CJ′1G1 
                 FcG1 
               
               
                 SEQ ID NO: 206 
                 SEQ ID NO: 50 
                 SEQ ID NO: 323 
                 SEQ ID NO: 53 
                 SEQ ID NO: 302 
               
               
                 Design_7_Pre_TCR_Conjunction′_1_Cys15 LC 
                 LCJB 
                 CPreAlpha 
               
               
                   
                   
                 (S11C)(N50Q) 
               
               
                 SEQ ID NO: 204 
                 SEQ ID NO: 309 
                 SEQ ID NO: 313 
               
               
                 Design_8_Pre_TCR_Conjunction′_1_Cys1_4L4T_1 HC 
                 HCJB 
                 Cbeta(S56C)(N69Q) 
                 CJ′1G1 
                 FcG1 
               
               
                 SEQ ID NO: 208 
                 SEQ ID NO: 50 
                 SEQ ID NO: 34 
                 SEQ ID NO: 53 
                 SEQ ID NO: 302 
               
               
                 Design_8_Pre_TCR_Conjunction′_1_Cys1_4L4T_1 LC 
                 LCJB 
                 CPreAlpha 
               
               
                   
                   
                 (S62C)(N50Q) 
               
               
                 SEQ ID NO: 207 
                 SEQ ID NO: 309 
                 SEQ ID NO: 314 
               
               
                 Design_9_Pre_TCR_Conjunction′_1_Cys2_4L4T_2 HC 
                 HCJB 
                 Cbeta(S56C)(N69Q) 
                 CJ′1G1 
                 FcG1 
               
               
                 SEQ ID NO: 207 
                 SEQ ID NO: 50 
                 SEQ ID NO: 34 
                 SEQ ID NO: 53 
                 SEQ ID NO: 302 
               
               
                 Design_9_Pre_TCR_Conjunction′_1_Cys2_4L4T_2 LC 
                 LCJB 
                 CPreAlpha 
               
               
                   
                   
                 (T65C)(N50Q) 
               
               
                 SEQ ID NO: 209 
                 SEQ ID NO: 309 
                 SEQ ID NO: 315 
               
               
                 Design_10_Pre_TCR_Conjunction′_1_Cys4 HC 
                 HCJB 
                 Cbeta(A11C)(N69Q) 
                 CJ′1G1 
                 FcG1 
               
               
                 SEQ ID NO: 211 
                 SEQ ID NO: 50 
                 SEQ ID NO: 324 
                 SEQ ID NO: 53 
                 SEQ ID NO: 302 
               
               
                 Design_10_Pre_TCR_Conjunction′_1_Cys4 LC 
                 LCJB 
                 CPreAlpha 
               
               
                   
                   
                 (I16C)(N50Q) 
               
               
                 SEQ ID NO: 210 
                 SEQ ID NO: 309 
                 SEQ ID NO: 316 
               
               
                 PreTCR_Design_5_crossed_1 HC 
               
               
                 SEQ ID NO: 213 
                 HCJC 
                 CPreAlpha 
                 CJ′2G1 
                 FcG1 
               
               
                   
                   
                 (S11C)(N50Q) 
               
               
                 PreTCR_Design_5_crossed_1 LC 
                 SEQ ID NO: 132 
                 SEQ ID NO: 313 
                 SEQ ID NO: 134 
                 SEQ ID NO: 302 
               
               
                 SEQ ID NO: 212 
                 LCJC 
                 Cbeta (N69Q, 
               
               
                   
                   
                 S16C) 
               
               
                 PreTCR_Design_6_crossed_1 HC 
                 SEQ ID NO: 50 
                 SEQ ID NO: 
               
               
                   
                   
                 321 + 306 
               
               
                 SEQ ID NO: 213 
               
               
                 PreTCR_Design_6_crossed_1 LC 
                 HCJC 
                 CPreAlpha 
                 CJ′2G4 
                 FcG4 
               
               
                   
                   
                 (S11C)(N50Q) 
               
               
                 SEQ ID NO: 215 
                 SEQ ID NO: 132 
                 SEQ ID NO: 313 
                 SEQ ID NO: 134 
                 SEQ ID NO: 303 
               
               
                 PreTCR_Design_5_crossed_2 HC 
                 LCJC 
                 Cbeta(N69Q, 
               
               
                   
                   
                 A18C) 
               
               
                 SEQ ID NO: 214 
                 SEQ ID NO: 50 
                 SEQ ID NO: 
               
               
                   
                   
                 322 + 306 
               
               
                 PreTCR_Design_5_crossed_2 LC 
               
               
                 SEQ ID NO: 212 
                 HCJD 
                 CPreAlpha 
                 CJ′2G1 
                 FcG1 
               
               
                   
                   
                 (S11C)(N50Q) 
               
               
                 PreTCR_Design_6_crossed_2 HC 
                 SEQ ID NO: 133 
                 SEQ ID NO: 313 
                 SEQ ID NO: 134 
                 SEQ ID NO: 302 
               
               
                 SEQ ID NO: 214 
                 LCJC 
                 Cbeta (N69Q, 
               
               
                   
                   
                 S16C) 
               
               
                 PreTCR_Design_6_crossed_2 LC 
                 SEQ ID NO: 50 
                 SEQ ID NO: 
               
               
                   
                   
                 NO: 321 + 306 
               
               
                 SEQ ID NO: 215 
                 HCJD 
                 CPreAlpha 
                 CJ′2G1 
                 FcG1 
               
               
                   
                   
                 (S11C)(N50Q) 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 17 
               
             
            
               
                   
               
               
                 Designs and names of chimeric constant regions (CGamma/CDelta) 
               
            
           
           
               
               
               
            
               
                 Templates based on 
                   
                   
               
               
                 Table 13 (IgG1) 
                 Construct of Design 
                 SEQ ID NOs in HC/LC 
               
               
                   
               
               
                 dg_Design_1 
                 dg_Design_1 
                 234/233 
               
               
                 dg_Design_2 
                 dg_Design_2 
                 232/231 
               
               
                   
                 dg_Design_2_no_Glyco 
                 216/215 
               
               
                   
                 dg_Design_2_hypeCys1_no_Glyco 
                 218/217 
               
               
                   
                 dg_Design_2_hypeCys2_no_Glyco 
                 220/219 
               
               
                   
                 dg_Design_2_hypeCys3_no_Glyco 
                 222/221 
               
               
                   
                 dg_Design_2_Cys2_no_Glyco 
                 224/223 
               
               
                   
                 dg_Design_2_Cys1_no_Glyco 
                 226/225 
               
               
                   
                 dg_Design_2_Cys3_no_Glyco 
                 227/223 
               
               
                   
                 dg_Design_2_Cys4_no_Glyco 
                 229/228 
               
               
                   
                 dg_Design_2_Cys5_no_Glyco 
                 229/230 
               
               
                 dg_Design_3 
                 dg_crossed_Design_1 
                 236/235 
               
               
                 dg_Design_4 
                 dg_crossed_Deisng_2 
                 238/237 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 18 
               
             
            
               
                   
               
               
                 Domains and SEQ ID NOs of chimeric constant regions (CGamma/CDelta) 
               
            
           
           
               
               
            
               
                   
                 Domains from N-terminal to C-terminal and their SEQ ID NOs 
               
            
           
           
               
               
               
               
               
            
               
                   
                 First or Second 
                   
                 Third Conjunction 
                   
               
               
                 Complex name and 
                 Conjunction 
                 TCR Constant 
                 domain + 
                 Dimerization 
               
               
                 chain SEQ ID NOs: 
                 domain (CJ) 
                 Domain (C1 or C2) 
                 Hinge (CJ′) 
                 Domain (D) 
               
               
                   
               
               
                 dg_Design_1 HC 
                 HCJ4 
                 CGamma 
                 CJ′3G1 
                 FcG1 
               
               
                 SEQ ID NO: 234 
                 SEQ ID NO: 117 
                 SEQ ID NO: 113 
                 SEQ ID NO: 121 
                 SEQ ID NO: 302 
               
               
                 dg_Design_1 LC 
                 LCJ4 
                 CDelta 
               
               
                 SEQ ID NO: 233 
                 SEQ ID NO: 119 
                 SEQ ID NO: 310 
               
               
                 dg_Design_2 HC 
                 HCJ5 
                 CGamma 
                 CJ′3G1 
                 FcG1 
               
               
                 SEQ ID NO: 232 
                 SEQ ID NO: 118 
                 SEQ ID NO: 113 
                 SEQ ID NO: 121 
                 SEQ ID NO: 302 
               
               
                 dg_Design_2 LC 
                 LCJ5 
                 CDelta 
               
               
                 SEQ ID NO: 231 
                 SEQ ID NO: 120 
                 SEQ ID NO: 115 
               
               
                 dg_Design_2_no_Glyco HC 
                 HCJ5 
                 CGamma (N65Q) 
                 CJ′3G1 
                 FcG1 
               
               
                 SEQ ID NO: 216 
                 SEQ ID NO: 118 
                 SEQ ID NO: 114 
                 SEQ ID NO: 121 
                 SEQ ID NO: 302 
               
               
                 dg_Design_2_no_Glyco LC 
                 LCJ5 
                 CDelta(N16Q + N79Q) 
               
               
                 SEQ ID NO: 215 
                 SEQ ID NO: 120 
                 SEQ ID NO: 116 
               
               
                 dg_Design_2_hypeCys1_no_Glyco HC 
                 HCJ5 
                 CGamma(T12C) 
                 CJ′3G1 
                 FcG1 
               
               
                   
                   
                 (N65Q) 
               
               
                 SEQ ID NO: 218 
                 SEQ ID NO: 118 
                 SEQ ID NO: 333 
                 SEQ ID NO: 121 
                 SEQ ID NO: 302 
               
               
                 dg_Design_2_hypeCys1_no_Glyco LC 
                 LCJ5 
                 CDelta (N16C) 
               
               
                   
                   
                 (N79Q) 
               
               
                 SEQ ID NO: 217 
                 SEQ ID NO: 120 
                 SEQ ID NO: 325 
               
               
                 dg_Design_2_hypeCys2_no_Glyco HC 
                 HCJ5 
                 CGamma (Q57C) 
                 CJ′3G1 
                 FcG1 
               
               
                   
                   
                 (N65Q) 
               
               
                 SEQ ID NO: 220 
                 SEQ ID NO: 118 
                 SEQ ID NO: 334 
                 SEQ ID NO: 121 
                 SEQ ID NO: 302 
               
               
                 dg_Design_2_hypeCys2_no_Glyco LC 
                 LCJ5 
                 CDelta (V50C) 
               
               
                   
                   
                 (N16Q + N79Q) 
               
               
                 SEQ ID NO: 219 
                 SEQ ID NO: 120 
                 SEQ ID NO: 326 
               
               
                 dg_Design_2_hypeCys3_no_Glyco HC 
                 HCJ5 
                 CGamma (M62C) 
                 CJ′3G1 
                 FcG1 
               
               
                   
                   
                 (N65Q) 
               
               
                 SEQ ID NO: 222 
                 SEQ ID NO: 118 
                 SEQ ID NO: 335 
                 SEQ ID NO: 121 
                 SEQ ID NO: 302 
               
               
                 dg_Design_2_hypeCys3_no_Glyco LC 
                 LCJ5 
                 CDelta (D46C) 
               
               
                   
                   
                 (N16Q + N79Q) 
               
               
                 SEQ ID NO: 221 
                 SEQ ID NO: 120 
                 SEQ ID NO: 327 
               
               
                 dg_Design_2_Cys2_no_Glyco HC 
                 HCJ5 
                 CGamma(S17C) 
                 CJ′3G1 
                 FcG1 
               
               
                   
                   
                 (N65Q) 
               
               
                 SEQ ID NO: 224 
                 SEQ ID NO: 118 
                 SEQ ID NO: 336 
                 SEQ ID NO: 121 
                 SEQ ID NO: 302 
               
               
                 dg_Design_2_Cys2_no_Glyco LC 
                 LCJ5 
                 CDelta (F12C) 
               
               
                   
                   
                 (N16Q + N79Q) 
               
               
                 SEQ ID NO: 223 
                 SEQ ID NO: 120 
                 SEQ ID NO: 328 
               
               
                 dg_Design_2_Cys1_no_Glyco HC 
                 HCJ5 
                 CGamma(F14C) 
                 CJ′3G1 
                 FcG1 
               
               
                   
                   
                 (N65Q) 
               
               
                 SEQ ID NO: 226 
                 SEQ ID NO: 118 
                 SEQ ID NO: 337 
                 SEQ ID NO: 121 
                 SEQ ID NO: 302 
               
               
                 dg_Design_2_Cys1_no_Glyco LC 
                 LCJ5 
                 CDelta(M14C) 
               
               
                   
                   
                 (N16Q + N79Q) 
               
               
                 SEQ ID NO: 225 
                 SEQ ID NO: 120 
                 SEQ ID NO: 329 
               
               
                 dg_Design_2_Cys3_no_Glyco HC 
                 HCJ5 
                 CGamma (E20C) 
                 CJ′3G1 
                 FcG1 
               
               
                   
                   
                 (N65Q) 
               
               
                 SEQ ID NO: 227 
                 SEQ ID NO: 118 
                 SEQ ID NO: 338 
                 SEQ ID NO: 121 
                 SEQ ID NO: 302 
               
               
                 dg_Design_2_Cys3_no_Glyco LC 
                 LCJ5 
                 CDelta (F12C) 
               
               
                   
                   
                 (N16Q + N79Q) 
               
               
                 SEQ ID NO: 223 
                 SEQ ID NO: 120 
                 SEQ ID NO: 328 
               
               
                 dg_Design_2_Cys4_no_Glyco HC 
                 HCJ5 
                 CGamma (A19C) 
                 CJ′3G1 
                 FcG1 
               
               
                   
                   
                 (N65Q) 
               
               
                 SEQ ID NO: 229 
                 SEQ ID NO: 118 
                 SEQ ID NO: 339 
                 SEQ ID NO: 121 
                 SEQ ID NO: 302 
               
               
                 dg_Design_2_Cys4_no_Glyco LC 
                 LCJ5 
                 CDelta(F87C) (N16Q + 
               
               
                   
                   
                 N79Q) 
               
               
                 SEQ ID NO: 228 
                 SEQ ID NO: 120 
                 SEQ ID NO: 330 
               
               
                 dg_Design_2_Cys5_no_Glyco HC 
                 HCJ5 
                 CGamma (A19C) 
                 CJ′3G1 
                 FcG1 
               
               
                   
                   
                 (N65Q) 
               
               
                 SEQ ID NO: 229 
                 SEQ ID NO: 118 
                 SEQ ID NO: 339 
                 SEQ ID NO: 121 
                 SEQ ID NO: 302 
               
               
                 dg_Design_2_Cys5_no_Glyco LC 
                 LCJ5 
                 CDelta (E88C) 
               
               
                   
                   
                 (N16Q + N79Q) 
               
               
                 SEQ ID NO: 230 
                 SEQ ID NO: 120 
                 SEQ ID NO: 331 
               
               
                 dg_crossed_Design_1 HC 
                 HCJ6 
                 CDelta 
                 CJ′4G1 
                 FcG1 
               
               
                 SEQ ID NO: 236 
                 SEQ ID NO: 123 
                 SEQ ID NO: 332 
                 SEQ ID NO: 127 
                 SEQ ID NO: 302 
               
               
                 dg_crossed_Design_1 LC 
                 LCJ6 
                 CGamma 
               
               
                 SEQ ID NO: 235 
                 SEQ ID NO: 125 
                 SEQ ID NO: 340 
               
               
                 dg_crossed_Design_2 
                 HCJ7 
                 CDelta 
                 CJ′4G1 
                 FcG1 
               
               
                 HC 
               
               
                 SEQ ID NO: 238 
                 SEQ ID NO: 124 
                 SEQ ID NO: 332 
                 SEQ ID NO: 127 
                 SEQ ID NO: 302 
               
               
                 dg_crossed_Design_2 LC 
                 LCJ7 
                 CGamma 
               
               
                 SEQ ID NO: 237 
                 SEQ ID NO: 126 
                 SEQ ID NO: 340 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 19 
               
               
                   
               
               
                 Sequences for exemplary chimeric constant regions 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 Template 
                   
                 SEQ 
                   
                   
               
               
                 Designs 
                 Chain 
                 ID 
                   
                   
               
               
                 CAlpha/CBeta 
                 Type 
                 NO 
                   
                 Sequences 
               
               
                   
               
               
                 Design_1 
                 LC 
                 176 
                 LCJ1- 
                 KRTVAAPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSN 
               
               
                 (IgG1) 
                   
                   
                 CAlpha(T49C) 
                 SAVAWSNKSDFACANAFNNSIIPEDTFFPSPESS 
               
               
                 normal 
                 HC 
                 177 
                 HCJ1- 
                 SSASKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQ 
               
               
                   
                   
                   
                 CBeta(S56C)- 
                 PLKEQPALNDSRYALSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSA 
               
               
                   
                   
                   
                 CJ′ 1G1-Fc(G1) 
                 EAWGRASDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF 
               
               
                   
                   
                   
                   
                 NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS 
               
               
                   
                   
                   
                   
                 KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD 
               
               
                   
                   
                   
                   
                 SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 
               
               
                   
               
               
                 Design_2 
                 LC 
                 178 
                 LCJ2- 
                 KPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSN 
               
               
                 (IgG1) 
                   
                   
                 CAlpha(T49C) 
                 SAVAWSNKSDFACANAFNNSIIPEDTFFPSPESS 
               
               
                 normal 
                 HC 
                 179 
                 HCJ2- 
                 LEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQ 
               
               
                   
                   
                   
                 CBeta(S56C)- 
                 PLKEQPALNDSRYALSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSA 
               
               
                   
                   
                   
                 CJ′ 1G1- Fc(G1) 
                 EAWGRASDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF 
               
               
                   
                   
                   
                   
                 NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS 
               
               
                   
                   
                   
                   
                 KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD 
               
               
                   
                   
                   
                   
                 SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 
               
               
                   
               
               
                 Design_5 
                 LC 
                 176 
                 LCJ1- 
                 KRTVAAPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSN 
               
               
                 (IgG1) 
                   
                   
                 CAlpha(T49C) 
                 SAVAWSNKSDFACANAFNNSIIPEDTFFPSPESS 
               
               
                 normal 
                 HC 
                 180 
                 HCJ1- 
                 SSASKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQ 
               
               
                   
                   
                   
                 CBeta(S56C)(F 
                 PLKEQPALNDSRYALSSRLRVSATFWQNPRNHFRCQVQFYPSNQIVSAEAWGRASDKTHTCP 
               
               
                   
                   
                   
                 G-DE+)- 
                 PCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT 
               
               
                   
                   
                   
                 CJ′ 1G1- Fc(G1) 
                 KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL 
               
               
                   
                   
                   
                   
                 PPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD 
               
               
                   
                   
                   
                   
                 KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 
               
               
                   
               
               
                 Design_6 
                 LC 
                 178 
                 LCJ2- 
                 KPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSN 
               
               
                 (IgG1) 
                   
                   
                 CAlpha(T49C) 
                 SAVAWSNKSDFACANAFNNSIIPEDTFFPSPESS 
               
               
                 normal 
                 HC 
                 181 
                 HCJ2- 
                 LEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQ 
               
               
                   
                   
                   
                 CBeta(S56C)(F 
                 PLKEQPALNDSRYALSSRLRVSATFWQNPRNHFRCQVQFYPSNQIVSAEAWGRASDKTHTCP 
               
               
                   
                   
                   
                 G-DE+)- 
                 PCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT 
               
               
                   
                   
                   
                 CJ′ 1G1-Fc(G1) 
                 KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL 
               
               
                   
                   
                   
                   
                 PPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD 
               
               
                   
                   
                   
                   
                 KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 
               
               
                   
               
               
                 Design_6a 
                 LC 
                 178 
                 LCJ2- 
                 KPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSN 
               
               
                 (IgG1) 
                   
                   
                 CAlpha(T49C) 
                 SAVAWSNKSDFACANAFNNSIIPEDTFFPSPESS 
               
               
                 normal 
                 HC 
                 182 
                 HCJ2- 
                 LEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQ 
               
               
                   
                   
                   
                 CBeta(S56C)(F 
                 PLKEQSGRYALSSRLRVSATFWQNPRNHFRCQVQFYPSNQIVSAEAWGRASDKTHTCPPCPA 
               
               
                   
                   
                   
                 G-DE+)- 
                 PEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE 
               
               
                   
                   
                   
                 CJ′ 1G1-Fc(G1) 
                 EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR 
               
               
                   
                   
                   
                   
                 EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW 
               
               
                   
                   
                   
                   
                 QQGNVFSCSVMHEALHNHYTQKSLSLSPGK 
               
               
                   
               
               
                 Design_3 
                 LC 
                 183 
                 LCJ3- 
                 KLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDP 
               
               
                 (IgG1) 
                   
                   
                 CBeta(S56C) 
                 QPLKEQPALNDSRYALSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVS 
               
               
                 crossed 
                   
                   
                   
                 AEAWGRA 
               
               
                   
                 HC 
                 184 
                 HCJ2- 
                 SSASIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKS 
               
               
                   
                   
                   
                 CBeta(S56C)(F 
                 NSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSSDKTHTCPPCPAPEAAGGPSVFLFPPK 
               
               
                   
                   
                   
                 G-DE-)- 
                 PKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV 
               
               
                   
                   
                   
                 CJ′ 1G1-Fc(G1) 
                 LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVK 
               
               
                   
                   
                   
                   
                 GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL 
               
               
                   
                   
                   
                   
                 HNHYTQKSLSLSPGK 
               
               
                   
               
               
                 Design_4 
                 LC 
                 183 
                 LCJ4- 
                 KLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDP 
               
               
                 (IgG1) 
                   
                   
                 CBeta(S56C) 
                 QPLKEQPALNDSRYALSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVS 
               
               
                 crossed 
                   
                   
                   
                 AEAWGRA 
               
               
                   
                 HC 
                 185 
                 HCJ4- 
                 SSPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKS 
               
               
                   
                   
                   
                 CAlpha(T49C)- 
                 NSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSSDKTHTCPPCPAPEAAGGPSVFLFPPK 
               
               
                   
                   
                   
                 CJ′2G1-Fc(G1) 
                 PKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV 
               
               
                   
                   
                   
                   
                 LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVK 
               
               
                   
                   
                   
                   
                 GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL 
               
               
                   
                   
                   
                   
                 HNHYTQKSLSLSPGK 
               
               
                   
               
               
                 Design _7 
                 LC 
                 186 
                 LCJ3- 
                 KLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDP 
               
               
                 (IgG1) 
                   
                   
                 CBeta(S56C)(F 
                 QPLKEQPALNDSRYALSSRLRVSATFWQNPRNHFRCQVQFYPSNQIVSAEAWGRA 
               
               
                 crossed 
                   
                   
                 G-DE+) 
                   
               
               
                   
                 HC 
                 184 
                 HCJ3- 
                 SSASIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKS 
               
               
                   
                   
                   
                 CAlpha(T49C)- 
                 NSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSSDKTHTCPPCPAPEAAGGPSVFLFPPK 
               
               
                   
                   
                   
                 CJ′2G1- Fc(G1) 
                 PKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV 
               
               
                   
                   
                   
                   
                 LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVK 
               
               
                   
                   
                   
                   
                 GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL 
               
               
                   
                   
                   
                   
                 HNHYTQKSLSLSPGK 
               
               
                   
               
               
                 Design _8 
                 LC 
                 186 
                 LCJ4- 
                 KLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDP 
               
               
                 (IgG1) 
                   
                   
                 CBeta(S56C)(F 
                 QPLKEQPALNDSRYALSSRLRVSATFWQNPRNHFRCQVQFYPSNQIVSAEAWGRA 
               
               
                 crossed 
                   
                   
                 G-DE+) 
                 SSPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKS 
               
               
                   
                 HC 
                 185 
                 HCJ4- 
                 NSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSSDKTHTCPPCPAPEAAGGPSVFLFPPK 
               
               
                   
                   
                   
                 CAlpha(T49C)- 
                 PKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV 
               
               
                   
                   
                   
                 CJ′2G1- Fc(G1) 
                 LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVK 
               
               
                   
                   
                   
                   
                 GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL 
               
               
                   
                   
                   
                   
                 HNHYTQKSLSLSPGK 
               
               
                   
               
               
                 Design 2- 
                 LC 
                 187 
                 LCJ2- 
                 KPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTQVSQSKDSDVYITDKCVLDMRSMDFKSN 
               
               
                 QQQQ 
                   
                   
                 CAlpha(T49C) 
                 SAVAWSQKSDFACANAFQNSIIPEDTFFPSPESS 
               
               
                 (IgG1) 
                   
                   
                 (N34Q+ N68Q+ 
                   
               
               
                   
                   
                   
                 N79Q) 
                 LEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQ 
               
               
                   
                 HC 
                 188 
                 HCJ2- 
                 PLKEQPALQDSRYALSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSA 
               
               
                   
                   
                   
                 CBeta(S56C) 
                 EAWGRASDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF 
               
               
                   
                   
                   
                 (N69Q)- 
                 NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS 
               
               
                   
                   
                   
                 CJ′ 1G1- Fc(G1) 
                 KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD 
               
               
                   
                   
                   
                   
                 SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 
               
               
                   
               
               
                 Design 2- 
                 LC 
                 189 
                 LCJ2- 
                 KPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTAVSQSKDSDVYITDKCVLDMRSMDFKSN 
               
               
                 AAAA 
                   
                   
                 CAlpha(T49C) 
                 SAVAWSAKSDFACANAFANSIIPEDTFFPSPESS 
               
               
                 (IgG1) 
                   
                   
                 (N34A + N68A + 
                   
               
               
                   
                   
                   
                 N79A) 
                   
               
               
                   
                 HC 
                 190 
                 HCJ2- 
                 LEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQ 
               
               
                   
                   
                   
                 CBeta(S56C) 
                 PLKEQPALADSRYALSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSA 
               
               
                   
                   
                   
                 (N69A)- 
                 EAWGRASDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF 
               
               
                   
                   
                   
                 CJ′ 1G1- Fc(G1) 
                 NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS 
               
               
                   
                   
                   
                   
                 KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD 
               
               
                   
                   
                   
                   
                 SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 
               
               
                   
               
               
                 Design 2- 
                 LC 
                 191 
                 LCJ2- 
                 KPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTQVSQSKDSDVYITDKCVLDMRSMDFKSN 
               
               
                 QSKE 
                   
                   
                 CAlpha(T49C) 
                 SAVAWSSKSDFACANAFKNSIIPEDTFFPSPESS 
               
               
                 (IgG1) 
                   
                   
                 (N34Q+ N68S+ 
                   
               
               
                   
                   
                   
                 N79K) 
                   
               
               
                   
                 HC 
                 192 
                 HCJ2- 
                 LEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQ 
               
               
                   
                   
                   
                 CBeta(S56C) 
                 PLKEQPALEDSRYALSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSA 
               
               
                   
                   
                   
                 (N69E)-CJ′ 1G1- 
                 EAWGRASDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF 
               
               
                   
                   
                   
                 Fc(G1) 
                 NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS 
               
               
                   
                   
                   
                   
                 KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD 
               
               
                   
                   
                   
                   
                 SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 
               
               
                   
               
               
                 Design 2- 
                 LC 
                 193 
                 LCJ2- 
                 KPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTAVSQSKDSDVYITDKCVLDMRSMDFKSN 
               
               
                 ASKE 
                   
                   
                 CAlpha(T49C) 
                 SAVAWSSKSDFACANAFKNSIIPEDTFFPSPESS 
               
               
                 (IgG1) 
                   
                   
                 (N34A + N68S+ 
                   
               
               
                   
                   
                   
                 N79K) 
                   
               
               
                   
                 HC 
                 192 
                 HCJ2- 
                 LEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQ 
               
               
                   
                   
                   
                 CBeta(S56C)(N 
                 PLKEQPALEDSRYALSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSA 
               
               
                   
                   
                   
                 69E)-CJ′ 1G1 - 
                 EAWGRASDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF 
               
               
                   
                   
                   
                 Fc(G1) 
                 NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS 
               
               
                   
                   
                   
                   
                 KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD 
               
               
                   
                   
                   
                   
                 SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 
               
               
                   
               
               
                 Design_2- 
                 LC 
                 194 
                 LCJ2- 
                 KPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTQVSQSKDSDVYITDKCVLDMRSMDFKSQ 
               
               
                 QQQQQ 
                   
                   
                 CAlpha(T49C) 
                 SAVAWSQKSDFACANAFQNSIIPEDTFFPSPESS 
               
               
                 (IgG1) 
                   
                   
                 (N34Q+ N68Q+ 
                   
               
               
                   
                   
                   
                 N79Q+ N61Q) 
                   
               
               
                   
                 HC 
                 195 
                 HCJ2- 
                 LEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQ 
               
               
                   
                   
                   
                 CBeta(S56C) 
                 PLKEQPALQDSRYALSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSA 
               
               
                   
                   
                   
                 (N69Q)- 
                 EAWGRASDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF 
               
               
                   
                   
                   
                 CJ′ 1G1- Fc(G1) 
                 NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS 
               
               
                   
                   
                   
                   
                 KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD 
               
               
                   
                   
                   
                   
                 SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 
               
               
                   
               
               
                 Design 2- 
                 LC 
                 187 
                   
                 KPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTQVSQSKDSDVYITDKCVLDMRSMDFKSN 
               
               
                 QQQQ 
                   
                   
                   
                 SAVAWSQKSDFACANAFQNSIIPEDTFFPSPESS 
               
               
                 (IgG4) 
                 HC 
                 196 
                 HCJ2- 
                 LEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQ 
               
               
                   
                   
                   
                 CBeta(S56C) 
                 PLKEQPALQDSRYALSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSA 
               
               
                   
                   
                   
                 (N69Q)- 
                 EAWGRYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWY 
               
               
                   
                   
                   
                 CJ′ 1G4- Fc(G4) 
                 VDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK 
               
               
                   
                   
                   
                   
                 GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG 
               
               
                   
                   
                   
                   
                 SFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 
               
               
                   
               
               
                   
                   
                 SEQ 
                   
                   
               
               
                 PreAlpha- 
                 Chain 
                 ID 
                   
                   
               
               
                 Beta Designs 
                 Name 
                 NO 
                   
                 Sequences 
               
               
                   
               
               
                 Design_1_Pr 
                 LC 
                 197 
                 LCJB- 
                 KPTGVGGTPFPSLAPPIMLLVDGKQQMVVVCLVLDVAPPGLDSPIWFSAGQGSALDAFTYGP 
               
               
                 e_TCR_Con 
                   
                   
                 CPreAlpha(N50 
                 SPATDGTWTNLAHLSLPSEELASWEPLVCHTGPGAEGHSRSTQPMHLSGEASTART 
               
               
                 junction′1 
                   
                   
                 Q) 
                   
               
               
                   
                 HC 
                 198 
                 HCJB- 
                 LEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQ 
               
               
                   
                   
                   
                 CBeta(N69Q)- 
                 PLKEQPALQDSRYALSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSA 
               
               
                   
                   
                   
                 CJ′ 1G1- Fc(G1)  
                 EAWGRASDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF 
               
               
                   
                   
                   
                   
                 NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS 
               
               
                   
                   
                   
                   
                 KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD 
               
               
                   
                   
                   
                   
                 SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 
               
               
                   
               
               
                 Design_2_Pr 
                 LC 
                 199 
                 LCJB- 
                 KPTGVGGTPFPSLAPPIMLLVDGKQQMVVVCLVLDVAPPGLDSPIWFSAGQGSALDAFTCGP 
               
               
                 e_TCR_Con 
                   
                   
                 CPreAlpha 
                 SPATDGTWTNLAHLSLPSEELASWEPLVCHTGPGAEGHSRSTQPMHLSGEASTART 
               
               
                 junction′1_ 
                   
                   
                 (Y59C)(N50Q) 
                   
               
               
                 Cys10 
                 HC 
                 200 
                 HCJB- 
                 LEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQ 
               
               
                   
                   
                   
                 CBeta(S76C) 
                 PLKEQPALQDSRYALCSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSA 
               
               
                   
                   
                   
                 (N69Q)- 
                 EAWGRASDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF 
               
               
                   
                   
                   
                 CJ′ 1G1- Fc(G1) 
                 NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS 
               
               
                   
                   
                   
                   
                 KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD 
               
               
                   
                   
                   
                   
                 SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 
               
               
                   
               
               
                 Design_3_Pr 
                 LC 
                 201 
                 VL(CD3)- 
                 KPTGVGGTPFPSLCPPIMLLVDGKQQMVVVCLVLDVAPPGLDSPIWFSAGQGSALDAFTYGP 
               
               
                 e_TCR_Con 
                   
                   
                 LCJB- 
                 SPATDGTWTNLAHLSLPSEELASWEPLVCHTGPGAEGHSRSTQPMHLSGEASTART 
               
               
                 junction′1_ 
                   
                   
                 CPreAlpha 
                   
               
               
                 Cys11 
                   
                   
                 (A13C)(N50Q) 
                   
               
               
                   
                 HC 
                 202 
                 HCJB- 
                 LEDLKNVFPPEVAVCEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQ 
               
               
                   
                   
                   
                 CBeta(F13C)  
                 PLKEQPALQDSRYALSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSA 
               
               
                   
                   
                   
                 (N69Q)- 
                 EAWGRASDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF 
               
               
                   
                   
                   
                 CJ′ 1G1- Fc(G1) 
                 NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS 
               
               
                   
                   
                   
                   
                 KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD 
               
               
                   
                   
                   
                   
                 SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 
               
               
                   
               
               
                 Design_4_Pr 
                 LC 
                 201 
                 VL(CD3)- 
                 KPTGVGGTPFPSLCPPIMLLVDGKQQMVVVCLVLDVAPPGLDSPIWFSAGQGSALDAFTYGP 
               
               
                 e_TCR_Con 
                   
                   
                 LCJB- 
                 SPATDGTWTNLAHLSLPSEELASWEPLVCHTGPGAEGHSRSTQPMHLSGEASTART 
               
               
                 junction′1_ 
                   
                   
                 CPreAlpha 
                   
               
               
                 Cys12 
                   
                   
                 (A13C)(N50Q) 
                   
               
               
                   
                 HC 
                 203 
                 HCJB- 
                 LEDLKNVFPPEVAVFEPCEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQ 
               
               
                   
                   
                   
                 CBeta(S16C) 
                 PLKEQPALQDSRYALSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSA 
               
               
                   
                   
                   
                 (N69Q)- 
                 EAWGRASDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF 
               
               
                   
                   
                   
                 CJ′ 1G1- Fc(G1) 
                 NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS 
               
               
                   
                   
                   
                   
                 KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD 
               
               
                   
                   
                   
                   
                 SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 
               
               
                   
               
               
                 Design_5_Pr 
                 LC 
                 204 
                 VL(CD3)- 
                 KPTGVGGTPFPCLAPPIMLLVDGKQQMVVVCLVLDVAPPGLDSPIWFSAGQGSALDAFTYGP 
               
               
                 e_TCR_Con 
                   
                   
                 LCJB- 
                 SPATDGTWTNLAHLSLPSEELASWEPLVCHTGPGAEGHSRSTQPMHLSGEASTART 
               
               
                 junction′1_ 
                   
                   
                 CPreAlpha 
                   
               
               
                 Cys13 
                   
                   
                 (S11C)(N50Q) 
                   
               
               
                   
                 HC 
                 203 
                 HCJB- 
                 LEDLKNVFPPEVAVFEPCEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQ 
               
               
                   
                   
                   
                 CBeta(S16C) 
                 PLKEQPALQDSRYALSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSA 
               
               
                   
                   
                   
                 (N69Q)- 
                 EAWGRASDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF 
               
               
                   
                   
                   
                 CJ′ 1G1- Fc(G1) 
                 NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS 
               
               
                   
                   
                   
                   
                 KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD 
               
               
                   
                   
                   
                   
                 SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 
               
               
                   
               
               
                 Design_6_Pr 
                 LC 
                 204 
                 VL(CD3)- 
                 KPTGVGGTPFPCLAPPIMLLVDGKQQMVVVCLVLDVAPPGLDSPIWFSAGQGSALDAFTYGP 
               
               
                 e_TCR_Con 
                   
                   
                 LCJB- 
                 SPATDGTWTNLAHLSLPSEELASWEPLVCHTGPGAEGHSRSTQPMHLSGEASTART 
               
               
                 junction′1_ 
                   
                   
                 CPreAlpha 
                   
               
               
                 Cys14 
                   
                   
                 (S11C)(N50Q) 
                   
               
               
                   
                 HC 
                 205 
                 HCJB- 
                 LEDLKNVFPPEVAVFEPSECEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQ 
               
               
                   
                   
                   
                 CBeta(A18C) 
                 PLKEQPALQDSRYALSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSA 
               
               
                   
                   
                   
                 (N69Q)- 
                 EAWGRASDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF 
               
               
                   
                   
                   
                 CJ′ 1G1- Fc(G1) 
                 NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS 
               
               
                   
                   
                   
                   
                 KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD 
               
               
                   
                   
                   
                   
                 SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 
               
               
                   
               
               
                 Design_7_Pr 
                 LC 
                 204 
                 VL(CD3)- 
                 KPTGVGGTPFPCLAPPIMLLVDGKQQMVVVCLVLDVAPPGLDSPIWFSAGQGSALDAFTYGP 
               
               
                 e_TCR_Con 
                   
                   
                 LCJB- 
                 SPATDGTWTNLAHLSLPSEELASWEPLVCHTGPGAEGHSRSTQPMHLSGEASTART 
               
               
                 junction′1_ 
                   
                   
                 CPreAlpha 
                   
               
               
                 Cys15 
                   
                   
                 (S11C)(N50Q) 
                   
               
               
                   
                 HC 
                 206 
                 HCJB- 
                 LEDLKNVFPPEVAVFEPSEACISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQ 
               
               
                   
                   
                   
                 CBeta(E19C) 
                 PLKEQPALQDSRYALSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSA 
               
               
                   
                   
                   
                 (N69Q)- 
                 EAWGRASDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF 
               
               
                   
                   
                   
                 CJ′ 1G1- Fc(G1) 
                 NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS 
               
               
                   
                   
                   
                   
                 KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD 
               
               
                   
                   
                   
                   
                 SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 
               
               
                   
               
               
                 Design_8_Pr 
                 LC 
                 207 
                 VL(CD3)- 
                 KPTGVGGTPFPSLAPPIMLLVDGKQQMVVVCLVLDVAPPGLDSPIWFSAGQGSALDAFTYGP 
               
               
                 e_TCR_Con 
                   
                   
                 LCJB- 
                 CPATDGTWTNLAHLSLPSEELASWEPLVCHTGPGAEGHSRSTQPMHLSGEASTART 
               
               
                 junction′1_ 
                   
                   
                 CPreAlpha 
                   
               
               
                 Cys1_4L4T_ 
                   
                   
                 (S62C)(N50Q) 
                   
               
               
                 1 
                 HC 
                 208 
                 HCJB- 
                 LEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQ 
               
               
                   
                   
                   
                 CBeta(S56C) 
                 PLKEQPALQDSRYALSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSA 
               
               
                   
                   
                   
                 (N69Q)- 
                 EAWGRASDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF 
               
               
                   
                   
                   
                 CJ′ 1G1- Fc(G1) 
                 NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS 
               
               
                   
                   
                   
                   
                 KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD 
               
               
                   
                   
                   
                   
                 SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 
               
               
                   
               
               
                 Design_9_Pr 
                 LC 
                 209 
                 VL(CD3)- 
                 KPTGVGGTPFPSLAPPIMLLVDGKQQMVVVCLVLDVAPPGLDSPIWFSAGQGSALDAFTYGP 
               
               
                 e_TCR_Con 
                   
                   
                 LCJB- 
                 SPACDGTWTNLAHLSLPSEELASWEPLVCHTGPGAEGHSRSTQPMHLSGEASTART 
               
               
                 junction′1_ 
                   
                   
                 CPreAlpha 
                   
               
               
                 Cys2_4L4T_ 
                   
                   
                 (T65C)(N50Q) 
                   
               
               
                 2 
                 HC 
                 208 
                 HCJB- 
                 LEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQ 
               
               
                   
                   
                   
                 CBeta(556C) 
                 PLKEQPALQDSRYALSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSA 
               
               
                   
                   
                   
                 (N69Q)- 
                 EAWGRASDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF 
               
               
                   
                   
                   
                 CJ′ 1G1- Fc(G1) 
                 NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS 
               
               
                   
                   
                   
                   
                 KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD 
               
               
                   
                   
                   
                   
                 SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 
               
               
                   
               
               
                 Design_10_P 
                 LC 
                 210 
                 VL(CD3)- 
                 KPTGVGGTPFPSLAPPCMLLVDGKQQMVVVCLVLDVAPPGLDSPIWFSAGQGSALDAFTYGP 
               
               
                 re_TCR_Co 
                   
                   
                 LCJB- 
                 SPATDGTWTNLAHLSLPSEELASWEPLVCHTGPGAEGHSRSTQPMHLSGEASTART 
               
               
                 njunction′1_ 
                   
                   
                 CPreAlpha 
                   
               
               
                 Cys4 
                   
                   
                 (I16C)(N50Q) 
                   
               
               
                   
                 HC 
                 211 
                 HCJB- 
                 LEDLKNVFPPEVCVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQ 
               
               
                   
                   
                   
                 CBeta(A11C) 
                 PLKEQPALQDSRYALSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSA 
               
               
                   
                   
                   
                 (N69Q)- 
                 EAWGRASDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF 
               
               
                   
                   
                   
                 CJ′ 1G1- Fc(G1) 
                 NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS 
               
               
                   
                   
                   
                   
                 KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD 
               
               
                   
                   
                   
                   
                 SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 
               
               
                   
               
               
                 PreTCR_De 
                 Light 
                 212 
                 VL(CD3)- 
                 KLEDLKNVFPPEVAVFEPCEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQ 
               
               
                 sign5_crosse 
                   
                   
                 HCJB- CBeta 
                 PALQDSRYALSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRA 
               
               
                 d_1 
                   
                   
                 (N69Q, S16C)- 
                   
               
               
                   
                   
                   
                 CJ′ 1G 
                   
               
               
                   
                 Heav 
                 213 
                 HCJC- 
                 SSASGVGGTPFP C LAPPIMLLVDGKQQMVVVCLVLDVAPPGLDSPIWFSAG Q GSALDAFTYGPSPATD 
               
               
                   
                 y 
                   
                 CPreAlpha 
                 GTWTNLAHLSLPSEELASWEPLVCHTGPGAEGHSRSTQPMHLSGEASTARTSDKTHTCPPCPAPEAAG 
               
               
                   
                 (Conj 
                   
                 (S11C, N50Q) - 
                 GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS 
               
               
                   
                 unctio 
                   
                 CJ′2G1- Fc 
                 VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR E E M TKNQVSLTCLVKGF 
               
               
                   
                 n 
                   
                   
                 YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS 
               
               
                   
                 more 
                   
                   
                 LSLSPGK 
               
               
                   
                 antibo 
                   
                   
                   
               
               
                   
                 dy) 
                   
                   
                   
               
               
                   
               
               
                 Pre_TCR_De 
                 Light 
                 212 
                 VL(CD3)- 
                 KLEDLKNVFPPEVAVFEPCEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQ 
               
               
                 sign_5_cross 
                   
                   
                 HCJB- CBeta 
                 PALQDSRYALSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRA 
               
               
                 ed_2 
                   
                   
                 (N69Q, S16C)- 
                   
               
               
                   
                   
                   
                 CJ′ 1G 
                   
               
               
                   
                 Heav 
                 214 
                 HCJD- 
                 SSPTGVGGTPFP C LAPPIMLLVDGKQQMVVVCLVLDVAPPGLDSPIWFSAG Q GSALDAFTYGPSPATD 
               
               
                   
                 y 
                   
                 CPreAlpha 
                 GTWTNLAHLSLPSEELASWEPLVCHTGPGAEGHSRSTQPMHLSGEASTARTSDKTHTCPPCPAPEAAG 
               
               
                   
                 (Conj 
                   
                 (S11) -CJ′2G1- 
                 GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS 
               
               
                   
                 unctio 
                   
                 Fc 
                 VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR E E M TKNQVSLTCLVKGF 
               
               
                   
                 n 
                   
                   
                 YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS 
               
               
                   
                 more 
                   
                   
                 LSLSPGK 
               
               
                   
                 PreT 
                   
                   
                   
               
               
                   
                 CR) 
                   
                   
                   
               
               
                   
               
               
                 Pre_TCR_De 
                 Light 
                 215 
                 VL(CD3)- 
                 KLEDLKNVFPPEVAVFEPSECEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQ 
               
               
                 sign_6_cross 
                   
                   
                 LCJC- CBeta 
                 PALQDSRYALSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRA 
               
               
                 ed_1 
                   
                   
                 (A18C, N69Q) 
                   
               
               
                   
                 Heav 
                 213 
                   
                   
               
               
                   
                 y 
                   
                   
                   
               
               
                   
                 (Conj 
                   
                   
                   
               
               
                   
                 unctio 
                   
                   
                   
               
               
                   
                 n 
                   
                   
                   
               
               
                   
                 more 
                   
                   
                   
               
               
                   
                 antibo 
                   
                   
                   
               
               
                   
                 dy) 
                   
                   
                   
               
               
                   
               
               
                 Pre_TCR_De 
                 Light 
                 215 
                   
                 KLEDLKNVFPPEVAVFEPSECEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQ 
               
               
                 sign_6_cross 
                   
                   
                   
                 PALQDSRYALSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRA 
               
               
                 ed-2 
                 Heav 
                 214 
                   
                   
               
               
                   
                 y 
                   
                   
                   
               
               
                   
                 (Conj 
                   
                   
                   
               
               
                   
                 unctio 
                   
                   
                   
               
               
                   
                 n 
                   
                   
                   
               
               
                   
                 more 
                   
                   
                   
               
               
                   
                 TCR) 
               
               
                   
               
               
                 Delta- 
                   
                 SEQ 
                   
                   
               
               
                 Gamma 
                 Chain 
                 ID 
                   
                   
               
               
                 Designs 
                 Name 
                 NO 
                   
                 Sequences 
               
               
                   
               
               
                 dg_Design_2 
                 LC 
                 215 
                 VL(CD3)-LCJ5- 
                 EPRSQPHTKPSVFVMKQGTNVACLVKEFYPKDIRINLVSSKKITEFDPAIVISPSGKYNAVK 
               
               
                 no Glyco 
                   
                   
                 CDelta 
                 LGKYEDSNSVTCSVQHDQKTVHSTDFE 
               
               
                   
                   
                   
                 (N16Q+ N79Q) 
                   
               
               
                   
                 HC 
                 216 
                 HCJ5-CGamma- 
                 TDKQLDADVSPKPTIFLPSIAETKLQKAGTYLCLLEKFFPDVIKIHWQEKKSNTILGSQEGN 
               
               
                   
                   
                   
                 CJ′3G1- Fc(G1) 
                 TMKTQDTYMKFSWLTVPEESLDKEHRCIVRHENNKNGVDQEIIFPPIKSDKTHTCPPCPAPE 
               
               
                   
                   
                   
                 (N65Q) 
                 AAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ 
               
               
                   
                   
                   
                   
                 YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE 
               
               
                   
                   
                   
                   
                 MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ 
               
               
                   
                   
                   
                   
                 GNVFSCSVMHEALHNHYTQKSLSLSPGK 
               
               
                   
               
               
                 dg_Design_2 
                 LC 
                 217 
                 VL(CD3)-LCJ5- 
                 EPRSQPHTKPSVFVMKCGTNVACLVKEFYPKDIRINLVSSKKITEFDPAIVISPSGKYNAVK 
               
               
                 _hypeCys1_ 
                   
                   
                 CDelta (N16C) 
                 LGKYEDSNSVTCSVQHDQKTVHSTDFE 
               
               
                 no_Glyco 
                   
                   
                 (N79Q) 
                   
               
               
                   
                 HC 
                 218 
                 HCJ5-CGamma 
                 TDKQLDADVSPKPCIFLPSIAETKLQKAGTYLCLLEKFFPDVIKIHWQEKKSNTILGSQEGN 
               
               
                   
                   
                   
                 (T12C)(N65Q)- 
                 TMKTQDTYMKFSWLTVPEESLDKEHRCIVRHENNKNGVDQEIIFPPIKSDKTHTCPPCPAPE 
               
               
                   
                   
                   
                 CJ′3G1- Fc(G1) 
                 AAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ 
               
               
                   
                   
                   
                   
                 YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE 
               
               
                   
                   
                   
                   
                 MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ 
               
               
                   
                   
                   
                   
                 GNVFSCSVMHEALHNHYTQKSLSLSPGK 
               
               
                   
               
               
                 dg_Design_2 
                 LC 
                 219 
                 VL(CD3)-LCJ5- 
                 EPRSQPHTKPSVFVMKQGTNVACLVKEFYPKDIRINLVSSKKITEFDPAICISPSGKYNAVK 
               
               
                 _hypeCys2_ 
                   
                   
                 CDelta (V50C) 
                 LGKYEDSNSVTCSVQHDQKTVHSTDFE 
               
               
                 no_Glyco 
                   
                   
                 (N16Q+ N79Q) 
                   
               
               
                   
                 HC 
                 220 
                 HCJ5-CGamma 
                 TDKQLDADVSPKPTIFLPSIAETKLQKAGTYLCLLEKFFPDVIKIHWQEKKSNTILGSCEGN 
               
               
                   
                   
                   
                 (Q57C)(N65Q)- 
                 TMKTQDTYMKFSWLTVPEESLDKEHRCIVRHENNKNGVDQEIIFPPIKSDKTHTCPPCPAPE 
               
               
                   
                   
                   
                 CJ′3G1- Fc(G1) 
                 AAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ 
               
               
                   
                   
                   
                   
                 YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE 
               
               
                   
                   
                   
                   
                 MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ 
               
               
                   
                   
                   
                   
                 GNVFSCSVMHEALHNHYTQKSLSLSPGK 
               
               
                   
               
               
                 dg_Design_2 
                 LC 
                 221 
                 VL(CD3)-LCJ5- 
                 EPRSQPHTKPSVFVMKQGTNVACLVKEFYPKDIRINLVSSKKITEFCPAIVISPSGKYNAVK 
               
               
                 _hypeCys3_ 
                   
                   
                 CDelta (D46C) 
                 LGKYEDSNSVTCSVQHDQKTVHSTDFE 
               
               
                 no_Glyco 
                   
                   
                 (N16Q+ N79Q) 
                   
               
               
                   
                 HC 
                 222 
                 HCJ5-CGamma 
                 TDKQLDADVSPKPTIFLPSIAETKLQKAGTYLCLLEKFFPDVIKIHWQEKKSNTILGSQEGN 
               
               
                   
                   
                   
                 (M62C) 
                 TCKTQDTYMKFSWLTVPEESLDKEHRCIVRHENNKNGVDQEIIFPPIKSDKTHTCPPCPAPE 
               
               
                   
                   
                   
                 (N65Q)- 
                 AAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ 
               
               
                   
                   
                   
                 CJ′3G1- Fc(G1) 
                 YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE 
               
               
                   
                   
                   
                   
                 MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ 
               
               
                   
                   
                   
                   
                 GNVFSCSVMHEALHNHYTQKSLSLSPGK 
               
               
                   
               
               
                 dg_Design_2 
                 LC 
                 223 
                 VL(CD3)-LCJ5- 
                 EPRSQPHTKPSVCVMKQGTNVACLVKEFYPKDIRINLVSSKKITEFDPAIVISPSGKYNAVK 
               
               
                 _Cys2_no_G 
                   
                   
                 CDelta (F12C) 
                 LGKYEDSNSVTCSVQHDQKTVHSTDFE 
               
               
                 lyco 
                   
                   
                 (N16Q+ N79Q) 
                   
               
               
                   
                 HC 
                 224 
                 HCJ5-CGamma 
                 TDKQLDADVSPKPTIFLPCIAETKLQKAGTYLCLLEKFFPDVIKIHWQEKKSNTILGSQEGN 
               
               
                   
                   
                   
                 (S17C)(N65Q)- 
                 TMKTQDTYMKFSWLTVPEESLDKEHRCIVRHENNKNGVDQEIIFPPIKSDKTHTCPPCPAPE 
               
               
                   
                   
                   
                 CJ′3G1- Fc(G1) 
                 AAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ 
               
               
                   
                   
                   
                   
                 YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE 
               
               
                   
                   
                   
                   
                 MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ 
               
               
                   
                   
                   
                   
                 GNVFSCSVMHEALHNHYTQKSLSLSPGK 
               
               
                   
               
               
                 dg_Design_2 
                 LC 
                 225 
                 VL(CD3)-LCJ5- 
                 EPRSQPHTKPSVFVCKQGTNVACLVKEFYPKDIRINLVSSKKITEFDPAIVISPSGKYNAVK 
               
               
                 _Cys1_no_G 
                   
                   
                 CDelta (M14C) 
                 LGKYEDSNSVTCSVQHDQKTVHSTDFE 
               
               
                 lyco 
                   
                   
                 (N16Q+ N79Q) 
                   
               
               
                   
                 HC 
                 226 
                 HCJ5-CGamma 
                 TDKQLDADVSPKPTICLPSIAETKLQKAGTYLCLLEKFFPDVIKIHWQEKKSNTILGSQEGN 
               
               
                   
                   
                   
                 (F14C)(N65Q)- 
                 TMKTQDTYMKFSWLTVPEESLDKEHRCIVRHENNKNGVDQEIIFPPIKSDKTHTCPPCPAPE 
               
               
                   
                   
                   
                 CJ′3G1- Fc(G1) 
                 AAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ 
               
               
                   
                   
                   
                   
                 YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE 
               
               
                   
                   
                   
                   
                 MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ 
               
               
                   
                   
                   
                   
                 GNVFSCSVMHEALHNHYTQKSLSLSPGK 
               
               
                   
               
               
                 dg_Design_2 
                 LC 
                 223 
                 VL(CD3)-LCJ5- 
                 EPRSQPHTKPSVCVMKQGTNVACLVKEFYPKDIRINLVSSKKITEFDPAIVISPSGKYNAVK 
               
               
                 _Cys3_no_G 
                   
                   
                 CDelta (F12C) 
                 LGKYEDSNSVTCSVQHDQKTVHSTDFE 
               
               
                 lyco 
                   
                   
                 (N16Q+ N79Q) 
                   
               
               
                   
                 HC 
                 227 
                 HCJ5-CGamma 
                 TDKQLDADVSPKPTIFLPSIACTKLQKAGTYLCLLEKFFPDVIKIHWQEKKSNTILGSQEGN 
               
               
                   
                   
                   
                 (E20C)(N65Q)- 
                 TMKTQDTYMKFSWLTVPEESLDKEHRCIVRHENNKNGVDQEIIFPPIKSDKTHTCPPCPAPE 
               
               
                   
                   
                   
                 CJ′3G1- Fc(G1) 
                 AAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ 
               
               
                   
                   
                   
                   
                 YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE 
               
               
                   
                   
                   
                   
                 MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ 
               
               
                   
                   
                   
                   
                 GNVFSCSVMHEALHNHYTQKSLSLSPGK 
               
               
                   
               
               
                 dg_Design_2 
                 LC 
                 228 
                 VL(CD3)-LCJ5- 
                 EPRSQPHTKPSVFVMKQGTNVACLVKEFYPKDIRINLVSSKKITEFDPAIVISPSGKYNAVK 
               
               
                 _Cys4_no_G 
                   
                   
                 CDelta (F87C) 
                 LGKYEDSNSVTCSVQHDQKTVHSTDCE 
               
               
                 lyco 
                   
                   
                 (N16Q+ N79Q) 
                   
               
               
                   
                 HC 
                 229 
                 HCJ5-CGamma 
                 TDKQLDADVSPKPTIFLPSICETKLQKAGTYLCLLEKFFPDVIKIHWQEKKSNTILGSQEGN 
               
               
                   
                   
                   
                 (A19C)(N65Q)- 
                 TMKTQDTYMKFSWLTVPEESLDKEHRCIVRHENNKNGVDQEIIFPPIKSDKTHTCPPCPAPE 
               
               
                   
                   
                   
                 CJ′3G1- Fc(G1) 
                 AAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ 
               
               
                   
                   
                   
                   
                 YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE 
               
               
                   
                   
                   
                   
                 MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ 
               
               
                   
                   
                   
                   
                 GNVFSCSVMHEALHNHYTQKSLSLSPGK 
               
               
                   
               
               
                 dg_Design_2 
                 LC 
                 230 
                 VL(CD3)-LCJ5- 
                 EPRSQPHTKPSVFVMKQGTNVACLVKEFYPKDIRINLVSSKKITEFDPAIVISPSGKYNAVK 
               
               
                 _Cys5_no_G 
                   
                   
                 CDelta (E88C) 
                 LGKYEDSNSVTCSVQHDQKTVHSTDFC 
               
               
                 lyco 
                   
                   
                 (N16Q+ N79Q) 
                   
               
               
                   
                 HC 
                 229 
                 HCJ5-CGamma 
                 TDKQLDADVSPKPTIFLPSICETKLQKAGTYLCLLEKFFPDVIKIHWQEKKSNTILGSQEGN 
               
               
                   
                   
                   
                 (A19C)(N65Q)- 
                 TMKTQDTYMKFSWLTVPEESLDKEHRCIVRHENNKNGVDQEIIFPPIKSDKTHTCPPCPAPE 
               
               
                   
                   
                   
                 CJ′3G1- Fc(G1) 
                 AAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ 
               
               
                   
                   
                   
                   
                 YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE 
               
               
                   
                   
                   
                   
                 MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ 
               
               
                   
                   
                   
                   
                 GNVFSCSVMHEALHNHYTQKSLSLSPGK 
               
               
                   
               
               
                 dg_Design_2 
                 LC 
                 231 
                 VL(CD3)-LCJ5- 
                 EPRSQPHTKPSVFVMKNGTNVACLVKEFYPKDIRINLVSSKKITEFDPAIVISPSGKYNAVK 
               
               
                   
                   
                   
                 CDelta 
                 LGKYEDSNSVTCSVQHDNKTVHSTDFE 
               
               
                   
                 HC 
                 232 
                 HCJ5-CGamma- 
                 TDKQLDADVSPKPTIFLPSIAETKLQKAGTYLCLLEKFFPDVIKIHWQEKKSNTILGSQEGN 
               
               
                   
                   
                   
                 CJ′3G1- Fc(G1) 
                 TMKTNDTYMKFSWLTVPEESLDKEHRCIVRHENNKNGVDQEIIFPPIKSDKTHTCPPCPAPE 
               
               
                   
                   
                   
                   
                 AAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ 
               
               
                   
                   
                   
                   
                 YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE 
               
               
                   
                   
                   
                   
                 MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ 
               
               
                   
                   
                   
                   
                 GNVFSCSVMHEALHNHYTQKSLSLSPGK 
               
               
                   
               
               
                 dg_Design_1 
                 LC 
                 233 
                 VL(CD3)-LCJ4- 
                 KPRSQPHTKPSVFVMKNGTNVACLVKEFYPKDIRINLVSSKKITEFDPAIVISPSGKYNAVK 
               
               
                   
                   
                   
                 CDelta 
                 LGKYEDSNSVTCSVQHDNKTVHSTDFE 
               
               
                   
                 HC 
                 234 
                 HCJ4-CGamma- 
                 SSASLDADVSPKPTIFLPSIAETKLQKAGTYLCLLEKFFPDVIKIHWQEKKSNTILGSQEGN 
               
               
                   
                   
                   
                 CJ′3G1- Fc(G1) 
                 TMKTNDTYMKFSWLTVPEESLDKEHRCIVRHENNKNGVDQEIIFPPIKSDKTHTCPPCPAPE 
               
               
                   
                   
                   
                   
                 AAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ 
               
               
                   
                   
                   
                   
                 YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE 
               
               
                   
                   
                   
                   
                 MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ 
               
               
                   
                   
                   
                   
                 GNVFSCSVMHEALHNHYTQKSLSLSPGK 
               
               
                   
               
               
                 dg_crossed_ 
                 LC 
                 235 
                 VL(CD3)-LCJ6- 
                 KDKQLDADVSPKPTIFLPSIAETKLQKAGTYLCLLEKFFPDVIKIHWQEKKSNTILGSCEGN 
               
               
                 Design_1 
                   
                   
                 CGamma 
                 TMKT Q DTYMKFSWLTVPEESLDKEHRCIVRHENNKNGVDQEIIFPPIKTDVITMD 
               
               
                   
                 HC 
                 236 
                 HCJ6- CDelta-  
                 SSRSQPHTKP SVFVMK Q GTNVACLVKEFYPKDIRINLVSSKKITEFDPAI C ISPSGKYNAVK   
               
               
                   
                   
                   
                 CJ′4G1- Fc(G1) 
                   LGKYEDSNSVTCSVQHD Q KTVHSTD EPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLM 
               
               
                   
                   
                   
                   
                 ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL 
               
               
                   
                   
                   
                   
                 NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSD 
               
               
                   
                   
                   
                   
                 IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ 
               
               
                   
                   
                   
                   
                 KSLSLSPGK 
               
               
                   
               
               
                 dg_crossed_ 
                 LC 
                 237 
                 VL(CD3)-LCJ7- 
                 KDKQLDADVSPKPTIFLPSIAETKLQKAGTYLCLLEKFFPDVIKIHWQEKKSNTILGSCEGN 
               
               
                 Design_2 
                   
                   
                 CGamma 
                 TMKT Q DTYMKFSWLTVPEESLDKEHRCIVRHENNKNGVDQEIIFPPIKTDVITMD 
               
               
                   
                 HC 
                 238 
                 HCJ7- CDelta- 
                 EPRSQPHTKP SVFVMK Q GTNVACLVKEFYPKDIRINLVSSKKITEFDPAI C ISPSGKYNAVK   
               
               
                   
                   
                   
                 CJ′4G1- Fc(G1) 
                   LGKYEDSNSVTCSVQHD Q KTVHSTD EPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLM 
               
               
                   
                   
                   
                   
                 ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL 
               
               
                   
                   
                   
                   
                 NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSD 
               
               
                   
                   
                   
                   
                 IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ 
               
               
                   
                   
                   
                   
                 KSLSLSPGK 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 20 
               
               
                   
               
               
                 Sequences for exemplary polypeptide complexes 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                 SEQ 
                   
                   
               
               
                 Alpha-Beta 
                 Chain 
                 ID 
                   
                   
               
               
                 Designs 
                 Name 
                 No 
                   
                 Sequences 
               
               
                   
               
               
                 T3-Design_1 
                 LC 
                 1 
                 VL(CD3)- 
                 DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQQKPGQPPKLLIY 
               
               
                 (IgG1) 
                   
                   
                 LCJ1- 
                 WASTRQSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTQSHTLRTFGGGTKVE 
               
               
                 normal 
                   
                   
                 CAlpha(T49C) 
                 IKRTVAAPDPAVYQLRDSKSSDKSVCLFTDFDSQT N VSQSKDSDVYITDK C VLDM 
               
               
                   
                   
                   
                   
                 RSMDFKSNSAVAWS N KSDFACANAF N NSIIPEDTFFPSPESS 
               
               
                   
                 HC 
                 2 
                 VH(CD3)- 
                 QVQLVQSGAEVKKPGSSVKVSCKASGFAFTDYYIHWVRQAPGQGLEWMGWISPGN 
               
               
                   
                   
                   
                 HCJ1- 
                 VNTKYNENFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDGYSLYYFDYWG 
               
               
                   
                   
                   
                 CBeta(S56C)- 
                 QGTLVTVSSASKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWV 
               
               
                   
                   
                   
                 CJ′ 1G1-Fc(G1) 
                 NGKEVHSGV C TDPQPLKEQPAL N DSRYALSSRLRVSATFWQNPRNHFRCQVQFYG 
               
               
                   
                   
                   
                   
                 LSENDEWTQDRAKPVTQIVSAEAWGRASDKTHTCPPCPAPEAAGGPSVFLFPPKP 
               
               
                   
                   
                   
                   
                 KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV 
               
               
                   
                   
                   
                   
                 VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE 
               
               
                   
                   
                   
                   
                 MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV 
               
               
                   
                   
                   
                   
                 DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 
               
               
                   
               
               
                 T3-Design_2 
                 LC 
                 3 
                 VL(CD3)- 
                 DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQQKPGQPPKLLIY 
               
               
                 (IgG1) 
                   
                   
                 LCJ2- 
                 WASTRQSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTQSHTLRTFGGGTKVE 
               
               
                 normal 
                   
                   
                 CAlpha(T49C) 
                 IKPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQT N VSQSKDSDVYITDK C VLDM 
               
               
                   
                   
                   
                   
                 RSMDFKSNSAVAWS N KSDFACANAF N NSIIPEDTFFPSPESS 
               
               
                   
                 HC 
                 4 
                 VH(CD3)- 
                 QVQLVQSGAEVKKPGSSVKVSCKASGFAFTDYYIHWVRQAPGQGLEWMGWISPGN 
               
               
                   
                   
                   
                 HCJ2- 
                 VNTKYNENFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDGYSLYYFDYWG 
               
               
                   
                   
                   
                 CBeta(S56C)- 
                 QGTLVTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWV 
               
               
                   
                   
                   
                 CJ′ 1G1- 
                 NGKEVHSGV C TDPQPLKEQPAL N DSRYALSSRLRVSATFWQNPRNHFRCQVQFYG 
               
               
                   
                   
                   
                 Fc(G1) 
                 LSENDEWTQDRAKPVTQIVSAEAWGRASDKTHTCPPCPAPEAAGGPSVFLFPPKP 
               
               
                   
                   
                   
                   
                 KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV 
               
               
                   
                   
                   
                   
                 VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE 
               
               
                   
                   
                   
                   
                 MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV 
               
               
                   
                   
                   
                   
                 DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 
               
               
                   
               
               
                 T3-Design_5 
                 LC 
                 1 
                 VL(CD3)- 
                 DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQQKPGQPPKLLIY 
               
               
                 (IgG1) 
                   
                   
                 LCJ1- 
                 WASTRQSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTQSHTLRTFGGGTKVE 
               
               
                 normal 
                   
                   
                 CAlpha(T49C) 
                 IKRTVAAPDPAVYQLRDSKSSDKSVCLFTDFDSQT N VSQSKDSDVYITDK C VLDM 
               
               
                   
                   
                   
                   
                 RSMDFKSNSAVAWS N KSDFACANAF N NSIIPEDTFFPSPESS 
               
               
                   
                 HC 
                 5 
                 VH(CD3) - 
                 QVQLVQSGAEVKKPGSSVKVSCKASGFAFTDYYIHWVRQAPGQGLEWMGWISPGN 
               
               
                   
                   
                   
                 HCJ1- 
                 VNTKYNENFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDGYSLYYFDYWG 
               
               
                   
                   
                   
                 CBeta(S56C)(F 
                 QGTLVTVSSASKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWV 
               
               
                   
                   
                   
                 G-DE+)- 
                 NGKEVHSGV C TDPQPLKEQPAL N DSRYALSSRLRVSATFWQNPRNHFRCQVQFYP 
               
               
                   
                   
                   
                 CJ′ 1G1- 
                 SNQIVSAEAWGRASDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTC 
               
               
                   
                   
                   
                 Fc(G1) 
                 VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG 
               
               
                   
                   
                   
                   
                 KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKG 
               
               
                   
                   
                   
                   
                 FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS 
               
               
                   
                   
                   
                   
                 VMHEALHNHYTQKSLSLSPGK 
               
               
                   
               
               
                 T3-Design_6 
                 LC 
                 3 
                 VL(CD3)- 
                 DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQQKPGQPPKLLIY 
               
               
                 (IgG1) 
                   
                   
                 LCJ2- 
                 WASTRQSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTQSHTLRTFGGGTKVE 
               
               
                 normal 
                   
                   
                 CAlpha(T49C) 
                 IKPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQT N VSQSKDSDVYITDK C VLDM 
               
               
                   
                   
                   
                   
                 RSMDFKSNSAVAWS N KSDFACANAF N NSIIPEDTFFPSPESS 
               
               
                   
                 HC 
                 6 
                 VH(CD3)- 
                 QVQLVQSGAEVKKPGSSVKVSCKASGFAFTDYYIHWVRQAPGQGLEWMGWISPGN 
               
               
                   
                   
                   
                 HCJ2- 
                 VNTKYNENFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDGYSLYYFDYWG 
               
               
                   
                   
                   
                 CBeta(S56C)(F 
                 QGTLVTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWV 
               
               
                   
                   
                   
                 G-DE+)- 
                 NGKEVHSGV C TDPQPLKEQPAL N DSRYALSSRLRVSATFWQNPRNHFRCQVQFYP 
               
               
                   
                   
                   
                 CJ′ 1G1-Fc(G1) 
                 SNQIVSAEAWGRASDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTC 
               
               
                   
                   
                   
                   
                 VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG 
               
               
                   
                   
                   
                   
                 KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKG 
               
               
                   
                   
                   
                   
                 FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS 
               
               
                   
                   
                   
                   
                 VMHEALHNHYTQKSLSLSPGK 
               
               
                   
               
               
                   
                 LC 
                 3 
                 VL(CD3)- 
                 DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQQKPGQPPKLLIY 
               
               
                 T3-Design_6a 
                   
                   
                 LCJ2- 
                 WASTRQSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTQSHTLRTFGGGTKVE 
               
               
                 (IgG1) 
                   
                   
                 CAlpha(T49C) 
                 IKPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQT N VSQSKDSDVYITDK C VLDM 
               
               
                 normal 
                   
                   
                   
                 RSMDFKSNSAVAWS N KSDFACANAF N NSIIPEDTFFPSPESS 
               
               
                   
                 HC 
                 7 
                 VH(CD3)- 
                 QVQLVQSGAEVKKPGSSVKVSCKASGFAFTDYYIHWVRQAPGQGLEWMGWISPGN 
               
               
                   
                   
                   
                 HCJ2- 
                 VNTKYNENFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDGYSLYYFDYWG 
               
               
                   
                   
                   
                 CBeta(S56C)(F 
                 QGTLVTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWV 
               
               
                   
                   
                   
                 G-DE-)- 
                 NGKEVHSGV C TDPQPLKEQ --SG--   
               
               
                   
                   
                   
                 CJ′ 1G1-Fc(G1) 
                 RYALSSRLRVSATFWQNPRNHFRCQVQFYPSNQIVSAEAWGRASDKTHTCPPCPA 
               
               
                   
                   
                   
                   
                 PEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN 
               
               
                   
                   
                   
                   
                 AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG 
               
               
                   
                   
                   
                   
                 QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP 
               
               
                   
                   
                   
                   
                 VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 
               
               
                   
               
               
                 T3-Design_3 
                 LC 
                 8 
                 VL(CD3)- 
                 DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQQKPGQPPKLLIY 
               
               
                 (IgG1) 
                   
                   
                 LCJ3- 
                 WASTRQSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTQSHTLRTFGGGTKVE 
               
               
                 crossed 
                   
                   
                 CBeta(S56C) 
                 IKLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEV 
               
               
                   
                   
                   
                   
                 HSGV C TDPQPLKEQPAL N DSRYALSSRLRVSATFWQNPRNHFRCQVQFYGLSEND 
               
               
                   
                   
                   
                   
                 EWTQDRAKPVTQIVSAEAWGRA 
               
               
                   
                 HC 
                 9 
                 VH(CD3)- 
                 QVQLVQSGAEVKKPGSSVKVSCKASGFAFTDYYIHWVRQAPGQGLEWMGWISPGN 
               
               
                   
                   
                   
                 HCJ3- 
                 VNTKYNENFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDGYSLYYFDYWG 
               
               
                   
                   
                   
                 CAlpha(T49C)- 
                 QGTLVTVSSASIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQT N VSQSKDSDVYIT 
               
               
                   
                   
                   
                 CJ′2G1-Fc(G1) 
                 DK C VLDMRSMDFKSNSAVAWS N KSDFACANAF N NSIIPEDTFFPSPESSSDKTHT 
               
               
                   
                   
                   
                   
                 CPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD 
               
               
                   
                   
                   
                   
                 GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT 
               
               
                   
                   
                   
                   
                 ISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN 
               
               
                   
                   
                   
                   
                 YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 
               
               
                   
                   
                   
                   
                 GK 
               
               
                   
               
               
                 T3-Design_4 
                 LC 
                 8 
                 VL(CD3)- 
                 DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQQKPGQPPKLLIY 
               
               
                 (IgG1) 
                   
                   
                 LCJ4- 
                 WASTRQSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTQSHTLRTFGGGTKVE 
               
               
                 crossed 
                   
                   
                 CBeta(S56C) 
                 IKLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEV 
               
               
                   
                   
                   
                   
                 HSGV C TDPQPLKEQPAL N DSRYALSSRLRVSATFWQNPRNHFRCQVQFYGLSEND 
               
               
                   
                   
                   
                   
                 EWTQDRAKPVTQIVSAEAWGRA 
               
               
                   
                 HC 
                 10 
                 VH(CD3) 
                 QVQLVQSGAEVKKPGSSVKVSCKASGFAFTDYYIHWVRQAPGQGLEWMGWISPGN 
               
               
                   
                   
                   
                 HCJ4- 
                 VNTKYNENFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDGYSLYYFDYWG 
               
               
                   
                   
                   
                 CAlpha(T49C)- 
                 QGTLVTVSSPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQT N VSQSKDSDVYIT 
               
               
                   
                   
                   
                 CJ′2G1- 
                 DK C VLDMRSMDFKSNSAVAWS N KSDFACANAF N NSIIPEDTFFPSPESSSDKTHT 
               
               
                   
                   
                   
                 Fc(G1) 
                 CPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD 
               
               
                   
                   
                   
                   
                 GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT 
               
               
                   
                   
                   
                   
                 ISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN 
               
               
                   
                   
                   
                   
                 YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 
               
               
                   
                   
                   
                   
                 GK 
               
               
                   
               
               
                 T3-Design_7 
                 LC 
                 11 
                 VL(CD3)- 
                 DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQQKPGQPPKLLIY 
               
               
                 (IgG1) 
                   
                   
                 LCJ3- 
                 WASTRQSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTQSHTLRTFGGGTKVE 
               
               
                 crossed 
                   
                   
                 CBeta(S56C)(F 
                 IKLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEV 
               
               
                   
                   
                   
                 G-DE+) 
                 HSGV C TDPQPLKEQPAL N DSRYALSSRLRVSATFWQNPRNHFRCQVQFYPSNQIV 
               
               
                   
                   
                   
                   
                 SAEAWGRA 
               
               
                   
                 HC 
                 9 
                 VH(CD3)- 
                 QVQLVQSGAEVKKPGSSVKVSCKASGFAFTDYYIHWVRQAPGQGLEWMGWISPGN 
               
               
                   
                   
                   
                 HCJ3- 
                 VNTKYNENFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDGYSLYYFDYWG 
               
               
                   
                   
                   
                 CAlpha(T49C)- 
                 QGTLVTVSSASIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQT N VSQSKDSDVYIT 
               
               
                   
                   
                   
                 CJ′2G1- 
                 DK C VLDMRSMDFKSNSAVAWS N KSDFACANAF N NSIIPEDTFFPSPESSSDKTHT 
               
               
                   
                   
                   
                 Fc(G1) 
                 CPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD 
               
               
                   
                   
                   
                   
                 GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT 
               
               
                   
                   
                   
                   
                 ISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN 
               
               
                   
                   
                   
                   
                 YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 
               
               
                   
                   
                   
                   
                 GK 
               
               
                   
               
               
                 T3-Design_8 
                 LC 
                 11 
                 VL(CD3)- 
                 DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQQKPGQPPKLLIY 
               
               
                 (IgG1) 
                   
                   
                 LCJ4- 
                 WASTRQSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTQSHTLRTFGGGTKVE 
               
               
                 crossed 
                   
                   
                 CBeta(S56C)(FI 
                 KLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEV 
               
               
                   
                   
                   
                 G-DE+) 
                 HSGV C TDPQPLKEQPAL N DSRYALSSRLRVSATFWQNPRNHFRCQVQFYPSNQIV 
               
               
                   
                   
                   
                   
                 SAEAWGRA 
               
               
                   
                 HC 
                 10 
                 VH(CD3)- 
                 QVQLVQSGAEVKKPGSSVKVSCKASGFAFTDYYIHWVRQAPGQGLEWMGWISPGN 
               
               
                   
                   
                   
                 HCJ4- 
                 VNTKYNENFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDGYSLYYFDYWG 
               
               
                   
                   
                   
                 CAlpha(T49C)- 
                 QGTLVTVSSPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQT N VSQSKDSDVYIT 
               
               
                   
                   
                   
                 CJ′2G1- 
                 DK C VLDMRSMDFKSNSAVAWS N KSDFACANAF N NSIIPEDTFFPSPESSSDKTHT 
               
               
                   
                   
                   
                 Fc(G1) 
                 CPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD 
               
               
                   
                   
                   
                   
                 GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT 
               
               
                   
                   
                   
                   
                 ISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN 
               
               
                   
                   
                   
                   
                 YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 
               
               
                   
                   
                   
                   
                 GK 
               
               
                   
               
               
                 T3-Design_2- 
                 LC 
                 12 
                 VL(CD3)- 
                 DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQQKPGQPPKLLIY 
               
               
                 QQQQ (IgG1) 
                   
                   
                 LCJ2- 
                 WASTRQSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTQSHTLRTFGGGTKVE 
               
               
                   
                   
                   
                 CAlpha(T49C) 
                 IKPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQT Q VSQSKDSDVYITDK C VLDM 
               
               
                   
                   
                   
                 (N34Q + N68Q + 
                 RSMDFKSNSAVAWS Q KSDFACANAF Q NSIIPEDTFFPSPESS 
               
               
                   
                   
                   
                 N79Q) 
                   
               
               
                   
                 HC 
                 13 
                 VH(CD3)- 
                 QVQLVQSGAEVKKPGSSVKVSCKASGFAFTDYYIHWVRQAPGQGLEWMGWISPGN 
               
               
                   
                   
                   
                 HCJ2- 
                 VNTKYNENFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDGYSLYYFDYWG 
               
               
                   
                   
                   
                 CBeta(S56C) 
                 QGTLVTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWV 
               
               
                   
                   
                   
                 (N69Q)- 
                 NGKEVHSGV C TDPQPLKEQPAL Q DSRYALSSRLRVSATFWQNPRNHFRCQVQFYG 
               
               
                   
                   
                   
                 CJ′ 1G1- 
                 LSENDEWTQDRAKPVTQIVSAEAWGRASDKTHTCPPCPAPEAAGGPSVFLFPPKP 
               
               
                   
                   
                   
                 Fc(G1) 
                 KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV 
               
               
                   
                   
                   
                   
                 VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE 
               
               
                   
                   
                   
                   
                 MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV 
               
               
                   
                   
                   
                   
                 DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 
               
               
                   
               
               
                   
                 LC 
                 14 
                 VL(CD3)- 
                 DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQQKPGQPPKLLIY 
               
               
                 T3-Design_2- 
                   
                   
                 LCJ2- 
                 WASTRQSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTQSHTLRTFGGGTKVE 
               
               
                 AAAA (IgG1) 
                   
                   
                 CAlpha(T49C) 
                 IKPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQT A VSQSKDSDVYITDK C VLDM 
               
               
                   
                   
                   
                 (N34A + N68A + 
                 RSMDFKSNSAVAWS A KSDFACANAF A NSIIPEDTFFPSPESS 
               
               
                   
                   
                   
                 N79A) 
                   
               
               
                   
                 HC 
                 15 
                 VH(CD3)- 
                 QVQLVQSGAEVKKPGSSVKVSCKASGFAFTDYYIHWVRQAPGQGLEWMGWISPGN 
               
               
                   
                   
                   
                 HCJ2- 
                 VNTKYNENFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDGYSLYYFDYWG 
               
               
                   
                   
                   
                 CBeta(S56C) 
                 QGTLVTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWV 
               
               
                   
                   
                   
                 (N69A)- 
                 NGKEVHSGV C TDPQPLKEQPAL A DSRYALSSRLRVSATFWQNPRNHFRCQVQFYG 
               
               
                   
                   
                   
                 CJ′ 1G1- 
                 LSENDEWTQDRAKPVTQIVSAEAWGRASDKTHTCPPCPAPEAAGGPSVFLFPPKP 
               
               
                   
                   
                   
                 Fc(G1) 
                 KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV 
               
               
                   
                   
                   
                   
                 VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE 
               
               
                   
                   
                   
                   
                 MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV 
               
               
                   
                   
                   
                   
                 DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 
               
               
                   
               
               
                 T3-Design_2- 
                 LC 
                 16 
                 VL(CD3)- 
                 DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQQKPGQPPKLLIY 
               
               
                 QSKE (IgG1) 
                   
                   
                 LCJ2- 
                 WASTRQSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTQSHTLRTFGGGTKVE 
               
               
                   
                   
                   
                 CAlpha(T49C) 
                 IKPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQT Q VSQSKDSDVYITDK C VLDM 
               
               
                   
                   
                   
                 (N34Q + N68S + 
                 RSMDFKSNSAVAWS S KSDFACANAF K NSIIPEDTFFPSPESS 
               
               
                   
                   
                   
                 N79K) 
                   
               
               
                   
                 HC 
                 17 
                 VH(CD3)- 
                 QVQLVQSGAEVKKPGSSVKVSCKASGFAFTDYYIHWVRQAPGQGLEWMGWISPGN 
               
               
                   
                   
                   
                 HCJ2- 
                 VNTKYNENFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDGYSLYYFDYWG 
               
               
                   
                   
                   
                 CBeta(S56C) 
                 QGTLVTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWV 
               
               
                   
                   
                   
                 (N69E)- 
                 NGKEVHSGV C TDPQPLKEQPAL E DSRYALSSRLRVSATFWQNPRNHFRCQVQFYG 
               
               
                   
                   
                   
                 CJ′ 1G1- 
                 LSENDEWTQDRAKPVTQIVSAEAWGRASDKTHTCPPCPAPEAAGGPSVFLFPPKP 
               
               
                   
                   
                   
                 Fc(G1) 
                 KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV 
               
               
                   
                   
                   
                   
                 VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE 
               
               
                   
                   
                   
                   
                 MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV 
               
               
                   
                   
                   
                   
                 DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 
               
               
                   
               
               
                 T3-Design_2- 
                 LC 
                 18 
                 VL(CD3)- 
                 DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQQKPGQPPKLLIY 
               
               
                 ASKE (IgG1) 
                   
                   
                 LCJ2- 
                 WASTRQSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTQSHTLRTFGGGTKVE 
               
               
                   
                   
                   
                 CAlpha(T49C) 
                 IKPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQT A VSQSKDSDVYITDK C VLDM 
               
               
                   
                   
                   
                 (N34A + N68S + 
                 RSMDFKSNSAVAWS S KSDFACANAF K NSIIPEDTFFPSPESS 
               
               
                   
                   
                   
                 N79K) 
                   
               
               
                   
                 HC 
                 17 
                 VH(CD3)- 
                 QVQLVQSGAEVKKPGSSVKVSCKASGFAFTDYYIHWVRQAPGQGLEWMGWISPGN 
               
               
                   
                   
                   
                 HCJ2- 
                 VNTKYNENFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDGYSLYYFDYWG 
               
               
                   
                   
                   
                 CBeta(S56C)(N 
                 QGTLVTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWV 
               
               
                   
                   
                   
                 69E)-CJ′ 1G1 - 
                 NGKEVHSGV C TDPQPLKEQPAL E DSRYALSSRLRVSATFWQNPRNHFRCQVQFYG 
               
               
                   
                   
                   
                 Fc(G1) 
                 LSENDEWTQDRAKPVTQIVSAEAWGRASDKTHTCPPCPAPEAAGGPSVFLFPPKP 
               
               
                   
                   
                   
                   
                 KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV 
               
               
                   
                   
                   
                   
                 VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE 
               
               
                   
                   
                   
                   
                 MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV 
               
               
                   
                   
                   
                   
                 DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 
               
               
                   
               
               
                 T3-Design_2- 
                 LC 
                 19 
                 VL(CD3)- 
                 DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQQKPGQPPKLLIY 
               
               
                 QQQQQ 
                   
                   
                 LCJ2- 
                 WASTRQSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTQSHTLRTFGGGTKVE 
               
               
                 (IgG1) 
                   
                   
                 CAlpha(T49C) 
                 IKPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQT Q VSQSKDSDVYITDK C VLDM 
               
               
                   
                   
                   
                 (N34Q + N68Q + 
                 RSMDFKS Q SAVAWS Q KSDFACANAF Q NSIIPEDTFFPSPESS 
               
               
                   
                   
                   
                 N79Q + N61Q) 
                   
               
               
                   
                 HC 
                 20 
                 VH(CD3)- 
                 QVQLVQSGAEVKKPGSSVKVSCKASGFAFTDYYIHWVRQAPGQGLEWMGWISPGN 
               
               
                   
                   
                   
                 HCJ2- 
                 VNTKYNENFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDGYSLYYFDYWG 
               
               
                   
                   
                   
                 CBeta(S56C) 
                 QGTLVTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWV 
               
               
                   
                   
                   
                 (N69Q)- 
                 NGKEVHSGV C TDPQPLKEQPAL Q DSRYALSSRLRVSATFWQNPRNHFRCQVQFYG 
               
               
                   
                   
                   
                 CJ′ 1G1- 
                 LSENDEWTQDRAKPVTQIVSAEAWGRASDKTHTCPPCPAPEAAGGPSVFLFPPKP 
               
               
                   
                   
                   
                 Fc(G1) 
                 KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV 
               
               
                   
                   
                   
                   
                 VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE 
               
               
                   
                   
                   
                   
                 MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV 
               
               
                   
                   
                   
                   
                 DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 
               
               
                   
               
               
                 T3-Design_2- 
                 LC 
                 12 
                   
                 DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQQKPGQPPKLLIY 
               
               
                 QQQQ (IgG4) 
                   
                   
                   
                 WASTRQSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTQSHTLRTFGGGTKVE 
               
               
                   
                   
                   
                   
                 IKPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQT Q VSQSKDSDVYITDK C VLDM 
               
               
                   
                   
                   
                   
                 RSMDFKSNSAVAWS Q KSDFACANAF Q NSIIPEDTFFPSPESS 
               
               
                   
                 HC 
                 21 
                 VH(CD3)- 
                 QVQLVQSGAEVKKPGSSVKVSCKASGFAFTDYYIHWVRQAPGQGLEWMGWISPGN 
               
               
                   
                   
                   
                 HCJ2- 
                 VNTKYNENFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDGYSLYYFDYWG 
               
               
                   
                   
                   
                 CBeta(S56C) 
                 QGTLVTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWV 
               
               
                   
                   
                   
                 (N69Q)- 
                 NGKEVHSGV C TDPQPLKEQPAL Q DSRYALSSRLRVSATFWQNPRNHFRCQVQFYG 
               
               
                   
                   
                   
                 CJ′ 1G4- 
                 LSENDEWTQDRAKPVTQIVSAEAWGRYGPPCPPCPAPEFLGGPSVFLFPPKPKDT 
               
               
                   
                   
                   
                 Fc(G4) 
                 LMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSV 
               
               
                   
                   
                   
                   
                 LTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTK 
               
               
                   
                   
                   
                   
                 NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKS 
               
               
                   
                   
                   
                   
                 RWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 
               
               
                   
               
               
                 E17-Design_2- 
                 T3-LC 
                 12 
                 VL(CD3)- 
                 DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQQKPGQPPKLLIY 
               
               
                 QQQQ (IgG1) 
                   
                   
                 LCJ2- 
                 WASTRQSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTQSHTLRTFGGGTKVE 
               
               
                   
                   
                   
                 CAlpha(T49C) 
                 IKPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQT Q VSQSKDSDVYITDK C VLDM 
               
               
                   
                   
                   
                 (N34Q + N68Q + 
                 RSMDFKSNSAVAWS Q KSDFACANAF Q NSIIPEDTFFPSPESS 
               
               
                   
                   
                   
                 N79Q) 
                   
               
               
                   
                 T3-HC 
                 22 
                 VH(CD3)- 
                 QVQLVQSGAEVKKPGSSVKVSCKASGFAFTDYYIHWVRQAPGQGLEWMGWISPGN 
               
               
                   
                   
                   
                 HCJ2- 
                 VNTKYNENFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDGYSLYYFDYWG 
               
               
                   
                   
                   
                 CBeta(S56C) 
                 QGTLVTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWV 
               
               
                   
                   
                   
                 (N69Q)- 
                 NGKEVHSGV C TDPQPLKEQPAL Q DSRYALSSRLRVSATFWQNPRNHFRCQVQFYG 
               
               
                   
                   
                   
                 CJ′ 1G1- 
                 LSENDEWTQDRAKPVTQIVSAEAWGRASDKTHTCPPCPAPEAAGGPSVFLFPPKP 
               
               
                   
                   
                   
                 Fc(G1)(Knob) 
                 KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV 
               
               
                   
                   
                   
                   
                 VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP C REE 
               
               
                   
                   
                   
                   
                 MTKNQVSL W CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV 
               
               
                   
                   
                   
                   
                 DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 
               
               
                   
                 U4-LC 
                 23 
                 VL(CD19)-CL 
                 DIQLTQSPSFLSASVGDRVTITCSASSTVNYMHWYQQKPGKAPKLLIYSTSNLAS 
               
               
                   
                   
                   
                   
                 GVPSRFSGSGSGTEFTLTISSLQPEDFATYYCHQWSSYPYTFGQGTKLEIKRTVA 
               
               
                   
                   
                   
                   
                 APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ 
               
               
                   
                   
                   
                   
                 DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 
               
               
                   
                 U4-HC 
                 24 
                 VH(CD19)- 
                 QMQLVQSGPEVKKPGTSVKVSCKASGYAFTSYNMYWVRQARGQRLEWIGYIDPYN 
               
               
                   
                   
                   
                 CH1-Fc(G1) 
                 GDTTYNQKFKGRVTITRDMSTSTAYMELSSLRSEDTAVYYCLTTAYAMDYWGQGT 
               
               
                   
                   
                   
                 (Hole) 
                 LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG 
               
               
                   
                   
                   
                   
                 VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCD 
               
               
                   
                   
                   
                   
                 KTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN 
               
               
                   
                   
                   
                   
                 WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP 
               
               
                   
                   
                   
                   
                 IEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQ 
               
               
                   
                   
                   
                   
                 PENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL 
               
               
                   
                   
                   
                   
                 SLSPGK 
               
               
                   
               
               
                 E17-Design_2- 
                 T3-LC 
                 12 
                 VL(CD3)- 
                 DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQQKPGQPPKLLIY 
               
               
                 QQQQ (IgG4) 
                   
                   
                 LCJ2- 
                 WASTRQSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTQSHTLRTFGGGTKVE 
               
               
                   
                   
                   
                 CAlpha(T49C) 
                 IKPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQT Q VSQSKDSDVYITDK C VLDM 
               
               
                   
                   
                   
                 (N34Q + N68Q + 
                 RSMDFKSNSAVAWS Q KSDFACANAF Q NSIIPEDTFFPSPESS 
               
               
                   
                   
                   
                 N79Q) 
                   
               
               
                   
                 T3-HC 
                 25 
                 VH(CD3)- 
                 QVQLVQSGAEVKKPGSSVKVSCKASGFAFTDYYIHWVRQAPGQGLEWMGWISPGN 
               
               
                   
                   
                   
                   
                 VNTKYNENFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDGYSLYYFDYWG 
               
               
                   
                   
                   
                 HCJ2- 
                 QGTLVTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWV 
               
               
                   
                   
                   
                 CBeta(S56C) 
                 NGKEVHSGV C TDPQPLKEQPAL Q DSRYALSSRLRVSATFWQNPRNHFRCQVQFYG 
               
               
                   
                   
                   
                 (N69Q)- 
                 LSENDEWTQDRAKPVTQIVSAEAWGRYGPPCPPCPAPEFLGGPSVFLFPPKPKDT 
               
               
                   
                   
                   
                 CJ′ 1G4- 
                 LMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSV 
               
               
                   
                   
                   
                 Fc(G4)(Knob)  
                 LTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPP C QEEMTK 
               
               
                   
                   
                   
                   
                 NQVSL W CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKS 
               
               
                   
                   
                   
                   
                 RWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 
               
               
                   
                 U4-LC 
                 23 
                 VL(CD19)-CL 
                 DIQLTQSPSFLSASVGDRVTITCSASSTVNYMHWYQQKPGKAPKLLIYSTSNLAS 
               
               
                   
                   
                   
                   
                 GVPSRFSGSGSGTEFTLTISSLQPEDFATYYCHQWSSYPYTFGQGTKLEIKRTVA 
               
               
                   
                   
                   
                   
                 APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ 
               
               
                   
                   
                   
                   
                 DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 
               
               
                   
                 U4-HC 
                 26 
                 VH(CD19)- 
                 QMQLVQSGPEVKKPGTSVKVSCKASGYAFTSYNMYWVRQARGQRLEWIGYIDPYN 
               
               
                   
                   
                   
                 CH1- 
                 GDTTYNQKFKGRVTITRDMSTSTAYMELSSLRSEDTAVYYCLTTAYAMDYWGQGT 
               
               
                   
                   
                   
                 Fc(G4)(Hole) 
                 LVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSG 
               
               
                   
                   
                   
                   
                 VHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGP 
               
               
                   
                   
                   
                   
                 PCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV 
               
               
                   
                   
                   
                   
                 DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEK 
               
               
                   
                   
                   
                   
                 TISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPEN 
               
               
                   
                   
                   
                   
                 NYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLS 
               
               
                   
                   
                   
                   
                 LGK 
               
               
                   
               
               
                 F16-Design_2- 
                 T3-LC 
                 12 
                   
                 DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQQKPGQPPKLLIY 
               
               
                 QQQQ (IgG4) 
                   
                   
                   
                 WASTRQSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTQSHTLRTFGGGTKVE 
               
               
                   
                   
                   
                   
                 IKPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQT Q VSQSKDSDVYITDK C VLDM 
               
               
                   
                   
                   
                   
                 RSMDFKSNSAVAWS Q KSDFACANAF Q NSIIPEDTFFPSPESS 
               
               
                   
                 T3-HC 
                 25 
                   
                 QVQLVQSGAEVKKPGSSVKVSCKASGFAFTDYYIHWVRQAPGQGLEWMGWISPGN 
               
               
                   
                   
                   
                   
                 VNTKYNENFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDGYSLYYFDYWG 
               
               
                   
                   
                   
                   
                 QGTLVTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWV 
               
               
                   
                   
                   
                   
                 NGKEVHSGV C TDPQPLKEQPAL Q DSRYALSSRLRVSATFWQNPRNHFRCQVQFYG 
               
               
                   
                   
                   
                   
                 LSENDEWTQDRAKPVTQIVSAEAWGRYGPPCPPCPAPEFLGGPSVFLFPPKPKDT 
               
               
                   
                   
                   
                   
                 LMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSV 
               
               
                   
                   
                   
                   
                 LTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPP C QEEMTK 
               
               
                   
                   
                   
                   
                 NQVSL W CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKS 
               
               
                   
                   
                   
                   
                 RWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 
               
               
                   
               
               
                   
                 U4-LC 
                 23 
                 VL(CD19)-CL 
                 DIQLTQSPSFLSASVGDRVTITCSASSTVNYMHWYQQKPGKAPKLLIYSTSNLAS 
               
               
                   
                   
                   
                   
                 GVPSRFSGSGSGTEFTLTISSLQPEDFATYYCHQWSSYPYTFGQGTKLEIKRTVA 
               
               
                   
                   
                   
                   
                 APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ 
               
               
                   
                   
                   
                   
                 DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 
               
               
                   
                 U4-HC 
                 27 
                 VH(CD19)- 
                 QMQLVQSGPEVKKPGTSVKVSCKASGYAFTSYNMYWVRQARGQRLEWIGYIDPYN 
               
               
                   
                   
                   
                 CH1- Spacer- 
                 GDTTYNQKFKGRVTITRDMSTSTAYMELSSLRSEDTAVYYCLTTAYAMDYWGQGT 
               
               
                   
                   
                   
                 VH(CD19)- 
                 LVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSG 
               
               
                   
                   
                   
                 CH1- Fc(G4) 
                 VHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVGGGGSG 
               
               
                   
                   
                   
                 (Hole) 
                 GGGSQMQLVQSGPEVKKPGTSVKVSCKASGYAFTSYNMYWVRQARGQRLEWIGYI 
               
               
                   
                   
                   
                   
                 DPYNGDTTYNQKFKGRVTITRDMSTSTAYMELSSLRSEDTAVYYCLTTAYAMDYW 
               
               
                   
                   
                   
                   
                 GQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGA 
               
               
                   
                   
                   
                   
                 LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVES 
               
               
                   
                   
                   
                   
                 KYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQF 
               
               
                   
                   
                   
                   
                 NWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPS 
               
               
                   
                   
                   
                   
                 SIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNG 
               
               
                   
                   
                   
                   
                 QPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKS 
               
               
                   
                   
                   
                   
                 LSLSLGK 
               
               
                   
               
               
                 Fc-IgG1 
                   
                 304 
                   
                 SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE 
               
               
                 (knob) 
                   
                   
                   
                 EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV 
               
               
                   
                   
                   
                   
                 YTLPP C REEMTKNQVSL W CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS 
               
               
                   
                   
                   
                   
                 FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 
               
               
                   
               
               
                 Fc-IgG4 
                   
                 305 
                   
                 SVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPRE 
               
               
                 (knob) 
                   
                   
                   
                 EQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQV 
               
               
                   
                   
                   
                   
                 YTLPP C QEEMTKNQVSL W CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS 
               
               
                   
                   
                   
                   
                 FFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 
               
               
                   
               
               
                 T3-Fab- 
                 LC 
                 3 
                 VL(CD3)- 
                 DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQQKPGQPPKLLIY 
               
               
                 Design_2.his1 
                   
                   
                 LCJ2- 
                 WASTRQSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTQSHTLRTFGGGTKVE 
               
               
                   
                   
                   
                 CAlpha(T49C) 
                 IKPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQT N VSQSKDSDVYITDK C VLDM 
               
               
                   
                   
                   
                   
                 RSMDFKSNSAVAWS N KSDFACANAF N NSIIPEDTFFPSPESS 
               
               
                   
                 HC 
                 28 
                 VH(CD3)- 
                 QVQLVQSGAEVKKPGSSVKVSCKASGFAFTDYYIHWVRQAPGQGLEWMGWISPGN 
               
               
                   
                   
                   
                 HCJ2- 
                 VNTKYNENFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDGYSLYYFDYWG 
               
               
                   
                   
                   
                 CBeta(556C)C 
                 QGTLVTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWV 
               
               
                   
                   
                   
                 onjunction′ 
                 NGKEVHSGV C TDPQPLKEQPAL N DSRYALSSRLRVSATFWQNPRNHFRCQVQFYG 
               
               
                   
                   
                   
                   
                 LSENDEWTQDRAKPVTQIVSAEAWGR 
               
               
                   
               
               
                 T3-Fab- 
                 LC 
                 3 
                   
                 DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQQKPGQPPKLLIY 
               
               
                 Design_2.his2 
                   
                   
                   
                 WASTRQSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTQSHTLRTFGGGTKVE 
               
               
                   
                   
                   
                   
                 IKPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQT N VSQSKDSDVYITDK C VLDM 
               
               
                   
                   
                   
                   
                 RSMDFKSNSAVAWS N KSDFACANAF N NSIIPEDTFFPSPESS 
               
               
                   
                 HC 
                 29 
                 VH(CD3)- 
                 QVQLVQSGAEVKKPGSSVKVSCKASGFAFTDYYIHWVRQAPGQGLEWMGWISPGN 
               
               
                   
                   
                   
                 HCJ2- 
                 VNTKYNENFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDGYSLYYFDYWG 
               
               
                   
                   
                   
                 CBeta(S56C)C 
                 QGTLVTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWV 
               
               
                   
                   
                   
                 onjunction′ 
                 NGKEVHSGV C TDPQPLKEQPAL N DSRYALSSRLRVSATFWQNPRNHFRCQVQFYG 
               
               
                   
                   
                   
                   
                 LSENDEWTQDRAKPVTQIVSAEAWGRAD 
               
               
                   
               
               
                 T3-Fab- 
                 LC 
                 12 
                 VL(CD3)- 
                 DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQQKPGQPPKLLIY 
               
               
                 Design_2- 
                   
                   
                 LCJ2- 
                 WASTRQSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTQSHTLRTFGGGTKVE 
               
               
                 QQQQ.his1 
                   
                   
                 CAlpha(T49C) 
                 IKPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQT Q VSQSKDSDVYITDK C VLDM 
               
               
                   
                   
                   
                 (N34Q + N68Q + 
                 RSMDFKSNSAVAWS Q KSDFACANAF Q NSIIPEDTFFPSPESS 
               
               
                   
                   
                   
                 N79Q) 
                   
               
               
                   
                 HC 
                 30 
                 VH(CD3)- 
                 QVQLVQSGAEVKKPGSSVKVSCKASGFAFTDYYIHWVRQAPGQGLEWMGWISPGN 
               
               
                   
                   
                   
                 HCJ2- 
                 VNTKYNENFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDGYSLYYFDYWG 
               
               
                   
                   
                   
                 CBeta(S56C) 
                 QGTLVTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWV 
               
               
                   
                   
                   
                 (N69Q)-His1 
                 NGKEVHSGV C TDPQPLKEQPAL Q DSRYALSSRLRVSATFWQNPRNHFRCQVQFYG 
               
               
                   
                   
                   
                   
                 LSENDEWTQDRAKPVTQIVSAEAWGR 
               
               
                   
               
               
                 T3-Fab- 
                 LC 
                 12 
                   
                 DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQQKPGQPPKLLIY 
               
               
                 Design_2- 
                   
                   
                   
                 WASTRQSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTQSHTLRTFGGGTKVE 
               
               
                 QQQQ.his2 
                   
                   
                   
                 IKPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQT Q VSQSKDSDVYITDK C VLDM 
               
               
                   
                   
                   
                   
                 RSMDFKSNSAVAWS Q KSDFACANAF Q NSIIPEDTFFPSPESS 
               
               
                   
                 HC 
                 31 
                 VH(CD3)- 
                 QVQLVQSGAEVKKPGSSVKVSCKASGFAFTDYYIHWVRQAPGQGLEWMGWISPGN 
               
               
                   
                   
                   
                 HCJ2- 
                 VNTKYNENFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDGYSLYYFDYWG 
               
               
                   
                   
                   
                 CBeta(S56C) 
                 QGTLVTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWV 
               
               
                   
                   
                   
                 (N69Q)-His2 
                 NGKEVHSGV C TDPQPLKEQPAL Q DSRYALSSRLRVSATFWQNPRNHFRCQVQFYG 
               
               
                   
                   
                   
                   
                 LSENDEWTQDRAKPVTQIVSAEAWGRAD 
               
               
                   
               
               
                 CBeta 
                 CBeta_1_n 
                 32 
                 CBeta (C74A) 
                 EVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGV S TDPQPLK 
               
               
                   
                 oCys 
                   
                   
                 EQPAL N DSRY A LSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQ 
               
               
                   
                   
                   
                   
                 IVSAEA 
               
               
                   
                 CBeta_1 
                 33 
                 CBeta(S56C) 
                 EVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGV C TDPQPLK 
               
               
                   
                   
                   
                 (C74A) 
                 EQPAL N DSRY A LSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQ 
               
               
                   
                   
                   
                   
                 IVSAEA 
               
               
                   
                 CBeta_1-Q 
                 34 
                 CBeta(S56C) 
                 EVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGV C TDPQPLK 
               
               
                   
                   
                   
                 (N69Q)(C74A)  
                 EQPAL Q DSRY A LSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQ 
               
               
                   
                   
                   
                   
                 IVSAEA 
               
               
                   
                 CBeta_1-A 
                 35 
                 CBeta(S56C) 
                 EVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGV C TDPQPLK 
               
               
                   
                   
                   
                 (N69A)(C74A)  
                 EQPAL A DSRY A LSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQ 
               
               
                   
                   
                   
                   
                 IVSAEA 
               
               
                   
                 CBeta_1-E 
                 36 
                 CBeta(S56C) 
                 EVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGV C TDPQPLK 
               
               
                   
                   
                   
                 (N69E)(C74A)  
                 EQPAL E DSRY A LSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQ 
               
               
                   
                   
                   
                   
                 IVSAEA 
               
               
                   
                 CBeta_2 
                 37 
                 CBeta(S56C)(F 
                 EVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGV C TDPQPLK 
               
               
                   
                   
                   
                 G-)(C74A) 
                 EQPAL N DSRY A LSSRLRVSATFWQNPRNHFRCQVQFYPSN-------------- 
               
               
                   
                   
                   
                   
                 QIVSAEA 
               
               
                   
                 CBeta_2-Q 
                 38 
                 CBeta(S56C) 
                 EVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGV C TDPQPLK 
               
               
                   
                   
                   
                 (N69Q)(FG-) 
                 EQPAL Q DSRY A LSSRLRVSATFWQNPRNHFRCQVQFYPSN-------------- 
               
               
                   
                   
                   
                 (C74A) 
                 QIVSAEA 
               
               
                   
                 CBeta_2-A 
                 39 
                 CBeta(S56C) 
                 EVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGV C TDPQPLK 
               
               
                   
                   
                   
                 (N69A)(FG-) 
                 EQPAL A DSRY A LSSRLRVSATFWQNPRNHFRCQVQFYPSN-------------- 
               
               
                   
                   
                   
                 (C74A) 
                 QIVSAEA 
               
               
                   
                 CBeta_2-E 
                 40 
                 CBeta(S56C) 
                 EVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGV C TDPQPLK 
               
               
                   
                   
                   
                 (N69E)(FG-) 
                 EQPAL E DSRY A LSSRLRVSATFWQNPRNHFRCQVQFYPSN-------------- 
               
               
                   
                   
                   
                 (C74A) 
                 QIVSAEA 
               
               
                   
                 CBeta_3 
                 41 
                 CBeta(S56C)(F 
                 EVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGV C TDPQPLK 
               
               
                   
                   
                   
                 G-DE-)(C74A)  
                 EQ---S-GRY A LSSRLRVSATFWQNPRNHFRCQVQFYPSN-------------- 
               
               
                   
                   
                   
                   
                 QIVSAEA 
               
               
                   
               
               
                 CAlpha 
                 CAlpha_1 
                 42 
                 CAlpha 
                 AVYQLRDSKSSDKSVCLFTDFDSQT N VSQSKDSDVYITDK T VLDMRSMDFKSNSA 
               
               
                   
                 noCys 
                   
                   
                 VAWS N KSDFACANAF N NSIIPEDTFFPSPESS 
               
               
                   
                 CAlpha_1 
                 43 
                 CAlpha(T49C) 
                 AVYQLRDSKSSDKSVCLFTDFDSQT N VSQSKDSDVYITDK C VLDMRSMDFKSNSA 
               
               
                   
                   
                   
                   
                 VAWS N KSDFACANAF N NSIIPEDTFFPSPESS 
               
               
                   
                 CAlpha_1- 
                 44 
                 CAlpha(T49C) 
                 AVYQLRDSKSSDKSVCLFTDFDSQT Q VSQSKDSDVYITDK C VLDMRSMDFKSNSA 
               
               
                   
                 QQQ 
                   
                 (N34Q + N68Q + 
                 VAWS Q KSDFACANAF Q NSIIPEDTFFPSPESS 
               
               
                   
                   
                   
                 N79Q) 
                   
               
               
                   
                 CAlpha_1- 
                 45 
                 CAlpha(T49C) 
                 AVYQLRDSKSSDKSVCLFTDFDSQT A VSQSKDSDVYITDK C VLDMRSMDFKSNSA 
               
               
                   
                 AAA 
                   
                 (N34A + N68A + 
                 VAWS A KSDFACANAF A NSIIPEDTFFPSPESS 
               
               
                   
                   
                   
                 N79A) 
                   
               
               
                   
                 CAlpha_1- 
                 46 
                 CAlpha(T49C) 
                 AVYQLRDSKSSDKSVCLFTDFDSQT Q VSQSKDSDVYITDK C VLDMRSMDFKSNSA 
               
               
                   
                 QSK 
                   
                 (N34Q + N68S + 
                 VAWS S KSDFACANAF K NSIIPEDTFFPSPESS 
               
               
                   
                   
                   
                 N79K) 
                   
               
               
                   
                 CAlpha_1- 
                 47 
                 CAlpha(T49C) 
                 AVYQLRDSKSSDKSVCLFTDFDSQT A VSQSKDSDVYITDK C VLDMRSMDFKSNSA 
               
               
                   
                 ASK 
                   
                 (N34A + N685 + 
                 VAWS S KSDFACANAF K NSIIPEDTFFPSPESS 
               
               
                   
                   
                   
                 N79K) 
                   
               
               
                   
                 CAlpha_1- 
                 48 
                 CAlpha(T49C) 
                 AVYQLRDSKSSDKSVCLFTDFDSQT Q VSQSKDSDVYITDK C VLDMRSMDFKS Q SA 
               
               
                   
                 QQQQ 
                   
                 (N34Q + N68Q + 
                 VAWS Q KSDFACANAF Q NSIIPEDTFFPSPESS 
               
               
                   
                   
                   
                 N79Q + N61Q) 
                   
               
               
                   
               
               
                 ConjunctionX 
                 HConjunct 
                 49 
                 VH-CBetaCJ1 
                 SSASKNVFPP 
               
               
                   
                 ion_1 
                   
                   
                   
               
               
                   
                 HConjunct 
                 50 
                 VH-CBetaCJ2 
                 LEDLKNVFPP 
               
               
                   
                 ion_2 
                   
                   
                   
               
               
                   
               
               
                 ConjunctionZ 
                 LConjuncti 
                 51 
                 VL-CAlphaCJ1 
                 KRTVAAPDP 
               
               
                   
                 on_1 
                   
                   
                   
               
               
                   
                 LConjuncti 
                 52 
                 VL-CAlphaCJ2 
                 KPDIQNPDP 
               
               
                   
                 on_2 
                   
                   
                   
               
               
                   
               
               
                 Conjunction′ 
                 Conjunction 
                 53 
                 CBeta- 
                 WGRASDKTHTCPPCPAPEAAGGP 
               
               
                 Y 
                 ′IgG1 
                   
                 Conjunction′ 
                   
               
               
                   
                   
                   
                 (IgG1)CJ1 
                   
               
               
                   
                 Conjunction 
                 54 
                 CBeta- 
                 WGR---YGPPCPPCPAPEFLGGP 
               
               
                   
                 ′ IgG4 
                   
                 Conjunction′ 
                   
               
               
                   
                   
                   
                 (IgG4)CJ1 
                   
               
               
                   
               
               
                   
                   
                 307 
                   
                 FE 
               
               
                   
               
               
                   
                   
                 308 
                 VH-CBetaCJ2 
                   K LEDLKNVFPP 
               
               
                   
               
               
                   
                   
                 309 
                 VL-Cpre- 
                   K PTGVGGTP 
               
               
                   
                   
                   
                 AlphaCJB 
                   
               
               
                   
               
               
                   
                   
                 306 
                 Cter (crossed 
                 WGRA 
               
               
                   
                   
                   
                 light chain 
                   
               
               
                   
                   
                   
                 cter) 
               
               
                   
               
               
                   
                   
                 SEQ 
                   
                   
               
               
                 PreAlpha- 
                 Chain 
                 ID 
                   
                   
               
               
                 Beta Designs 
                 Name 
                 No 
                   
                 Sequences 
               
               
                   
               
               
                 Design_1_Pre 
                 LC 
                 64 
                 VL(CD3)- 
                 DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQQKPGQPPKLLIY 
               
               
                 _TCR_Conju 
                   
                   
                 LCJB- 
                 WASTRQSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTQSHTLRTFGGGTKVE 
               
               
                 nction′1 
                   
                   
                 CPreAlpha(N50 
                 I KPTGVGGTPFP S   LAPPIMLLVDGKQQMVVVCLVLDVAPPGLDSPIWFSAG Q GSA 
               
               
                   
                   
                   
                 Q) 
                 LDAFTYGPSPATDGTWTNLAHLSLPSEELASWEPLVCHTGPGAEGHSRSTQPMHL 
               
               
                   
                   
                   
                   
                 SGEASTART 
               
               
                   
                 HC 
                 65 
                 VH(CD3)- 
                 QVQLVQSGAEVKKPGSSVKVSCKASGFAFTDYYIHWVRQAPGQGLEWMGWISPGN 
               
               
                   
                   
                   
                 HCJB- 
                 VNTKYNENFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDGYSLYYFDYWG 
               
               
                   
                   
                   
                 CB eta(N69Q)- 
                 QGTLVTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWV 
               
               
                   
                   
                   
                 CJ′ 1G1- 
                 NGKEVHSGV S TDPQPLKEQPAL Q DSRYAL S SRLRVSATFWQNPRNHFRCQVQFYG 
               
               
                   
                   
                   
                 Fc(G1) 
                 LSENDEWTQDRAKPVTQIVSAEAWGRASDKTHTCPPCPAPEAAGGPSVFLFPPKP 
               
               
                   
                   
                   
                   
                 KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV 
               
               
                   
                   
                   
                   
                 VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE 
               
               
                   
                   
                   
                   
                 MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV 
               
               
                   
                   
                   
                   
                 DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 
               
               
                   
               
               
                 Design_2_Pre 
                 LC 
                 66 
                 VL(CD3)- 
                 DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQQKPGQPPKLLIY 
               
               
                 _TCR_Conju 
                   
                   
                 LCJB- 
                 WASTRQSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTQSHTLRTFGGGTKVE 
               
               
                 nction′1_Cys1 
                   
                   
                 CPreAlpha 
                 I KPTGVGGTPFPS LAPPIMLLVDGKQQMVVVCLVLDVAPPGLDSPIWFSAG Q GSA 
               
               
                 0 
                   
                   
                 (Y59C)(N50Q) 
                 LDAFT C GPSPATDGTWTNLAHLSLPSEELASWEPLVCHTGPGAEGHSRSTQPMHL 
               
               
                   
                   
                   
                   
                 SGEASTART 
               
               
                   
                 HC 
                 67 
                 VH(CD3)- 
                 QVQLVQSGAEVKKPGSSVKVSCKASGFAFTDYYIHWVRQAPGQGLEWMGWISPGN 
               
               
                   
                   
                   
                 HCJB- 
                 VNTKYNENFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDGYSLYYFDYWG 
               
               
                   
                   
                   
                 CBeta(S76C)  
                 QGTLVTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWV 
               
               
                   
                   
                   
                 (N69Q)- 
                 NGKEVHSGVSTDPQPLKEQPAL Q DSRYAL C SRLRVSATFWQNPRNHFRCQVQFYG 
               
               
                   
                   
                   
                 CJ′ 1G1- 
                 LSENDEWTQDRAKPVTQIVSAEAWGRASDKTHTCPPCPAPEAAGGPSVFLFPPKP 
               
               
                   
                   
                   
                 Fc(G1) 
                 KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV 
               
               
                   
                   
                   
                   
                 VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE 
               
               
                   
                   
                   
                   
                 MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV 
               
               
                   
                   
                   
                   
                 DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 
               
               
                   
               
               
                 Design_3_Pre 
                 LC 
                 68 
                 VL(CD3)- 
                 DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQQKPGQPPKLLIY 
               
               
                 _TCR_Conju 
                   
                   
                 LCJB- 
                 WASTRQSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTQSHTLRTFGGGTKVE 
               
               
                 nction′1_Cys1 
                   
                   
                 CPreAlpha 
                 I KPTGVGGTPFPS L C PPIMLLVDGKQQMVVVCLVLDVAPPGLDSPIWFSAG Q GSA 
               
               
                 1 
                   
                   
                 (A13C)(N50Q) 
                 LDAFTYGPSPATDGTWTNLAHLSLPSEELASWEPLVCHTGPGAEGHSRSTQPMHL 
               
               
                   
                   
                   
                   
                 SGEASTART 
               
               
                   
                 HC 
                 69 
                 VH(CD3)- 
                 QVQLVQSGAEVKKPGSSVKVSCKASGFAFTDYYIHWVRQAPGQGLEWMGWISPGN 
               
               
                   
                   
                   
                 HCJB- 
                 VNTKYNENFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDGYSLYYFDYWG 
               
               
                   
                   
                   
                 CBeta(F13C) 
                 QGTLVTVLEDLKNVFPPEVAV C EPSEAEISHTQKATLVCLATGFYPDHVELSWWV 
               
               
                   
                   
                   
                 (N69Q)- 
                 NGKEVHSGVSTDPQPLKEQPAL Q DSRYALSSRLRVSATFWQNPRNHFRCQVQFYG 
               
               
                   
                   
                   
                 CJ′ 1G1- 
                 LSENDEWTQDRAKPVTQIVSAEAWGRASDKTHTCPPCPAPEAAGGPSVFLFPPKP 
               
               
                   
                   
                   
                 Fc(G1) 
                 KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV 
               
               
                   
                   
                   
                   
                 VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE 
               
               
                   
                   
                   
                   
                 MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV 
               
               
                   
                   
                   
                   
                 DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 
               
               
                   
               
               
                 Design_4_Pre 
                 LC 
                 68 
                 VL(CD3)- 
                 DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQQKPGQPPKLLIY 
               
               
                 _TCR_Conju 
                   
                   
                 LCJB- 
                 WASTRQSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTQSHTLRTFGGGTKVE 
               
               
                 nction′1_Cys1 
                   
                   
                 CPreAlpha 
                 I KPTGVGGTPFPS L C PPIMLLVDGKQQMVVVCLVLDVAPPGLDSPIWFSAG Q GSA 
               
               
                 2 
                   
                   
                 (A13 C)(N50Q) 
                 LDAFTYGPSPATDGTWTNLAHLSLPSEELASWEPLVCHTGPGAEGHSRSTQPMHL 
               
               
                   
                   
                   
                   
                 SGEASTART 
               
               
                   
                 HC 
                 70 
                 VH(CD3)- 
                 QVQLVQSGAEVKKPGSSVKVSCKASGFAFTDYYIHWVRQAPGQGLEWMGWISPGN 
               
               
                   
                   
                   
                 HCJB- 
                 VNTKYNENFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDGYSLYYFDYWG 
               
               
                   
                   
                   
                 CBeta(S16C) 
                 QGTLVTVLEDLKNVFPPEVAVFEP C EAEISHTQKATLVCLATGFYPDHVELSWWV 
               
               
                   
                   
                   
                 (N69Q)- 
                 NGKEVHSGVSTDPQPLKEQPAL Q DSRYALSSRLRVSATFWQNPRNHFRCQVQFYG 
               
               
                   
                   
                   
                 CJ′ 1G1- 
                 LSENDEWTQDRAKPVTQIVSAEAWGRASDKTHTCPPCPAPEAAGGPSVFLFPPKP 
               
               
                   
                   
                   
                 Fc(G1) 
                 KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV 
               
               
                   
                   
                   
                   
                 VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE 
               
               
                   
                   
                   
                   
                 MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV 
               
               
                   
                   
                   
                   
                 DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 
               
               
                   
               
               
                 Design_5_Pre 
                 LC 
                 71 
                 VL(CD3)- 
                 DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQQKPGQPPKLLIY 
               
               
                 _TCR_Conju 
                   
                   
                 LCJB- 
                 WASTRQSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTQSHTLRTFGGGTKVE 
               
               
                 nction′1_Cys1 
                   
                   
                 CPreAlpha 
                 I KPTGVGGTPFP   C LAPPIMLLVDGKQQMVVVCLVLDVAPPGLDSPIWFSAG Q GSA 
               
               
                 3 
                   
                   
                 (S11C)(N50Q) 
                 LDAFTYGPSPATDGTWTNLAHLSLPSEELASWEPLVCHTGPGAEGHSRSTQPMHL 
               
               
                   
                   
                   
                   
                 SGEASTART 
               
               
                   
                 HC 
                 70 
                 VH(CD3)- 
                 QVQLVQSGAEVKKPGSSVKVSCKASGFAFTDYYIHWVRQAPGQGLEWMGWISPGN 
               
               
                   
                   
                   
                 HCJB- 
                 VNTKYNENFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDGYSLYYFDYWG 
               
               
                   
                   
                   
                 CBeta(S16C) 
                 QGTLVTVLEDLKNVFPPEVAVFEP C EAEISHTQKATLVCLATGFYPDHVELSWWV 
               
               
                   
                   
                   
                 (N69Q)- 
                 NGKEVHSGVSTDPQPLKEQPAL Q DSRYALSSRLRVSATFWQNPRNHFRCQVQFYG 
               
               
                   
                   
                   
                 CJ′ 1G1- 
                 LSENDEWTQDRAKPVTQIVSAEAWGRASDKTHTCPPCPAPEAAGGPSVFLFPPKP 
               
               
                   
                   
                   
                 Fc(G1) 
                 KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV 
               
               
                   
                   
                   
                   
                 VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE 
               
               
                   
                   
                   
                   
                 MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV 
               
               
                   
                   
                   
                   
                 DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 
               
               
                   
               
               
                 Design_6_Pre 
                 LC 
                 71 
                 VL(CD3)- 
                 DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQQKPGQPPKLLIY 
               
               
                 _TCR_Conju 
                   
                   
                 LCJB- 
                 WASTRQSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTQSHTLRTFGGGTKVE 
               
               
                 nction′1_Cys1 
                   
                   
                 CPreAlpha 
                 I KPTGVGGTPFP   C LAPPIMLLVDGKQQMVVVCLVLDVAPPGLDSPIWFSAG Q GSA 
               
               
                 4 
                   
                   
                 (S11C)(N50Q) 
                 LDAFTYGPSPATDGTWTNLAHLSLPSEELASWEPLVCHTGPGAEGHSRSTQPMHL 
               
               
                   
                   
                   
                   
                 SGEASTART 
               
               
                   
                 HC 
                 72 
                 VH(CD3)- 
                 QVQLVQSGAEVKKPGSSVKVSCKASGFAFTDYYIHWVRQAPGQGLEWMGWISPGN 
               
               
                   
                   
                   
                 HCJB- 
                 VNTKYNENFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDGYSLYYFDYWG 
               
               
                   
                   
                   
                 CBeta(A18C) 
                 QGTLVTVLEDLKNVFPPEVAVFEPSE C EISHTQKATLVCLATGFYPDHVELSWWV 
               
               
                   
                   
                   
                 (N69Q)- 
                 NGKEVHSGVSTDPQPLKEQPAL Q DSRYALSSRLRVSATFWQNPRNHFRCQVQFYG 
               
               
                   
                   
                   
                 CJ′ 1G1- 
                 LSENDEWTQDRAKPVTQIVSAEAWGRASDKTHTCPPCPAPEAAGGPSVFLFPPKP 
               
               
                   
                   
                   
                 Fc(G1) 
                 KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV 
               
               
                   
                   
                   
                   
                 VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE 
               
               
                   
                   
                   
                   
                 MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV 
               
               
                   
                   
                   
                   
                 DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 
               
               
                   
               
               
                 Design_7_Pre 
                 LC 
                 71 
                 VL(CD3)- 
                 DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQQKPGQPPKLLIY 
               
               
                 _TCR_Conju 
                   
                   
                 LCJB- 
                 WASTRQSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTQSHTLRTFGGGTKVE 
               
               
                 nction′1_Cys1 
                   
                   
                 CPreAlpha 
                 I KPTGVGGTPFP   C LAPPIMLLVDGKQQMVVVCLVLDVAPPGLDSPIWFSAG Q GSA 
               
               
                 5 
                   
                   
                 (S11C)(N50Q) 
                 LDAFTYGPSPATDGTWTNLAHLSLPSEELASWEPLVCHTGPGAEGHSRSTQPMHL 
               
               
                   
                   
                   
                   
                 SGEASTART 
               
               
                   
                 HC 
                 73 
                 VH(CD3)- 
                 QVQLVQSGAEVKKPGSSVKVSCKASGFAFTDYYIHWVRQAPGQGLEWMGWISPGN 
               
               
                   
                   
                   
                 HCJB- 
                 VNTKYNENFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDGYSLYYFDYWG 
               
               
                   
                   
                   
                 CBeta(E19C) 
                 QGTLVTVLEDLKNVFPPEVAVFEPSEA C ISHTQKATLVCLATGFYPDHVELSWWV 
               
               
                   
                   
                   
                 (N69Q)- 
                 NGKEVHSGVSTDPQPLKEQPAL Q DSRYALSSRLRVSATFWQNPRNHFRCQVQFYG 
               
               
                   
                   
                   
                 CJ′ 1G1- 
                 LSENDEWTQDRAKPVTQIVSAEAWGRASDKTHTCPPCPAPEAAGGPSVFLFPPKP 
               
               
                   
                   
                   
                 Fc(G1) 
                 KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV 
               
               
                   
                   
                   
                   
                 VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE 
               
               
                   
                   
                   
                   
                 MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV 
               
               
                   
                   
                   
                   
                 DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 
               
               
                   
               
               
                 Design_8_Pre 
                 LC 
                 74 
                 VL(CD3)- 
                 DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQQKPGQPPKLLIY 
               
               
                 _TCR_Conju 
                   
                   
                 LCJB- 
                 WASTRQSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTQSHTLRTFGGGTKVE 
               
               
                 nction′1_Cys1 
                   
                   
                 CPreAlpha 
                 I KPTGVGGTPFP SLAPPIMLLVDGKQQMVVVCLVLDVAPPGLDSPIWFSAG Q GSA 
               
               
                 _4L4T_1 
                   
                   
                 (S62C)(N50Q) 
                 LDAFTYGP C PATDGTWTNLAHLSLPSEELASWEPLVCHTGPGAEGHSRSTQPMHL 
               
               
                   
                   
                   
                   
                 SGEASTART 
               
               
                   
                 HC 
                 75 
                 VH(CD3)- 
                 QVQLVQSGAEVKKPGSSVKVSCKASGFAFTDYYIHWVRQAPGQGLEWMGWISPGN 
               
               
                   
                   
                   
                 HCJB- 
                 VNTKYNENFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDGYSLYYFDYWG 
               
               
                   
                   
                   
                 CBeta(S56C) 
                 QGTLVTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWV 
               
               
                   
                   
                   
                 (N69Q)- 
                 NGKEVHSGV C TDPQPLKEQPAL Q DSRYALSSRLRVSATFWQNPRNHFRCQVQFYG 
               
               
                   
                   
                   
                 CJ′ 1G1- 
                 LSENDEWTQDRAKPVTQIVSAEAWGRASDKTHTCPPCPAPEAAGGPSVFLFPPKP 
               
               
                   
                   
                   
                 Fc(G1) 
                 KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV 
               
               
                   
                   
                   
                   
                 VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE 
               
               
                   
                   
                   
                   
                 MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV 
               
               
                   
                   
                   
                   
                 DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 
               
               
                   
               
               
                 Design_9_Pre 
                 LC 
                 76 
                 VL(CD3)- 
                 DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQQKPGQPPKLLIY 
               
               
                 _TCR_Conju 
                   
                   
                 LCJB- 
                 WASTRQSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTQSHTLRTFGGGTKVE 
               
               
                 nction′1_Cys2 
                   
                   
                 CPreAlpha 
                 I KPTGVGGTPFP SLAPPIMLLVDGKQQMVVVCLVLDVAPPGLDSPIWFSAG Q GSA 
               
               
                 _4L4T_2 
                   
                   
                 (T65C)(N50Q) 
                 LDAFTYGPSPA C DGTWTNLAHLSLPSEELASWEPLVCHTGPGAEGHSRSTQPMHL 
               
               
                   
                   
                   
                   
                 SGEASTART 
               
               
                   
                 HC 
                 75 
                 VH(CD3)- 
                 QVQLVQSGAEVKKPGSSVKVSCKASGFAFTDYYIHWVRQAPGQGLEWMGWISPGN 
               
               
                   
                   
                   
                 HCJB- 
                 VNTKYNENFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDGYSLYYFDYWG 
               
               
                   
                   
                   
                 CBeta(S56C) 
                 QGTLVTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWV 
               
               
                   
                   
                   
                 (N69Q)- 
                 NGKEVHSGV C TDPQPLKEQPAL Q DSRYALSSRLRVSATFWQNPRNHFRCQVQFYG 
               
               
                   
                   
                   
                 CJ′ 1G1- 
                 LSENDEWTQDRAKPVTQIVSAEAWGRASDKTHTCPPCPAPEAAGGPSVFLFPPKP 
               
               
                   
                   
                   
                 Fc(G1) 
                 KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV 
               
               
                   
                   
                   
                   
                 VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE 
               
               
                   
                   
                   
                   
                 MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV 
               
               
                   
                   
                   
                   
                 DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 
               
               
                   
               
               
                 Design_10_Pr 
                 LC 
                 77 
                 VL(CD3)- 
                 DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQQKPGQPPKLLIY 
               
               
                 e_TCR_Conju 
                   
                   
                 LCJB- 
                 WASTRQSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTQSHTLRTFGGGTKVE 
               
               
                 nction′1_Cys4 
                   
                   
                 CPreAlpha 
                 I KPTGVGGTPFP SLAPP C MLLVDGKQQMVVVCLVLDVAPPGLDSPIWFSAG Q GSA 
               
               
                   
                   
                   
                 (I16C)(N50Q) 
                 LDAFTYGPSPATDGTWTNLAHLSLPSEELASWEPLVCHTGPGAEGHSRSTQPMHL 
               
               
                   
                   
                   
                   
                 SGEASTART 
               
               
                   
                 HC 
                 78 
                 VH(CD3)- 
                 QVQLVQSGAEVKKPGSSVKVSCKASGFAFTDYYIHWVRQAPGQGLEWMGWISPGN 
               
               
                   
                   
                   
                 HCJB- 
                 VNTKYNENFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDGYSLYYFDYWG 
               
               
                   
                   
                   
                 CBeta(A11C) 
                 QGTLVTVLEDLKNVFPPEV C VFEPSEAEISHTQKATLVCLATGFYPDHVELSWWV 
               
               
                   
                   
                   
                 (N69Q)- 
                 NGKEVHSGVSTDPQPLKEQPAL Q DSRYALSSRLRVSATFWQNPRNHFRCQVQFYG 
               
               
                   
                   
                   
                 CJ′ 1G1- 
                 LSENDEWTQDRAKPVTQIVSAEAWGRASDKTHTCPPCPAPEAAGGPSVFLFPPKP 
               
               
                   
                   
                   
                 Fc(G1) 
                 KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV 
               
               
                   
                   
                   
                   
                 VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE 
               
               
                   
                   
                   
                   
                 MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV 
               
               
                   
                   
                   
                   
                 DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 
               
               
                   
               
               
                 Design_11_Pr 
                 LC 
                 58 
                   
                 DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQQKPGQPPKLLIY 
               
               
                 e_TCR_Conju 
                   
                   
                   
                 WASTRQSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTQSHTLRTFGGGTKVE 
               
               
                 nction′2_CTe 
                   
                   
                   
                 I KPTGVGGTPFP SLAPPIMLLVDGKQQMVVVCLVLDVAPPGLDSPIWFSAG Q GSA 
               
               
                 rminal 
                   
                   
                   
                 LDAFTYGPSPATDGTWTNLAHLSLPSEELASWEPLVCHTGPGAEGHSRSTQPMHL 
               
               
                   
                   
                   
                   
                 SGEASTART CPQEPLRGTPGG   
               
               
                   
                 HC 
                 79 
                   
                 QVQLVQSGAEVKKPGSSVKVSCKASGFAFTDYYIHWVRQAPGQGLEWMGWISPGN 
               
               
                   
                   
                   
                   
                 VNTKYNENFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDGYSLYYFDYWG 
               
               
                   
                   
                   
                   
                 QGTLVTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWV 
               
               
                   
                   
                   
                   
                 NGKEVHSGVSTDPQPLKEQPAL Q DSRYALSSRLRVSATFWQNPRNHFRCQVQFYG 
               
               
                   
                   
                   
                   
                 LSENDEWTQDRAKPVTQIVSAEAWGRADCDKTHTCPPCPAPEAAGGPSVFLFPPK 
               
               
                   
                   
                   
                   
                 PKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR 
               
               
                   
                   
                   
                   
                 VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE 
               
               
                   
                   
                   
                   
                 EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT 
               
               
                   
                   
                   
                   
                 VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 
               
               
                   
               
               
                 Design_12_Pr 
                 LC 
                 63 
                   
                 DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQQKPGQPPKLLIY 
               
               
                 e_TCR_Conju 
                   
                   
                   
                 WASTRQSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTQSHTLRTFGGGTKVE 
               
               
                 nction′3C 
                   
                   
                   
                 I KPTGVGGTPFP SLAPPIMLLVDGKQQMVVVCLVLDVAPPGLDSPIWFSAG Q GSA 
               
               
                   
                   
                   
                   
                 LDAFTYGPSPATDGTWTNLAHLSLPSEELASWEPLVCHTGPGAEGHSRSTQPMHL 
               
               
                   
                   
                   
                   
                 SGEASTARTC 
               
               
                   
                 HC 
                 80 
                   
                 QVQLVQSGAEVKKPGSSVKVSCKASGFAFTDYYIHWVRQAPGQGLEWMGWISPGN 
               
               
                   
                   
                   
                   
                 VNTKYNENFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDGYSLYYFDYWG 
               
               
                   
                   
                   
                   
                 QGTLVTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWV 
               
               
                   
                   
                   
                   
                 NGKEVHSGVSTDPQPLKEQPAL Q DSRYALSSRLRVSATFWQNPRNHFRCQVQFYG 
               
               
                   
                   
                   
                   
                 LSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVCPPCPAPEAAGGPSVFLFPPK 
               
               
                   
                   
                   
                   
                 PKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR 
               
               
                   
                   
                   
                   
                 VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE 
               
               
                   
                   
                   
                   
                 EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT 
               
               
                   
                   
                   
                   
                 VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 
               
               
                   
               
               
                 Conjunction 
                 LConjuncti 
                 81 
                 VL-Cpre- 
                 PTGVGGTP 
               
               
                 W 
                 on_3 
                   
                 AlphaCJB 
                   
               
               
                   
               
               
                 CPreAlpha 
                 CPreAlpha 
                 82 
                   
                 FPSLAPPIMLLVDGKQQMVVVCLVLDVAPPGLDSPIWFSAG N GSALDAFTYGPSP 
               
               
                   
                   
                   
                   
                 ATDGTWTNLAHLSLPSEELASWEPLVCHTGPGAEGHSRSTQPMHLSGEASTART 
               
               
                   
                 CPreAlpha- 
                 83 
                 (N50Q) 
                 FPSLAPPIMLLVDGKQQMVVVCLVLDVAPPGLDSPIWFSAG Q GSALDAFTYGPSP 
               
               
                   
                 noGlyco 
                   
                   
                 ATDGTWTNLAHLSLPSEELASWEPLVCHTGPGAEGHSRSTQPMHLSGEASTART 
               
               
                   
                 (Design1) 
                   
                   
                   
               
               
                   
                 CPreAlpha- 
                 311 
                 (N50Q, Y59C) 
                 FPSLAPPIMLLVDGKQQMVVVCLVLDVAPPGLDSPIWFSAG Q GSALDAFTCGPSP 
               
               
                   
                 noGlyco- 
                   
                   
                 ATDGTWTNLAHLSLPSEELASWEPLVCHTGPGAEGHSRSTQPMHLSGEASTART 
               
               
                   
                 Cys 
                   
                   
                   
               
               
                   
                 (Design2) 
                   
                   
                   
               
               
                   
                 CPreAlpha- 
                 312 
                 (N50Q, A13C) 
                 FPSLCPPIMLLVDGKQQMVVVCLVLDVAPPGLDSPIWFSAG Q GSALDAFTYGPSP 
               
               
                   
                 noGlyco- 
                   
                   
                 ATDGTWTNLAHLSLPSEELASWEPLVCHTGPGAEGHSRSTQPMHLSGEASTART 
               
               
                   
                 Cys 
                   
                   
                   
               
               
                   
                 (Design3) 
                   
                   
                   
               
               
                   
                 CPreAlpha- 
                 312 
                 (N50Q, A13C) 
                   
               
               
                   
                 noGlyco-Cys 
                   
                   
                   
               
               
                   
                 (Design4) 
                   
                   
                   
               
               
                   
                 CPreAlpha- 
                 313 
                 (N50Q, S11C) 
                 FPCLAPPIMLLVDGKQQMVVVCLVLDVAPPGLDSPIWFSAG Q GSALDAFTYGPSP 
               
               
                   
                 noGlyco- 
                   
                   
                 ATDGTWTNLAHLSLPSEELASWEPLVCHTGPGAEGHSRSTQPMHLSGEASTART 
               
               
                   
                 Cys 
                   
                   
                   
               
               
                   
                 (Design5) 
                   
                   
                   
               
               
                   
                 CPreAlpha- 
                 313 
                 (N50Q, S11C) 
                   
               
               
                   
                 noGlyco-Cys 
                   
                   
                   
               
               
                   
                 (Design6) 
                   
                   
                   
               
               
                   
                 CPreAlpha- 
                 313 
                 (N50Q, S11C) 
                   
               
               
                   
                 noGlyco-Cys 
                   
                   
                   
               
               
                   
                 (Design7) 
                   
                   
                   
               
               
                   
                 CPreAlpha- 
                 314 
                 (N50Q, S62C) 
                 FPSLAPPIMLLVDGKQQMVVVCLVLDVAPPGLDSPIWFSAG Q GSALDAFTYGPCP 
               
               
                   
                 noGlyco- 
                   
                   
                 ATDGTWTNLAHLSLPSEELASWEPLVCHTGPGAEGHSRSTQPMHLSGEASTART 
               
               
                   
                 Cys 
                   
                   
                   
               
               
                   
                 (Design8) 
                   
                   
                   
               
               
                   
                 CPreAlpha- 
                 315 
                 (N50Q, T65C) 
                 FPSLAPPIMLLVDGKQQMVVVCLVLDVAPPGLDSPIWFSAG Q GSALDAFTYGPSP 
               
               
                   
                 noGlyco- 
                   
                   
                 ACDGTWTNLAHLSLPSEELASWEPLVCHTGPGAEGHSRSTQPMHLSGEASTART 
               
               
                   
                 Cys 
                   
                   
                   
               
               
                   
                 (Design9) 
                   
                   
                   
               
               
                   
                 CPreAlpha- 
                 316 
                 (N50Q, I16C) 
                 FPSLAPPCMLLVDGKQQMVVVCLVLDVAPPGLDSPIWFSAG Q GSALDAFTYGPSP 
               
               
                   
                 noGlyco- 
                   
                   
                 ATDGTWTNLAHLSLPSEELASWEPLVCHTGPGAEGHSRSTQPMHLSGEASTART 
               
               
                   
                 Cys 
                   
                   
                   
               
               
                   
                 (Design10) 
                   
                   
                   
               
               
                   
                 CPreAlpha- 
                 317 
                 (N50Q, 
                 FPSLAPPIMLLVDGKQQMVVVCLVLDVAPPGLDSPIWFSAG Q GSALDAFTYGPSP 
               
               
                   
                 noGlyco 
                   
                 ter_residues) 
                 ATDGTWTNLAHLSLPSEELASWEPLVCHTGPGAEGHSRSTQPMHLSGEASTART C   
               
               
                   
                 (Design11) 
                   
                   
                 
                   PQEPLRGTPGG 
                 
               
               
                   
                 CPreAlpha- 
                 318 
                 (N50Q, 
                 FPSLAPPIMLLVDGKQQMVVVCLVLDVAPPGLDSPIWFSAG Q GSALDAFTYGPSP 
               
               
                   
                 noGlyco 
                   
                 Cter_only) 
                 ATDGTWTNLAHLSLPSEELASWEPLVCHTGPGAEGHSRSTQPMHLSGEASTART C   
               
               
                   
                 (Design12) 
                   
                   
                   
               
               
                   
               
               
                 Cbeta for_Cp 
                 CBeta for 
                 84 
                 N69Q 
                 EVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLK 
               
               
                 reAlpha 
                 PreAlpha 
                   
                   
                 EQPAL Q DSRY A LSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQ 
               
               
                   
                   
                   
                   
                 IVSAEA 
               
               
                   
                 Design_1 
                 84 
                 N69Q 
                   
               
               
                   
                 Design_2 
                 319 
                 N69Q, S76C 
                 EVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLK 
               
               
                   
                   
                   
                   
                 EQPAL Q DSRY A LCSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQ 
               
               
                   
                   
                   
                   
                 IVSAEA 
               
               
                   
                 Design_3 
                 320 
                 N69Q, F13C 
                 EVAVCEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLK 
               
               
                   
                   
                   
                   
                 EQPAL Q DSRY A LSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQ 
               
               
                   
                   
                   
                   
                 IVSAEA 
               
               
                   
                 Design_4 
                 321 
                 N69Q, S16C 
                 EVAVFEPCEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLK 
               
               
                   
                   
                   
                   
                 EQPAL Q DSRY A LSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQ 
               
               
                   
                   
                   
                   
                 IVSAEA 
               
               
                   
                 Design_5 
                 321 
                 N69Q, S16C 
                   
               
               
                   
                 Design_6 
                 322 
                 N69Q, A18C 
                 EVAVFEPSECEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLK 
               
               
                   
                   
                   
                   
                 EQPAL Q DSRY A LSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQ 
               
               
                   
                   
                   
                   
                 IVSAEA 
               
               
                   
                 Design_7 
                 323 
                 N69Q, E19C 
                 EVAVFEPSEACISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLK 
               
               
                   
                   
                   
                   
                 EQPAL Q DSRY A LSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQ 
               
               
                   
                   
                   
                   
                 IVSAEA 
               
               
                   
                 Design_8 
                 34 
                 N69Q, S56C 
                   
               
               
                   
                 Design_9 
                 34 
                 N69Q, S56C 
                   
               
               
                   
                 Design_10 
                 324 
                  N69Q, A11C 
                 EVCVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLK 
               
               
                   
                   
                   
                   
                 EQPAL Q DSRY A LSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQ 
               
               
                   
                   
                   
                   
                 IVSAEA 
               
               
                   
                 Design_11 
                 84 
                 N69Q 
                   
               
               
                   
                 Design_12 
                 84 
                 N69Q 
               
               
                   
               
               
                   
                   
                 SEQ 
                   
                   
               
               
                 Delta-Gamma 
                 Chain 
                 ID 
                   
                   
               
               
                 Designs 
                 Name 
                 NO 
                   
                 Sequences 
               
               
                   
               
               
                 dg_Design_2_ 
                 LC 
                 85 
                 VL(CD3)- 
                 DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQQKPGQPPKLLIY 
               
               
                 no_Glyco 
                   
                   
                 LCJ5-CDelta 
                 WASTRQSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTQSHTLRTFGGGTKVE 
               
               
                   
                   
                   
                 (N16Q + N79Q) 
                 IEPRSQPHTKPSVFVMKQGTNVACLVKEFYPKDIRINLVSSKKITEFDPAIVISP 
               
               
                   
                   
                   
                   
                 SGKYNAVKLGKYEDSNSVTCSVQHDQKTVHSTDFE 
               
               
                   
                 HC 
                 86 
                 VH(CD3)- 
                 QVQLVQSGAEVKKPGSSVKVSCKASGFAFTDYYIHWVRQAPGQGLEWMGWISPGN 
               
               
                   
                   
                   
                 HCJ5- 
                 VNTKYNENFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDGYSLYYFDYWG 
               
               
                   
                   
                   
                 CGamma- 
                 QGTLVTVTDKQLDADVSPKPTIFLPSIAETKLQKAGTYLCLLEKFFPDVIKIHWQ 
               
               
                   
                   
                   
                 CJ′3G1- 
                 EKKSNTILGSQEGNTMKTQDTYMKFSWLTVPEESLDKEHRCIVRHENNKNGVDQE 
               
               
                   
                   
                   
                 Fc(G1)(N65Q) 
                 IIFPPIKSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS 
               
               
                   
                   
                   
                   
                 HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK 
               
               
                   
                   
                   
                   
                 VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI 
               
               
                   
                   
                   
                   
                 AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL 
               
               
                   
                   
                   
                   
                 HNHYTQKSLSLSPGK 
               
               
                   
               
               
                 dg_Design_2_ 
                 LC 
                 89 
                 VL(CD3)- 
                 DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQQKPGQPPKLLIY 
               
               
                 hypeCys1_no_ 
                   
                   
                 LCJ5-CDelta 
                 WASTRQSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTQSHTLRTFGGGTKVE 
               
               
                 Glyco 
                   
                   
                 (N16C)(N79Q) 
                 IEPRSQPHTKPSVFVMKCGTNVACLVKEFYPKDIRINLVSSKKITEFDPAIVISP 
               
               
                   
                   
                   
                   
                 SGKYNAVKLGKYEDSNSVTCSVQHDQKTVHSTDFE 
               
               
                   
                 HC 
                 90 
                 VH(CD3)- 
                 QVQLVQSGAEVKKPGSSVKVSCKASGFAFTDYYIHWVRQAPGQGLEWMGWISPGN 
               
               
                   
                   
                   
                 HCJ5-CGamma 
                 VNTKYNENFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDGYSLYYFDYWG 
               
               
                   
                   
                   
                 (T12C) 
                 QGTLVTVTDKQLDADVSPKPCIFLPSIAETKLQKAGTYLCLLEKFFPDVIKIHWQ 
               
               
                   
                   
                   
                 (N65Q)- 
                 EKKSNTILGSQEGNTMKTQDTYMKFSWLTVPEESLDKEHRCIVRHENNKNGVDQE 
               
               
                   
                   
                   
                 CJ′3G1- 
                 IIFPPIKSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS 
               
               
                   
                   
                   
                 Fc(G1) 
                 HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK 
               
               
                   
                   
                   
                   
                 VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI 
               
               
                   
                   
                   
                   
                 AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL 
               
               
                   
                   
                   
                   
                 HNHYTQKSLSLSPGK 
               
               
                   
               
               
                 dg_Design_2_ 
                 LC 
                 91 
                 VL(CD3)- 
                 DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQQKPGQPPKLLIY 
               
               
                 hypeCys2_no_ 
                   
                   
                 LCJ5-CDelta 
                 WASTRQSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTQSHTLRTFGGGTKVE 
               
               
                 Glyco 
                   
                   
                 (V50C) 
                 IEPRSQPHTKPSVFVMKQGINVACLVKEFYPKDIRINLVSSKKITEFDPAICISP 
               
               
                   
                   
                   
                 (N16Q + N79Q) 
                 SGKYNAVKLGKYEDSNSVTCSVQHDQKTVHSTDFE 
               
               
                   
                 HC 
                 92 
                 VH(CD3)- 
                 QVQLVQSGAEVKKPGSSVKVSCKASGFAFTDYYIHWVRQAPGQGLEWMGWISPGN 
               
               
                   
                   
                   
                 HCJ5-CGamma 
                 VNTKYNENFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDGYSLYYFDYWG 
               
               
                   
                   
                   
                 (Q57C) 
                 QGTLVTVTDKQLDADVSPKPTIFLPSIAETKLQKAGTYLCLLEKFFPDVIKIHWQ 
               
               
                   
                   
                   
                 (N65Q)- 
                 EKKSNTILGSCEGNTMKTQDTYMKFSWLTVPEESLDKEHRCIVRHENNKNGVDQE 
               
               
                   
                   
                   
                 CJ′3G1- 
                 IIFPPIKSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS 
               
               
                   
                   
                   
                 Fc(G1) 
                 HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK 
               
               
                   
                   
                   
                   
                 VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI 
               
               
                   
                   
                   
                   
                 AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL 
               
               
                   
                   
                   
                   
                 HNHYTQKSLSLSPGK 
               
               
                   
               
               
                 dg_Design_2_ 
                 LC 
                 93 
                 VL(CD3)- 
                 DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQQKPGQPPKLLIY 
               
               
                 hypeCys3_no_ 
                   
                   
                 LCJ5-CDelta 
                 WASTRQSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTQSHTLRTFGGGTKVE 
               
               
                 Glyco 
                   
                   
                 (D46C) 
                 IEPRSQPHTKPSVFVMKQGINVACLVKEFYPKDIRINLVSSKKITEFCPAIVISP 
               
               
                   
                   
                   
                 (N16Q + N79Q) 
                 SGKYNAVKLGKYEDSNSVTCSVQHDQKTVHSTDFE 
               
               
                   
                 HC 
                 94 
                 VH(CD3)- 
                 QVQLVQSGAEVKKPGSSVKVSCKASGFAFTDYYIHWVRQAPGQGLEWMGWISPGN 
               
               
                   
                   
                   
                 HCJ5-CGamma 
                 VNTKYNENFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDGYSLYYFDYWG 
               
               
                   
                   
                   
                 (M62C) 
                 QGTLVTVTDKQLDADVSPKPTIFLPSIAETKLQKAGTYLCLLEKFFPDVIKIHWQ 
               
               
                   
                   
                   
                 (N65Q)- 
                 EKKSNTILGSQEGNTCKTQDTYMKFSWLTVPEESLDKEHRCIVRHENNKNGVDQE 
               
               
                   
                   
                   
                 CJ′3G1- 
                 IIFPPIKSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS 
               
               
                   
                   
                   
                 Fc(G1) 
                 HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK 
               
               
                   
                   
                   
                   
                 VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI 
               
               
                   
                   
                   
                   
                 AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL 
               
               
                   
                   
                   
                   
                 HNHYTQKSLSLSPGK 
               
               
                   
               
               
                 dg_Design_2_ 
                 LC 
                 95 
                 VL(CD3)- 
                 DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQQKPGQPPKLLIY 
               
               
                 Cys2_no_Glyc 
                   
                   
                 LCJ5-CDelta 
                 WASTRQSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTQSHTLRTFGGGTKVE 
               
               
                 o 
                   
                   
                 (F12C) 
                 IEPRSQPHTKPSVCVMKQGINVACLVKEFYPKDIRINLVSSKKITEFDPAIVISP 
               
               
                   
                   
                   
                 (N16Q + N79Q) 
                 SGKYNAVKLGKYEDSNSVTCSVQHDQKTVHSTDFE 
               
               
                   
                 HC 
                 96 
                 VH(CD3)- 
                 QVQLVQSGAEVKKPGSSVKVSCKASGFAFTDYYIHWVRQAPGQGLEWMGWISPGN 
               
               
                   
                   
                   
                 HCJ5-CGamma 
                 VNTKYNENFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDGYSLYYFDYWG 
               
               
                   
                   
                   
                 (S17C) 
                 QGTLVTVTDKQLDADVSPKPTIFLPCIAETKLQKAGTYLCLLEKFFPDVIKIHWQ 
               
               
                   
                   
                   
                 (N65Q)- 
                 EKKSNTILGSQEGNTMKTQDTYMKFSWLTVPEESLDKEHRCIVRHENNKNGVDQE 
               
               
                   
                   
                   
                 CJ′3G1- 
                 IIFPPIKSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS 
               
               
                   
                   
                   
                   
                 HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK 
               
               
                   
                   
                   
                 Fc(G1) 
                 VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI 
               
               
                   
                   
                   
                   
                 AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL 
               
               
                   
                   
                   
                   
                 HNHYTQKSLSLSPGK 
               
               
                   
               
               
                 dg_Design_2_ 
                 LC 
                 97 
                 VL(CD3)- 
                 DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQQKPGQPPKLLIY 
               
               
                 Cys1_no_Glyc 
                   
                   
                 LCJ5-CDelta 
                 WASTRQSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTQSHTLRTFGGGTKVE 
               
               
                 o 
                   
                   
                 (M14C) 
                 IEPRSQPHTKPSVFVCKQGTNVACLVKEFYPKDIRINLVSSKKITEFDPAIVISP 
               
               
                   
                   
                   
                 (N16Q + N79Q) 
                 SGKYNAVKLGKYEDSNSVTCSVQHDQKTVHSTDFE 
               
               
                   
                 HC 
                 98 
                 VH(CD3)- 
                 QVQLVQSGAEVKKPGSSVKVSCKASGFAFTDYYIHWVRQAPGQGLEWMGWISPGN 
               
               
                   
                   
                   
                 HCJ5-CGamma 
                 VNTKYNENFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDGYSLYYFDYWG 
               
               
                   
                   
                   
                 (F14C) 
                 QGTLVTVTDKQLDADVSPKPTICLPSIAETKLQKAGTYLCLLEKFFPDVIKIHWQ 
               
               
                   
                   
                   
                 (N65Q)- 
                 EKKSNTILGSQEGNTMKTQDTYMKFSWLTVPEESLDKEHRCIVRHENNKNGVDQE 
               
               
                   
                   
                   
                 CJ′3G1- 
                 IIFPPIKSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS 
               
               
                   
                   
                   
                 Fc(G1) 
                 HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK 
               
               
                   
                   
                   
                   
                 VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI 
               
               
                   
                   
                   
                   
                 AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL 
               
               
                   
                   
                   
                   
                 HNHYTQKSLSLSPGK 
               
               
                   
               
               
                 dg_Design_2_ 
                 LC 
                 95 
                 VL(CD3)- 
                 DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQQKPGQPPKLLIY 
               
               
                 Cys3_no_Glyc 
                   
                   
                 LCJ5-CDelta 
                 WASTRQSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTQSHTLRTFGGGTKVE 
               
               
                 o 
                   
                   
                 (F12C) 
                 IEPRSQPHTKPSVCVMKQGINVACLVKEFYPKDIRINLVSSKKITEFDPAIVISP 
               
               
                   
                   
                   
                 (N16Q + N79Q) 
                 SGKYNAVKLGKYEDSNSVTCSVQHDQKTVHSTDFE 
               
               
                   
                 HC 
                 99 
                 VH(CD3)- 
                 QVQLVQSGAEVKKPGSSVKVSCKASGFAFTDYYIHWVRQAPGQGLEWMGWISPGN 
               
               
                   
                   
                   
                 HCJ5-CGamma 
                 VNTKYNENFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDGYSLYYFDYWG 
               
               
                   
                   
                   
                 (E20C) 
                 QGTLVTVTDKQLDADVSPKPTIFLPSIACTKLQKAGTYLCLLEKFFPDVIKIHWQ 
               
               
                   
                   
                   
                 (N65Q)- 
                 EKKSNTILGSQEGNTMKTQDTYMKFSWLTVPEESLDKEHRCIVRHENNKNGVDQE 
               
               
                   
                   
                   
                 CJ′3G1- 
                 IIFPPIKSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS 
               
               
                   
                   
                   
                 Fc(G1) 
                 HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK 
               
               
                   
                   
                   
                   
                 VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI 
               
               
                   
                   
                   
                   
                 AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL 
               
               
                   
                   
                   
                   
                 HNHYTQKSLSLSPGK 
               
               
                   
               
               
                 dg_Design_2_ 
                 LC 
                 100 
                 VL(CD3)- 
                 DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQQKPGQPPKLLIY 
               
               
                 Cys4_no_Glyc 
                   
                   
                 LCJ5-CDelta 
                 WASTRQSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTQSHTLRTFGGGTKVE 
               
               
                 o 
                   
                   
                 (F87C) 
                 IEPRSQPHTKPSVFVMKQGINVACLVKEFYPKDIRINLVSSKKITEFDPAIVISP 
               
               
                   
                   
                   
                 (N16Q + N79Q) 
                 SGKYNAVKLGKYEDSNSVTCSVQHDQKTVHSTDCE 
               
               
                   
                 HC 
                 101 
                 VH(CD3)- 
                 QVQLVQSGAEVKKPGSSVKVSCKASGFAFTDYYIHWVRQAPGQGLEWMGWISPGN 
               
               
                   
                   
                   
                 HCJ5-CGamma 
                 VNTKYNENFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDGYSLYYFDYWG 
               
               
                   
                   
                   
                 (A19C) 
                 QGTLVTVTDKQLDADVSPKPTIFLPSICETKLQKAGTYLCLLEKFFPDVIKIHWQ 
               
               
                   
                   
                   
                 (N65Q)- 
                 EKKSNTILGSQEGNTMKTQDTYMKFSWLTVPEESLDKEHRCIVRHENNKNGVDQE 
               
               
                   
                   
                   
                 CJ′3G1- 
                 IIFPPIKSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS 
               
               
                   
                   
                   
                 Fc(G1) 
                 HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK 
               
               
                   
                   
                   
                   
                 VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI 
               
               
                   
                   
                   
                   
                 AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL 
               
               
                   
                   
                   
                   
                 HNHYTQKSLSLSPGK 
               
               
                   
               
               
                 dg_Design_2_ 
                 LC 
                 102 
                 VL(CD3)- 
                 DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQQKPGQPPKLLIY 
               
               
                 Cys5_no_Glyc 
                   
                   
                 LCJ5-CDelta 
                 WASTRQSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTQSHTLRTFGGGTKVE 
               
               
                 o 
                   
                   
                 (E88C) 
                 IEPRSQPHTKPSVFVMKQGTNVACLVKEFYPKDIRINLVSSKKITEFDPAIVISP 
               
               
                   
                   
                   
                 (N16Q + N79Q) 
                 SGKYNAVKLGKYEDSNSVTCSVQHDQKTVHSTDFC 
               
               
                   
                 HC 
                 101 
                 VH(CD3)- 
                 QVQLVQSGAEVKKPGSSVKVSCKASGFAFTDYYIHWVRQAPGQGLEWMGWISPGN 
               
               
                   
                   
                   
                 HCJ5-CGamma 
                 VNTKYNENFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDGYSLYYFDYWG 
               
               
                   
                   
                   
                 (A19C) 
                 QGTLVTVTDKQLDADVSPKPTIFLPSICETKLQKAGTYLCLLEKFFPDVIKIHWQ 
               
               
                   
                   
                   
                 (N65Q)- 
                 EKKSNTILGSQEGNTMKTQDTYMKFSWLTVPEESLDKEHRCIVRHENNKNGVDQE 
               
               
                   
                   
                   
                 CJ′3G1- 
                 IIFPPIKSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS 
               
               
                   
                   
                   
                 Fc(G1) 
                 HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK 
               
               
                   
                   
                   
                   
                 VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI 
               
               
                   
                   
                   
                   
                 AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL 
               
               
                   
                   
                   
                   
                 HNHYTQKSLSLSPGK 
               
               
                   
               
               
                 dg_Design_2 
                 LC 
                 105 
                 VL(CD3)- 
                 DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQQKPGQPPKLLIY 
               
               
                   
                   
                   
                 LCJ5-CDelta 
                 WASTRQSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTQSHTLRTFGGGTKVE 
               
               
                   
                   
                   
                   
                 IEPRSQPHTKPSVFVMKNGTNVACLVKEFYPKDIRINLVSSKKITEFDPAIVISP 
               
               
                   
                   
                   
                   
                 SGKYNAVKLGKYEDSNSVTCSVQHDNKTVHSTDFE 
               
               
                   
                 HC 
                 106 
                 VH(CD3)- 
                 QVQLVQSGAEVKKPGSSVKVSCKASGFAFTDYYIHWVRQAPGQGLEWMGWISPGN 
               
               
                   
                   
                   
                 HCJ5- 
                 VNTKYNENFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDGYSLYYFDYWG 
               
               
                   
                   
                   
                 CGamma- 
                 QGTLVTVTDKQLDADVSPKPTIFLPSIAETKLQKAGTYLCLLEKFFPDVIKIHWQ 
               
               
                   
                   
                   
                 CJ′3G1- 
                 EKKSNTILGSQEGNTMKTNDTYMKFSWLTVPEESLDKEHRCIVRHENNKNGVDQE 
               
               
                   
                   
                   
                 Fc(G1) 
                 IIFPPIKSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS 
               
               
                   
                   
                   
                   
                 HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK 
               
               
                   
                   
                   
                   
                 VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI 
               
               
                   
                   
                   
                   
                 AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL 
               
               
                   
                   
                   
                   
                 HNHYTQKSLSLSPGK 
               
               
                   
               
               
                 dg_Design_1 
                 LC 
                 107 
                 VL(CD3)- 
                 DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQQKPGQPPKLLIY 
               
               
                   
                   
                   
                 LCJ4-CDelta 
                 WASTRQSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTQSHTLRTFGGGTKVE 
               
               
                   
                   
                   
                   
                 IKPRSQPHTKPSVFVMKNGTNVACLVKEFYPKDIRINLVSSKKITEFDPAIVISP 
               
               
                   
                   
                   
                   
                 SGKYNAVKLGKYEDSNSVTCSVQHDNKTVHSTDFE 
               
               
                   
                 HC 
                 108 
                 VH(CD3)- 
                 QVQLVQSGAEVKKPGSSVKVSCKASGFAFTDYYIHWVRQAPGQGLEWMGWISPGN 
               
               
                   
                   
                   
                 HCJ4- 
                 VNTKYNENFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDGYSLYYFDYWG 
               
               
                   
                   
                   
                 CGamma- 
                 QGTLVTVSSASLDADVSPKPTIFLPSIAETKLQKAGTYLCLLEKFFPDVIKIHWQ 
               
               
                   
                   
                   
                 CJ′3G1- 
                 EKKSNTILGSQEGNTMKTNDTYMKFSWLTVPEESLDKEHRCIVRHENNKNGVDQE 
               
               
                   
                   
                   
                 Fc(G1) 
                 IIFPPIKSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS 
               
               
                   
                   
                   
                   
                 HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK 
               
               
                   
                   
                   
                   
                 VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI 
               
               
                   
                   
                   
                   
                 AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL 
               
               
                   
                   
                   
                   
                 HNHYTQKSLSLSPGK 
               
               
                   
               
               
                 dg_crossed_D 
                 LC 
                 109 
                 VL(CD3)- 
                 DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQQKPGQPPKLLIY 
               
               
                 esign_1 
                   
                   
                 LCJ6- 
                 WASTRQSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTQSHTLRTFGGGTKVE 
               
               
                   
                   
                   
                 CGamma 
                 IKDKQLDADVSPKPTIFLPSIAETKLQKAGTYLCLLEKFFPDVIKIHWQEKKSNT 
               
               
                   
                   
                   
                   
                 ILGS C EGNTMKT Q DTYMKFSWLTVPEESLDKEHRCIVRHENNKNGVDQEIIFPPI 
               
               
                   
                   
                   
                   
                 KTDVITMD 
               
               
                   
                 HC 
                 110 
                 VH(CD3)- 
                 QVQLVQSGAEVKKPGSSVKVSCKASGFAFTDYYIHWVRQAPGQGLEWMGWISPGN 
               
               
                   
                   
                   
                 HCJ6- CDelta- 
                 VNTKYNENFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDGYSLYYFDYWG 
               
               
                   
                   
                   
                 CJ′4G1- 
                 QGTLVTVSSRSQPHTKP SVFVMK Q GTNVACLVKEFYPKDIRINLVSSKKITEFDP   
               
               
                   
                   
                   
                 Fc(G1) 
                   AI C ISPSGKYNAVKLGKYEDSNSVTCSVQHD Q KTVHSTD EPKSCDKTHTCPPCPA 
               
               
                   
                   
                   
                   
                 PEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN 
               
               
                   
                   
                   
                   
                 AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG 
               
               
                   
                   
                   
                   
                 QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP 
               
               
                   
                   
                   
                   
                 VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 
               
               
                   
               
               
                 dg_crossed_D 
                 LC 
                 111 
                 VL(CD3)- 
                 DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQQKPGQPPKLLIY 
               
               
                 esign_2 
                   
                   
                 LCJ7- 
                 WASTRQSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTQSHTLRTFGGGTKVE 
               
               
                   
                   
                   
                 CGamma 
                 IKDKQLDADVSPKPTIFLPSIAETKLQKAGTYLCLLEKFFPDVIKIHWQEKKSNT 
               
               
                   
                   
                   
                   
                 ILGS C EGNTMKT Q DTYMKFSWLTVPEESLDKEHRCIVRHENNKNGVDQEIIFPPI 
               
               
                   
                   
                   
                   
                 KTDVITMD 
               
               
                   
                 HC 
                 112 
                 VH(CD3)- 
                 QVQLVQSGAEVKKPGSSVKVSCKASGFAFTDYYIHWVRQAPGQGLEWMGWISPGN 
               
               
                   
                   
                   
                 HCJ7- CDelta- 
                 VNTKYNENFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDGYSLYYFDYWG 
               
               
                   
                   
                   
                 CJ′4G1- 
                 QGTLVTVEPRSQPHTKP SVFVMK Q GTNVACLVKEFYPKDIRINLVSSKKITEFDP   
               
               
                   
                   
                   
                 Fc(G1) 
                   AI C ISPSGKYNAVKLGKYEDSNSVTCSVQHD Q KTVHSTD EPKSCDKTHTCPPCPA 
               
               
                   
                   
                   
                   
                 PEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN 
               
               
                   
                   
                   
                   
                 AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG 
               
               
                   
                   
                   
                   
                 QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP 
               
               
                   
                   
                   
                   
                 VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 
               
               
                   
               
               
                 CGamma 
                 CGamma_1 
                 113 
                 CGamma 
                 KPTIFLPSIAETKLQKAGTYLCLLEKFFPDVIKIHWQEKKSNTILGSQEGNTMKT 
               
               
                   
                   
                   
                   
                 NDTYMKFSWLTVPEESLDKEHRCIVRHENNKNGVDQEIIF 
               
               
                   
                 CGamma_1 
                 114 
                 CGamma 
                 KPTIFLPSIAETKLQKAGTYLCLLEKFFPDVIKIHWQEKKSNTILGSQEGNTMKT 
               
               
                   
                 no Glyco 
                   
                 (N65Q) 
                 QDTYMKFSWLTVPEESLDKEHRCIVRHENNKNGVDQEIIF 
               
               
                   
                 dg_Design_ 
                 113 
                 No mutations 
                   
               
               
                   
                 1 
                   
                   
                   
               
               
                   
                 dg_Design_ 
                 113 
                 No mutations 
                   
               
               
                   
                 2 
                   
                   
                   
               
               
                   
                 dg_Design_ 
                 114 
                 N65Q 
                   
               
               
                   
                 2_no_Glyco 
                   
                   
                   
               
               
                   
                 dg_Design_ 
                 333 
                 N65Q, T12C 
                 KP C IFLPSIAETKLQKAGTYLCLLEKFFPDVIKIHWQEKKSNTILGSQEGNTMKT 
               
               
                   
                 2_hypeCys 
                   
                   
                   Q DTYMKFSWLTVPEESLDKEHRCIVRHENNKNGVDQEIIF 
               
               
                   
                 1_no_Glyco 
                   
                   
                   
               
               
                   
                 dg_Design_ 
                 334 
                 N65Q, Q57C 
                 KPTIFLPSIAETKLQKAGTYLCLLEKFFPDVIKIHWQEKKSNTILGS C EGNTMKT 
               
               
                   
                 2_hypeCys 
                   
                   
                   Q DTYMKFSWLTVPEESLDKEHRCIVRHENNKNGVDQEIIF 
               
               
                   
                 2_no_Glyco 
                   
                   
                   
               
               
                   
                 dg_Design_ 
                 335 
                 N65Q, M62C 
                 KPTIFLPSIAETKLQKAGTYLCLLEKFFPDVIKIHWQEKKSNTILGSQEGNT C KT 
               
               
                   
                 2_hypeCys 
                   
                   
                   Q DTYMKFSWLTVPEESLDKEHRCIVRHENNKNGVDQEIIF 
               
               
                   
                 3_no_Glyco 
                   
                   
                   
               
               
                   
                 dg_Design_ 
                 336 
                 N65Q, S17C 
                 KPTIFLPCIAETKLQKAGTYLCLLEKFFPDVIKIHWQEKKSNTILGSQEGNTMKT 
               
               
                   
                 2_Cys2_no 
                   
                   
                   Q DTYMKFSWLTVPEESLD-KEHRCIVRHENNKNGVDQEIIF 
               
               
                   
                 _Glyco 
                   
                   
                   
               
               
                   
                 dg_Design_ 
                 337 
                 N65Q, F14C 
                 KPTI C LPSIAETKLQKAGTYLCLLEKFFPDVIKIHWQEKKSNTILGSQEGNTMKT 
               
               
                   
                 2_Cys1_no 
                   
                   
                   Q DTYMKFSWLTVPEESLDKEHRCIVRHENNKNGVDQEIIF 
               
               
                   
                 _Glyco 
                   
                   
                   
               
               
                   
                 dg_Design_ 
                 338 
                 N65Q, E20C 
                 KPTIFLPSIA C TKLQKAGTYLCLLEKFFPDVIKIHWQEKKSNTILGSQEGNTMKT 
               
               
                   
                 2_Cys3_no 
                   
                   
                   Q DTYMKFSWLTVPEESLDKEHRCIVRHENNKNGVDQEIIF 
               
               
                   
                 _Glyco 
                   
                   
                   
               
               
                   
                 dg_Design_ 
                 339 
                 N65Q, A19C 
                 KPTIFLPSI C ETKLQKAGTYLCLLEKFFPDVIKIHWQEKKSNTILGSQEGNTMKT 
               
               
                   
                 2_Cys4_no 
                   
                   
                   Q DTYMKFSWLTVPEESLDKEHRCIVRHENNKNGVDQEIIF 
               
               
                   
                 _Glyco 
                   
                   
                   
               
               
                   
                 dg_Design 
                 339 
                 N65Q, A19C 
                   
               
               
                   
                 2_Cys5_no 
                   
                   
                   
               
               
                   
                 Glyco 
                   
                   
                   
               
               
                   
                 dg_crossed 
                 340 
                   
                 KPTIFLPSIAETKLQKAGTYLCLLEKFFPDVIKIHWQEKKSNTILGS C EGNTMKT 
               
               
                   
                 _Design_1 
                   
                   
                   Q DTYMKFSWLTVPEESLDKEHRCIVRHENNKNGVDQEIIFPPIKTDVITMD 
               
               
                   
                 dg_crossed 
                 340 
                   
                   
               
               
                   
                 _Design_2 
                   
                   
                   
               
               
                   
               
               
                 CDelta 
                 CDelta_1 
                 115 
                 CDelta 
                 SVFVMKNGTNVACLVKEFYPKDIRINLVSSKKITEFDPAIVISPSGKYNAVKLGK 
               
               
                   
                   
                   
                   
                 YEDSNSVTCSVQHDNKTVHSTDFE 
               
               
                   
                 CDelta_1_n 
                 116 
                 CDelta(N16Q + 
                 SVFVMKQGTNVACLVKEFYPKDIRINLVSSKKITEFDPAIVISPSGKYNAVKLGK 
               
               
                   
                 o_Glyco 
                   
                 N79Q) 
                 YEDSNSVTCSVQHDQKTVHSTDFE 
               
               
                   
                   
                 310 
                 CDelta 
                   K SVFVMKNGTNVACLVKEFYPKDIRINLVSSKKITEFDPAIVISPSGKYNAVKLG 
               
               
                   
                   
                   
                   
                 KYEDSNSVTCSVQHDNKTVHSTDFE 
               
               
                   
                 dg_Design_ 
                 115 
                 No Mutations 
                   
               
               
                   
                 1 
                   
                   
                   
               
               
                   
                 dg_Design_ 
                 115 
                 No Mutations 
                   
               
               
                   
                 2 
                   
                   
                   
               
               
                   
                 dg_Design_ 
                 116 
                 N16Q, N79Q 
                   
               
               
                   
                 2_no_Glyco 
                   
                   
                   
               
               
                   
                 dg_Design_ 
                 325 
                 N16C, N79Q 
                 SVFVMK C GTNVACLVKEFYPKDIRINLVSSKKITEFDPAIVISPSGKYNAVKLGK 
               
               
                   
                 2_hypeCys 
                   
                   
                 YEDSNSVTCSVQHD Q KTVHSTDFE 
               
               
                   
                 1_no_Glyco 
                   
                   
                   
               
               
                   
                 dg_Design_ 
                 326 
                 N16Q, N79Q, 
                 SVFVMK Q GTNVACLVKEFYPKDIRINLVSSKKITEFDPAI C ISPSGKYNAVKLGK 
               
               
                   
                 2_hypeCys 
                   
                 V50C 
                 YEDSNSVTCSVQHD Q KTVHSTDFE 
               
               
                   
                 2_no_Glyco 
                   
                   
                   
               
               
                   
                 dg_Design_ 
                 327 
                 N16Q, N79Q, 
                 SVFVMK Q GTNVACLVKEFYPKDIRINLVSSKKITEF C PAIVISPSGKYNAVKLGK 
               
               
                   
                 2_hypeCys 
                   
                 D46C 
                 YEDSNSVTCSVQHD Q KTVHSTDFE 
               
               
                   
                 3_no_Glyco 
                   
                   
                   
               
               
                   
                 dg_Design_ 
                 328 
                 N16Q, N79Q, 
                 SV C VMK Q GTNVACLVKEFYPKDIRINLVSSKKITEFDPAIVISPSGKYNAVKLGK 
               
               
                   
                 2_Cys2_no 
                   
                 F12C 
                 YEDSNSVTCSVQHD Q KTVHSTDFE 
               
               
                   
                 _Glyco 
                   
                   
                   
               
               
                   
                 dg_Design_ 
                 329 
                 N16Q, N79Q, 
                 SVFV C K Q GTNVACLVKEFYPKDIRINLVSSKKITEFDPAIVISPSGKYNAVKLGK 
               
               
                   
                 2_Cysl_no 
                   
                 M14C 
                 YEDSNSVTCSVQHD Q KTVHSTDFE 
               
               
                   
                 Glyco 
                   
                   
                   
               
               
                   
                 dg_Design_ 
                 328 
                 N16Q, N79Q, 
                   
               
               
                   
                 2_Cys3_no 
                   
                 F12C 
                   
               
               
                   
                 _Glyco 
                   
                   
                   
               
               
                   
                 dg_Design_ 
                 330 
                 N16Q, N79Q, 
                 SVFVMK Q GTNVACLVKEFYPKDIRINLVSSKKITEFDPAIVISPSGKYNAVKLGK 
               
               
                   
                 2_Cys4_no 
                   
                 F87C 
                 YEDSNSVTCSVQHD Q KTVHSTD C E 
               
               
                   
                 _Glyco 
                   
                   
                   
               
               
                   
                 dg_Design_ 
                 331 
                 N16Q, N79Q, 
                 SVFVMK Q GTNVACLVKEFYPKDIRINLVSSKKITEFDPAIVISPSGKYNAVKLGK 
               
               
                   
                 2_Cys5_no 
                   
                 E88C 
                 YEDSNSVTCSVQHD Q KTVHSTDF C   
               
               
                   
                 _Glyco 
                   
                   
                   
               
               
                   
                 dg_crossed 
                 332 
                 N16Q, N79Q, 
                 SVFVMK Q GTNVACLVKEFYPKDIRINLVSSKKITEFDPAI C ISPSGKYNAVKLGK 
               
               
                   
                 _Design_1 
                   
                 V50C 
                 YEDSNSVTCSVQHD Q KTVHSTD-- 
               
               
                   
                 (the delta 
                   
                   
                   
               
               
                   
                 on heavy 
                   
                   
                   
               
               
                   
                 chain) 
                   
                   
                   
               
               
                   
                 dg_crossed 
                 332 
                   
                   
               
               
                   
                 _Design_2 
                   
                   
                   
               
               
                   
                 (the delta 
                   
                   
                   
               
               
                   
                 on heavy 
                   
                   
                   
               
               
                   
                 chain) 
                   
                   
                   
               
               
                   
               
               
                 ConjunctionR 
                 H_Conjunct 
                 117 
                 HCJ4 
                 SSASLDADVSP 
               
               
                   
                 ion_4 
                   
                   
                   
               
               
                   
                 H_Conjunct 
                 118 
                 HCJ5 
                 TDKQLDADVSP 
               
               
                   
                 ion_5 
                   
                   
                   
               
               
                   
               
               
                 ConjunctionT 
                 L_Conjuncti 
                 119 
                 LCJ4 
                 PRSQPHTKP 
               
               
                   
                 on_4 
                   
                   
                   
               
               
                   
                 L_Conjuncti 
                 120 
                 LCJ5 
                 EPRSQPHTKP 
               
               
                   
                 on_5 
                   
                   
                   
               
               
                   
               
               
                 Conjunction′S 
                 Conjunction 
                 121 
                 CJ′3G1 
                 PPIKSDKTHTCPPCPAPEAAGGP 
               
               
                   
                 ′3_IgG1 
                   
                   
                   
               
               
                   
                 Conjunction 
                 122 
                 CJ′3G4 
                 PPI---YGPPCPPCPAPEFLGGP 
               
               
                   
                 ′3_IG4 
                   
                   
                   
               
               
                   
               
               
                 ConjunctionH 
                 H_Conjunct 
                 123 
                 HCJ6 
                 SSRSQPHTKP 
               
               
                   
                 ion_6 
                   
                   
                   
               
               
                   
                 H_Conjunct 
                 124 
                 HCJ7 
                 EPRSQPHTKP 
               
               
                   
                 ion_7 
                   
                   
                   
               
               
                   
               
               
                 ConjunctionJ 
                 L_Conjuncti 
                 125 
                 LCJ6 
                 KDKLDADVSP 
               
               
                   
                 on_6 
                   
                   
                   
               
               
                   
                 L_Conjuncti 
                 126 
                 LCJ7 
                 TDKLDADVSP 
               
               
                   
                 on_7 
                   
                   
                   
               
               
                   
               
               
                 Conjunction′I 
                 Conjunction 
                 127 
                 CJ′4G1 
                 EPKSSDKTHTCPPCPAPEAAGGP 
               
               
                   
                 ′4_IgG1 
                   
                   
                   
               
               
                   
                 Conjunction 
                 128 
                 CJ′4G4 
                 ESK---YGPPCPPCPAPEFLGGP 
               
               
                   
                 ′4_IgG4 
                   
                   
                   
               
               
                   
               
               
                 Conjunction 
                 H_Conjunct 
                 129 
                 HCJ3 
                 SSASIQNPDP 
               
               
                 (Alpha-Beta, 
                 ion_3 
                   
                   
                   
               
               
                 crossed, more 
                 L_Conjuncti 
                 50 
                 LCJ3 
                   
               
               
                 antibody) 
                 on_3 
                   
                   
                   
               
               
                   
               
               
                 Conjunction 
                 H_Conjunct 
                 130 
                 HCJ4 
                 SSPDIQNPDP 
               
               
                 (Alpha-Beta, 
                 ion_4 
                   
                   
                   
               
               
                 crossed, more 
                 L_Conjuncti 
                 50 
                 LCJ3 
                   
               
               
                 TCR) 
                 on_3 
                   
                   
                   
               
               
                   
               
               
                 Conjunction 
                 Light chain 
                 131 
                 LCJA 
                 RTVAAGTP 
               
               
                 (PreAlpha- 
                 Conjunction 
                   
                   
                   
               
               
                 Beta, normal, 
                   
                   
                   
                   
               
               
                 more 
                   
                   
                   
                   
               
               
                 antibody) 
                   
                   
                   
                   
               
               
                   
               
               
                 Conjunction 
                 Light chain 
                 50 
                 LCJC 
                   
               
               
                 (PreAlpha- 
                 Conjunction 
                   
                   
                   
               
               
                 Beta, crossed, 
                 Heavy chain 
                 132 
                 HCJC 
                 SSASGVGGTP 
               
               
                 more 
                 Conjunction 
                   
                   
                   
               
               
                 antibody) 
                   
                   
                   
                   
               
               
                   
               
               
                 Conjunction 
                 Light chain 
                 50 
                 LCJD 
                   
               
               
                 (PreAlpha- 
                 Conjunction 
                   
                   
                   
               
               
                 Beta, crossed, 
                 Heavy chain 
                 133 
                 HCJD 
                 SSPTGVGGTP 
               
               
                 more TCR) 
                 Conjunction 
                   
                   
                   
               
               
                   
               
               
                 Conjunction′ 
                 Conjunction 
                 134 
                 CJ′2G1 
                 SDKTHTCPPCPAPEAAGGP 
               
               
                 (Alpha-Beta, 
                 ′ 
                   
                   
                   
               
               
                 PreAlpha- 
                 Conjunction 
                 135 
                 CJ′2G4 
                 --YGPPCPPCPAPEFLGGP 
               
               
                 Beta, crossed) 
                 ′ 
                   
                   
                   
               
               
                   
               
               
                 PreTCR_Desi 
                 Light 
                 136 
                 VL(CD3)- 
                 DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQQKPGQPPKLLIYWASTR 
               
               
                 gn5_crossed_1 
                   
                   
                 HCJB- CBeta 
                 QSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTQSHTLRTFGGGTKVEIKLEDLKNVF 
               
               
                   
                   
                   
                 (N69Q, S16C)- 
                 PPEVAVFEP C EAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQP 
               
               
                   
                   
                   
                 CJ′ 1G 
                 AL Q DSRYALSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWG 
               
               
                   
                   
                   
                   
                 RA 
               
               
                   
                 Heavy 
                 137 
                 VH(CD3)- 
                 QVQLVQSGAEVKKPGSSVKVSCKASGFAFTDYYIHWVRQAPGQGLEWMGWISPGNVNTKY 
               
               
                   
                 (Conjunctio 
                   
                 HCJC- 
                 NENFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDGYSLYYFDYWGQGTLVTVSSA 
               
               
                   
                 n more 
                   
                 CPreAlpha 
                 SGVGGTPFPCLAPPIMLLVDGKQQMVVVCLVLDVAPPGLDSPIWFSAG Q GSALDAFTYGP 
               
               
                   
                 antibody) 
                   
                 (S11C, N50Q) - 
                 SPATDGTWTNLAHLSLPSEELASWEPLVCHTGPGAEGHSRSTQPMHLSGEASTARTSDKT 
               
               
                   
                   
                   
                   
                 HTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE 
               
               
                   
                   
                   
                 CJ′2G1- Fc 
                 VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP 
               
               
                   
                   
                   
                   
                 REPQVYTLPPSR E E M TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS 
               
               
                   
                   
                   
                   
                 FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 
               
               
                   
               
               
                 PreTCR_Desi 
                 Light 
                 136 
                   
                   
               
               
                 gn_5_crossed_ 
                   
                   
                 VL(CD3)- 
                   
               
               
                 2 
                   
                   
                 HCJB- CBeta 
                   
               
               
                   
                   
                   
                 (N69Q, S16C)- 
                   
               
               
                   
                   
                   
                 CJ′ 1G 
                   
               
               
                   
                 Heavy 
                 138 
                 VH(CD3)- 
                 QVQLVQSGAEVKKPGSSVKVSCKASGFAFTDYYIHWVRQAPGQGLEWMGWISPGNVNTKY 
               
               
                   
                 (Conjunctio 
                   
                 HCJD- 
                 NENFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDGYSLYYFDYWGQGTLVTVSSP 
               
               
                   
                 n more 
                   
                 CPreAlpha 
                 TGVGGTPFPCLAPPIMLLVDGKQQMVVVCLVLDVAPPGLDSPIWFSAG Q GSALDAFTYGP 
               
               
                   
                 PreTCR) 
                   
                 (S11) -CJ′2G1- 
                 SPATDGTWTNLAHLSLPSEELASWEPLVCHTGPGAEGHSRSTQPMHLSGEASTARTSDKT 
               
               
                   
                   
                   
                 Fc Conjunction 
                 HTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE 
               
               
                   
                   
                   
                   
                 VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP 
               
               
                   
                   
                   
                   
                 REPQVYTLPPSR E E M TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS 
               
               
                   
                   
                   
                   
                 FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 
               
               
                   
               
               
                 PreTCR_Desi 
                 Light 
                 139 
                 VL(CD3)- 
                 DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQQKPGQPPKLLIYWASTR 
               
               
                 gn_6_crossed_ 
                   
                   
                 LCJC- CBeta 
                 QSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTQSHTLRTFGGGTKVEIKLEDLKNVF 
               
               
                 1 
                   
                   
                 (A18C, N69Q) 
                 PPEVAVFEPSECEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQP 
               
               
                   
                   
                   
                   
                 AL Q DSRYALSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWG 
               
               
                   
                   
                   
                   
                 RA 
               
               
                   
                 Heavy 
                 137 
                   
                   
               
               
                   
                 (Conjunctio 
                   
                   
                   
               
               
                   
                 n more 
                   
                   
                   
               
               
                   
                 antibody) 
                   
                   
                   
               
               
                   
               
               
                 PreTCR_Desi 
                 Light 
                 139 
                   
                   
               
               
                 gn_6_crossed_ 
                   
                   
                   
                   
               
               
                 2 
                 Heavy 
                 138 
                   
                   
               
               
                   
                 (Conjunctio 
                   
                   
                   
               
               
                   
                 n more 
                   
                   
                   
               
               
                   
                 TCR) 
                   
                   
                   
               
               
                   
               
               
                 Conjunction′ 
                 IgG1 
                 140 
                 CJ′2G1 
                 SDKTHTCPPCPAPEAAGGP 
               
               
                 (alpha-beta, 
                   
                   
                   
                   
               
               
                 crossed, IgG1) 
                   
                   
                   
                   
               
               
                   
               
               
                 Conjunction′ 
                 IgG4 
                 141 
                 CJ′2G4 
                 YGPPCPPCPAPEFLGGP 
               
               
                 (alpha-beta, 
                   
                   
                   
                   
               
               
                 crossed, IgG4) 
                   
                   
                   
                   
               
               
                   
               
               
                 Anti-CD3 
                   
                 300 
                   
                 QVQLVQSGAEVKKPGSSVKVSCKASGFAFTDYYIHWVRQAPGQGLEWMGWISPGN 
               
               
                 Antibody VH 
                   
                   
                   
                 VNTKYNENFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDGYSLYYFDYWG 
               
               
                   
                   
                   
                   
                 QGTLVTV 
               
               
                   
               
               
                 Anti-CD3 
                   
                 301 
                   
                 DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQQKPGQPPKLLIY 
               
               
                 Antibody VL 
                   
                   
                   
                 WASTRQSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTQSHTLRTFGGGTKVE 
               
               
                   
                   
                   
                   
                 I 
               
               
                   
               
               
                 Fc(G1) 
                   
                 302 
                   
                 SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE 
               
               
                   
                   
                   
                   
                 EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV 
               
               
                   
                   
                   
                   
                 YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS 
               
               
                   
                   
                   
                   
                 FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 
               
               
                   
               
               
                 Fc(G4) 
                   
                 303 
                   
                 SVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPRE 
               
               
                   
                   
                   
                   
                 EQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQV 
               
               
                   
                   
                   
                   
                 YTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS 
               
               
                   
                   
                   
                   
                 FFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 
               
               
                   
               
            
           
         
       
     
     EXAMPLES 
     Example 1: Design and Engineering of Antibody and TCR Chimeric Proteins 
     TCR Sequences 
     TCRs are heterodimeric proteins made up of two chains. About 95% human T cells have TCRs consisting of alpha and beta chains, whereas the rest 5% have TCRs composed of gamma and delta chains. The constant region of human alpha chain has only one gene TRAC. The constant region of human beta chain has two subclasses: gene TRBC1 and TRBC2. In Protein Data Bank (PDB), the number of crystal structures of TRBC1 is relatively more than those of TRBC2, so TRBC1 sequences were chosen as the major backbone to design the polypeptide complex disclosed herein (“WuXiBody”). A typical amino acid sequence of TRBC1 can be found in PDB structure 4L4T. 
     Interchain Disulphide-Bond of TCR 
     TCR crystal structures were used to guide our WuXiBody design. Unlike native TCR anchored on the membrane of T cell surface, soluble TCR molecules are less stable, although its 3D structure is very similar to antibody Fab. As a matter of fact, the instability of TCR in soluble condition used to be a big obstacle that prevents the elucidation of its crystal structure (Wang 2014, supra). We adopted a strategy of introducing a pair of Cys mutations in the TCR constant region and found it can significantly improve chain assembly and enhance expression. 
     Effects of Interchain Disulphide Bond on the Antibody Expression 
     To determine whether the disulphide bonds play a role in maintaining the WuXiBody structures, constructs with and/or without disulphide bonds in the TCR constant region of the chimeric antibodies were expressed. SDS-PAGE results of the expressed WuXiBody were shown in  FIGS. 15-17 . All the WuXiBody expressed were whole IgG-like construct with two identical arms. Expression of constructs with and without cysteine mutations between S56 in CBeta and T49 in CAlpha, expression of constructs with cysteine mutations between S16 or A18 in CBeta and S11 in CPre-Alpha, between Q57 in CGamma and V50 in CDelta, and between A19 in CGamma and E88 in CDelta were tested. 
     The result of constructs expression with disulphide bond absent in CBeta/CAlpha (SEQ ID NOs: 32/42) indicates that constructs absent of disulphide bonds were unable to maintain the antibody structure (see  FIG. 15B ). Expression of constructs with disulphide bond absent in CBeta/CPre-Alpha and CGamma/CDelta was also tested and show similar results. In contrast, constructs containing mutated cysteine residues were able to form interchain disulphide bonds, which were capable of maintaining the Ig-like structures (see  FIG. 15A ). 
     Design Chimeric Domains of WuXibody 
     The cysteine pair mutations (numbering in reference of the sequences in  FIGS. 19A-19E ) in the TCR constant regions were incorporated into different construct designs for the TCR chimeric antibodies, which were shown in Table 21. 
     
       
         
           
               
             
               
                 TABLE 21 
               
             
            
               
                   
               
               
                 Paired Cys mutations to introduce interchain disulphide bond 
               
            
           
           
               
               
               
            
               
                 Alpha-Beta 
                 PreAlpha-Beta 
                 Delta-Gamma 
               
            
           
           
               
               
               
               
               
            
               
                 Cys Pair 
                 Cys Pair 
                 Corresponding Protein Name 
                 Cys Pair 
                 Corresponding Protein Name 
               
               
                 Mutations 
                 Mutations 
                 SEQ ID NOs. in HC/LC 
                 Mutations 
                 SEQ ID NO. in HC/LC 
               
               
                   
               
               
                 Y11-S16 
                 S11-S16 
                 Design_5_Pre_TCR_Conjunction′1_Cys13 
                 F12-S17 
                 Design_2_Cys2_no_Glyco 
               
               
                   
                   
                 SEQ ID NOs: 70/71 
                   
                 SEQ ID NOs: 96/95 
               
               
                 L13-F13 
                 S11-A18 
                 Design_6_Pre_TCR_Conjunction′1_Cys14 
                 F12-E20 
                 Design_2_Cys3_no_Glyco 
               
               
                   
                   
                 SEQ ID NOs: 72/71 
                   
                 SEQ ID NOs: 99/95 
               
               
                 L13-S16 
                 S11-E19 
                 Design_7_Pre_TCR_Conjunction′1_Cys15 
                 M14-F14 
                 Design_2_Cys1_no_Glyco 
               
               
                   
                   
                 SEQ ID NOs: 73/71 
                   
                 SEQ ID NOs: 98/97 
               
               
                 S16-V12 
                 A13-F13 
                 Design_3_Pre_TCR_Conjunction′1_Cys11 
                 N16-T12 
                 Design_2_hypeCys1_no_Glyco 
               
               
                   
                   
                 SEQ ID NOs: 69/68 
                   
                 SEQ ID NOs: 90/89 
               
               
                 S16-E14 
                 A13-S16 
                 Design_4_Pre_TCR_Conjunction′1_Cys12 
                 D46-M62 
                 Design_2_hypeCys3_no_Glyco 
               
               
                   
                   
                 SEQ ID NOs: 70/68 
                   
                 SEQ ID NOs: 94/93 
               
               
                 V23-F13 
                 I16-A11 
                 Design_10_Pre_TCR_Conjunction′1_Cys4 
                 V50-Q57 
                 Design_2_hypeCys2_no_Glyco 
               
               
                   
                   
                 SEQ ID NOs: 78/77 
                   
                 SEQ ID NOs: 92/91 
               
               
                 Y44-L62 
                 S62-S56 
                 Design_8_Pre_TCR_Conjunction′1_Cys1_4L4T_1 
                 F87-A19 
                 Design_2_Cys4_no_Glyco 
               
               
                   
                   
                 SEQ ID NOs: 75/74 
                   
                 SEQ ID NOs:101/100 
               
               
                 T46-D58 
                 T65-S56 
                 Design_9_Pre_TCR_Conjunction′1_Cys2_4L4T_2 
                 E88-A19 
                 Design_2_Cys5_no_Glyco 
               
               
                   
                   
                 SEQ ID NOs: 75/76 
                   
                 SEQ ID NOs: 101/102 
               
               
                 T46-S76 
                 Y59-S76 
                 Design_2_Pre_TCR_Conjunction′1_Cys10 
               
               
                   
                   
                 SEQ ID NOs: 67/66 
               
               
                 T49-S56 
               
               
                 L51-S56 
               
               
                 S62-S56 
               
               
                 S62-R78 
               
               
                   
               
            
           
         
       
     
     For paired Cys mutations in TCR Alpha-Beta constant regions, T49C-S56C disulphide bond was used for all the designs. 
     The conjunctions connecting antibody variable and TCR constant domains, their relative fusion orientations, as well as the Fc-connecting conjunctions were all carefully fine-turned to make a stable and functional WuXiBody. As TCR structure is very similar to antibody Fab, we superimposed the antibody Fv homology model on TCR variable region (PDB 4L4T,  FIG. 2B ). The superimposed structure indicates that antibody Fv is structurally compatible with TCR constant domain. Based on this structural alignment and corresponding sequences, all the relevant engineering parameters were designed, as illustrated below. 
     Domain Orientation 
     As the fusion orientations of both VH-CBeta/VL-CAlpha and the crossed VH-CAlpha/VL-CBeta could correctly assemble the chimeric protein, we designed and tested both orientations. The sequence homology of VH-VL is closer to the TCR VBeta-VAlpha. We named VH-CBeta/VL-CAlpha formulas as “normal orientation”, and the VH-CAlpha/VL-CBeta as “crossed orientation”. 
     First and Second Conjunction Domains 
     We aligned the sequences of antibody and TCR based on structure alignment, and found the conjunctions defined in germline sequence are not always consistent to what it displays on the structure. For example, from sequence definition, the conjunctions connecting VH and the CH1 should start right after the last two residues “SS” in VH region. However, structurally, these two residues are already part of the conjunction. We defined our conjunctions based on structure rather than sequence. 
     Table 1 and Table 2 in the present disclosure showed the structure-based sequence alignment for two studied orientations. As it was challenging to predict which domain would be compatible with which conjunction domain, we checked how antibody and TCR conjunctions overlapped on the superimposed structures, and estimated the possible replacement using one to the other. The designs of the conjunction domains were listed in Table 1 and Table 2. 
     Third Conjunction Domains 
     Similar strategy as described above was used to align the human IgG1 and IgG4 hinge with TCR membrane proximal region (i.e. TCR hinge), and their overlap at the structural level was checked as well. Table 7 and Table 8 in the present disclosure listed designs of the third conjunction domains. 
     FG Loop and DE Loop 
     Aligning the structures of TCR constant region with that of antibody revealed that the FG and DE loop of TCR beta chain are longer than the corresponding region in antibody CH1.  FIGS. 3A-3B  show the differences of constant regions between T cell beta chain and antibody heavy chain. To test how these two loops could perturb the structure if CH1 was replaced by TCR beta, constructs with and without these two loops were designed. 
     With the above mentioned considerations, a total of nine constructs were designed by combining these parameters as listed in Table 22 and Table 23. 
     
       
         
           
               
             
               
                 TABLE 22 
               
             
            
               
                   
               
               
                 Design of chimeric proteins (CBeta/CAlpha) of WuXiBody 
               
            
           
           
               
               
               
               
               
               
            
               
                 SEQ ID NOs: 
                   
                   
                   
                   
                   
               
               
                 (Heavy Chain (HC)/ 
               
               
                 Light Chain (LC))-IgG1 
                 Orientation 
                 Conjunction 
                 Conjunction′ 
                 FG loop 
                 DE loop 
               
               
                   
               
               
                 Design_1 
                 Normal 
                 Conjunction_1 
                 Conjunction′_1 
                 Native 
                 Native 
               
               
                 SEQ ID NO: 2/1 
                   
                   
                 (IgGl, IgG4) 
               
               
                 Design_2 
                 Normal 
                 Conjunction_2 
                 Conjunction′_1 
                 Native 
                 Native 
               
               
                 SEQ ID NO: 4/3 
                   
                   
                 (IgG1, IgG4) 
               
               
                 Design_3 
                 Cross 
                 Conjunction_3 
                 Conjunction′_2 
                 Native 
                 Native 
               
               
                 SEQ ID NO: 9/8 
                   
                   
                 (IgG1, IgG4) 
               
               
                 Design_4 
                 Cross 
                 Conjunction_4 
                 Conjunction′_2 
                 Native 
                 Native 
               
               
                 SEQ ID NO: 10/8 
                   
                   
                 (IgG1, IgG4) 
               
               
                 Design_5 
                 Normal 
                 Conjunction_1 
                 Conjunction′_1 
                 Replaced 
                 Native 
               
               
                 SEQ ID NO: 5/1 
                   
                   
                 (IgG1, IgG4) 
               
               
                 Design_6 
                 Normal 
                 Conjunction_2 
                 Conjunction′_1 
                 Replaced 
                 Native 
               
               
                 SEQ ID NO: 6/3 
                   
                   
                 (IgG1, IgG4) 
               
               
                 Design_6a 
                 Normal 
                 Conjunction_2 
                 Conjunction′_1 
                 Replaced 
                 Replaced 
               
               
                 SEQ ID NO: 7/3 
                   
                   
                 (IgG1, IgG4) 
               
               
                 Design_7 
                 Cross 
                 Conjunction_3 
                 Conjunction′_2 
                 Replaced 
                 Native 
               
               
                 SEQ ID NO: 9/11 
                   
                   
                 (IgG1, IgG4) 
               
               
                 Design_8 
                 Cross 
                 Conjunction_4 
                 Conjunction′_2 
                 Replaced 
                 Native 
               
               
                 SEQ ID NO: 10/11 
                   
                   
                 (IgG1, IgG4) 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 23 
               
             
            
               
                   
               
               
                 Components and sequences of chimeric proteins (CBeta/CAlpha) of WuXiBody 
               
            
           
           
               
               
            
               
                   
                 Domains from N-terminal to C-terminal and their SEQ ID NOs 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 Antibody Heavy 
                 First or Second 
                   
                 Third Conjunction 
                   
               
               
                 Complex name and 
                 Chain Variable 
                 Conjunction 
                 TCR Constant 
                 domain + 
                 Dimerization 
               
               
                 chain SEQ ID NOs: 
                 Domain (VH or VL) 
                 domain (CJ) 
                 Domain (C1 or C2) 
                 Hinge (CJ′) 
                 Domain (D) 
               
               
                   
               
               
                 Design_1 HC 
                 VH(CD3) 
                 HCJ1 
                 Cbeta(S56C) 
                 CJ′1G1 
                 FcG1 
               
               
                 SEQ ID NO: 2 
                 SEQ ID NO: 300 
                 SEQ ID NO: 49 
                 SEQ ID NO: 33 
                 SEQ ID NO: 53 
                 SEQ ID NO: 302 
               
               
                 Design_1 LC 
                 VL(CD3) 
                 LCJ1 
                 CAlpha(T49C) 
               
               
                 SEQ ID NO: 1 
                 SEQ ID NO: 301 
                 SEQ ID NO: 51 
                 SEQ ID NO: 43 
               
               
                 Design_2 HC 
                 VH(CD3) 
                 HCJ2 
                 Cbeta(S56C) 
                 CJ′1G1 
                 FcG1 
               
               
                 SEQ ID NO: 4 
                 SEQ ID NO: 300 
                 SEQ ID NO: 50 
                 SEQ ID NO: 33 
                 SEQ ID NO: 53 
                 SEQ ID NO: 302 
               
               
                 Design_2 LC 
                 VL(CD3) 
                 LCJ2 
                 CAlpha(T49C) 
               
               
                 SEQ ID NO: 3 
                 SEQ ID NO: 301 
                 SEQ ID NO: 52 
                 SEQ ID NO: 43 
               
               
                 Design_3 HC 
                 VH(CD3) 
                 HCJ3 
                 CAlpha(T49C) 
                 CJ′2G1 
                 FcG1 
               
               
                 SEQ ID NO: 9 
                 SEQ ID NO: 300 
                 SEQ ID NO: 129 
                 SEQ ID NO: 43 
                 SEQ ID NO: 134 
                 SEQ ID NO: 302 
               
               
                 Design_3 LC 
                 VL(CD3) 
                 LCJ3 
                 Cbeta(S56C) 
               
               
                 SEQ ID NO: 8 
                 SEQ ID NO: 301 
                 SEQ ID NO: 308 
                 SEQ ID NO: 33 + 
               
               
                   
                   
                   
                 NO: 306 
               
               
                 Design_4 HC 
                 VH(CD3) 
                 HCJ4 
                 CAlpha(T49C) 
                 CJ′2G1 
                 FcG1 
               
               
                 SEQ ID NO: 10 
                 SEQ ID NO: 300 
                 SEQ ID NO: 130 
                 SEQ ID NO: 43 
                 SEQ ID NO: 134 
                 SEQ ID NO: 302 
               
               
                 Design_4 LC 
                 VL(CD3) 
                 LCJ3 
                 Cbeta(S56C) 
               
               
                 SEQ ID NO: 8 
                 SEQ ID NO: 301 
                 SEQ ID NO: 308 
                 SEQ ID NO: 33 + 
               
               
                   
                   
                   
                 NO: 306 
               
               
                 Design_5 HC 
                 VH(CD3) 
                 HCJ1 
                 Cbeta(S56C) (FG-) 
                 CJ′1G1 
                 FcG1 
               
               
                 SEQ ID NO: 5 
                 SEQ ID NO: 300 
                 SEQ ID NO: 49 
                 SEQ ID NO: 37 
                 SEQ ID NO: 53 
                 SEQ ID NO: 302 
               
               
                 Design_5 LC 
                 VL(CD3) 
                 LCJ1 
                 CAlpha(T49C) 
               
               
                 SEQ ID NO: 1 
                 SEQ ID NO: 301 
                 SEQ ID NO: 51 
                 SEQ ID NO: 43 
               
               
                 Design_6 HC 
                 VH(CD3) 
                 HCJ1 
                 CBeta(S56C)(FG-) 
                 CJ′1G1 
                 FcG1 
               
               
                 SEQ ID NO: 6 
                 SEQ ID NO: 300 
                 SEQ ID NO: 50 
                 SEQ ID NO: 37 
                 SEQ ID NO: 53 
                 SEQ ID NO: 302 
               
               
                 Design_6 LC 
                 VL(CD3) 
                 LCJ2 
                 CAlpha(T49C) 
               
               
                 SEQ ID NO: 3 
                 SEQ ID NO: 301 
                 SEQ ID NO: 52 
                 SEQ ID NO: 43 
               
               
                 Design_6a HC 
                 VH(CD3) 
                 HCJ2 
                 CBeta(S56C)(DE-FG-) 
                 CJ′1G1 
                 FcG1 
               
               
                 SEQ ID NO: 7 
                 SEQ ID NO: 300 
                 SEQ ID NO: 50 
                 SEQ ID NO: 41 
                 SEQ ID NO: 53 
                 SEQ ID NO: 302 
               
               
                 Design_6a LC 
                 VL(CD3) 
                 LCJ2 
                 CAlpha(T49C) 
               
               
                 SEQ ID NO: 3 
                 SEQ ID NO: 301 
                 SEQ ID NO: 52 
                 SEQ ID NO: 43 
               
               
                 Design_7 HC 
                 VH(CD3) 
                 HCJ3 
                 CAlpha(T49C) 
                 CJ′2G1 
                 FcG1 
               
               
                 SEQ ID NO: 9 
                 SEQ ID NO: 300 
                 SEQ ID NO: 129 
                 SEQ ID NO: 43 
                 SEQ ID NO: 134 
                 SEQ ID NO: 302 
               
               
                 Design_7 LC 
                 VL(CD3) 
                 LCJ3 
                 CBeta(S56C)(FG-) 
               
               
                 SEQ ID NO: 11 
                 SEQ ID NO: 301 
                 SEQ ID NO: 308 
                 SEQ ID NO: 37 + 
               
               
                   
                   
                   
                 NO: 306 
               
               
                 Design_8 HC 
                 VH(CD3) 
                 HCJ4 
                 CAlpha(T49C) 
                 CJ′2G1 
                 FcG1 
               
               
                 SEQ ID NO: 10 
                 SEQ ID NO: 300 
                 SEQ ID NO: 130 
                 SEQ ID NO: 43 
                 SEQ ID NO: 134 
                 SEQ ID NO: 302 
               
               
                 Design_8 LC 
                 VL(CD3) 
                 LCJ3 
                 CBeta(S56C)(FG-) 
               
               
                 SEQ ID NO: 11 
                 SEQ ID NO: 301 
                 SEQ ID NO: 308 
                 SEQ ID NO: 37 + 
               
               
                   
                   
                   
                 NO: 306 
               
               
                   
               
            
           
         
       
     
     Example 2: Generation and Characterization of Monospecific TCR/Antibody Chimeras 
     Before fusing TCR constant domains into bispecific antibody constructs, the feasibility of introducing them into a regular monospecific IgG was firstly evaluated. An anti-CD3 antibody developed in-house, named T3, was selected to conduct this Proof-of-Concept study. The constant domains CH1 and CL of T3 IgG were replaced by the corresponding TCR constant region (CAlpha and CBeta). All the nine different strategies listed in Table 22 (see above) were applied, and all constructs were expressed in Expi293 system. 
     Table 24 listed the expression level of the designed proteins in harvested supernatants quantified by Q-ELISA. In general, most of the “normal orientation” designs had better expression than the “crossed orientation” formats, and most of the “TCR conjunctions” are better than the “antibody conjunctions”. Two constructs of “normal orientation”, Design_5 and Design-6 had expression comparable to Design_2). For two constructs of “cross orientation”, Design_7, 8 had better expression than Design_3, 4. The low expression of extra-long FG loop in TCR CBeta was observed, suggesting that this FG loop might cause significant steric clashes with fused antibody VL domain. 
     
       
         
           
               
             
               
                 TABLE 24 
               
             
            
               
                   
               
               
                 The expression levels and CD3 binding 
               
               
                 of all the designs in supernatant 
               
            
           
           
               
               
               
            
               
                   
                 Expression Level 
                 Concentration (nM) in FACS 
               
            
           
           
               
               
               
               
               
            
               
                 Samples 
                 in Supernatant 
                 5.0 
                 0.4 
                 0.032 
               
            
           
           
               
               
               
            
               
                 (IgG1) 
                 (ug/mL) 
                 MFI 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 T3 
                 N/A 
                 5101 
                 2937 
                 408 
               
               
                 Design_1 
                 72.04 
                 5441 
                 2190 
                 292 
               
               
                 Design_2 
                 204.42 
                 5833 
                 2616 
                 380 
               
               
                 Design_3 
                 15.35 
                 5089 
                 982 
                 137 
               
               
                 Design_4 
                 26.11 
                 5438 
                 1213 
                 168 
               
               
                 Design_5 
                 113.68 
                 5388 
                 1865 
                 249 
               
               
                 Design_6 
                 178.56 
                 5789 
                 3914 
                 613 
               
               
                 Design_6a 
                 173.60 
                 5794 
                 2822 
                 405 
               
               
                 Design_7 
                 75.69 
                 6322 
                 1929 
                 259 
               
               
                 Design_8 
                 98.63 
                 6412 
                 1831 
                 243 
               
               
                   
               
            
           
         
       
     
     These results were completely different with what Wu et al. observed in their similar antibody-TCR chimeras design (Wu et al. 2015, supra): Their “crossed orientation” designs had low expression. Their “normal orientation” designs did not even express. 
     To confirm whether the expressed proteins had correct folding and retained the original function, we tested their binding on CD3 positive Jurkat cells. FACS bindings of all the samples were carried out at three different concentrations: 5.0, 0.4 and 0.032 nm. The original wild type antibody T3 was used as the positive control. The dose-dependent CD3 binding data was listed in column 3-5 in Table 24. Design_2, Design_6 and Design_6a showed best binding capability, comparable to native antibody T3. It is interesting that all these three constructs happened to be the best three expressed formats in mammalian cell. This strong correlation suggested that the level of expression or binding might result from the same molecular origin, i.e., the compatibility between antibody variable domain and TCR constant domains, which required careful designs of the components such as conjunction domains and interchain disulphide bond etc. 
     Based on the expression level and binding activity, Design_2 was selected as the final format to proceed. 
     Example 3: De-Glycosylation 
     Post translational modifications (PTM) like N-glycosylation sites on an antibody may cause heterogeneity of the proteins, becoming a challenge in development stages. Therefore, an attempt was conducted to remove the N-glycosylation sites on TCR constant region. There are total four N-glycosylation sites found in the TCR constant region. One is on CBeta (N69, see SEQ ID NO: 244), and the other three are on CAlpha (N34, N68 and N79, see SEQ ID NO: 241). The expression data of the present disclosure suggested that these sites, especially the sites on CAlpha, were indeed heavily glycosylated when the molecule was expressed in mammalian cell. 
     All the glycosylation sites on Design_2 were removed by substituting four Asn residues with Gln or Ala (refer to Design_2-QQQQ or -AAAA, see Table 25). Although this strategy is very general in protein engineering, it has been reported that Gln/Ala mutations may affect the expression level of TCR/antibody chimeric proteins (Wu et al., 2015, supra). To mitigate this risk, residues from Pre-TCR (N68S on CAlpha) and macaca TCR (N79 on CAlpha, N69E on CBeta) at the corresponding positions (refer to Design_2-QSKE and -ASKE) were also used (see Table 25). In addition, it was reported that there may exist an atypical glycosylation site on CAlpha (N61) (Wollscheid et al., Nature Biotechnology, 27(4), pp. 378-386 (2009) “Mass-spectrometric identification and relative quantification of N-linked cell surface glycoproteins.” Wollscheid B., Bausch-Fluck D., Henderson C., O&#39;Brien R., Bibel M., Schiess R., Aebersold R., Watts J. D., Nat. Biotechnol. 27:378-386(2009) [PubMed] [Europe PMC]). Therefore, this residue was also mutated to Gln (refer to Design_2-QQQQQ, see Table 25). All mutants were expressed in Expi293 for further tests. 
     
       
         
           
               
             
               
                 TABLE 25 
               
             
            
               
                   
               
               
                 The expression levels of all the de- 
               
               
                 glycosylation designs in supernatant 
               
            
           
           
               
               
               
               
            
               
                   
                   
                 Expression Level 
                 SEQ ID NO: 
               
               
                   
                   
                 in Supernatant 
                 (HC-CBeta/LC- 
               
               
                   
                 Sample 
                 (ug/mL) 
                 CAlpha) 
               
               
                   
                   
               
            
           
           
               
               
               
               
            
               
                   
                 Design_2-QQQQ 
                 334.39 
                 13/12 (IgG1) 
               
               
                   
                   
                   
                  21/12(IgG4) 
               
               
                   
                 Design_2-AAAA 
                 414.58 
                 15/14 (IgG1) 
               
               
                   
                 Design_2-QSKE 
                 311.48 
                 17/16 (IgG1) 
               
               
                   
                 Design_2-ASKE 
                 107.89 
                 17/18 (IgG1) 
               
               
                   
                 Design_2-QQQQQ 
                 213.31 
                 20/19 (IgG1) 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 26 
               
             
            
               
                   
               
               
                 Components of the de-glycosylation designs 
               
            
           
           
               
               
            
               
                   
                 Domains from N-terminal to C-terminal and their SEQ ID NOs 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 Antibody Heavy 
                 First or Second 
                   
                 Third 
                   
               
               
                   
                 Chain Variable 
                 Conjunction 
                 TCR Constant 
                 Conjunction 
                 Dimerization 
               
               
                 Complex name and 
                 Domain 
                 domain 
                 Domain 
                 domain + Hinge 
                 Domain 
               
               
                 chain SEQ ID NOs: 
                 (VH or VL) 
                 (CJ) 
                 (C1 or C2) 
                 (CJ′) 
                 (D) 
               
               
                   
               
               
                 Design_2-QQQQ 
                 VH(CD3) 
                 HCJ2 
                 CBeta(S56C) (N69Q) 
                 CJ′1G1 
                 FcG1 
               
               
                 (IgG1) HC 
               
               
                 SEQ ID NO: 13 
                 SEQ ID NO: 300 
                 SEQ ID NO: 50 
                 SEQ ID NO: 34 
                 SEQ ID NO: 53 
                 SEQ ID NO: 302 
               
               
                 Design_2-QQQQ 
                 VL(CD3) 
                 LCJ2 
                 CAlpha(T49C) 
               
               
                 (IgG1) LC 
                   
                   
                 (N34Q + N68Q + N79Q) 
               
               
                 SEQ ID NO: 12 
                 SEQ ID NO: 301 
                 SEQ ID NO: 52 
                 SEQ ID NO: 44 
               
               
                 Design_2-QQQQ 
                 VH(CD3) 
                 HCJ2 
                 CBeta(S56C) (N69Q) 
                 CJ′1G4 
                 FcG4 
               
               
                 (IgG4) HC 
               
               
                 SEQ ID NO: 21 
                 SEQ ID NO: 300 
                 SEQ ID NO: 50 
                 SEQ ID NO: 34 
                 SEQ ID NO: 54 
                 SEQ ID NO: 303 
               
               
                 Design_2-QQQQ 
                 VL(CD3) 
                 LCJ2 
                 CAlpha(T49C) 
               
               
                 (IgG4) LC 
                   
                   
                 (N34Q + N68Q + N79Q) 
               
               
                 SEQ ID NO: 12 
                 SEQ ID NO: 301 
                 SEQ ID NO: 52 
                 SEQ ID NO: 44 
               
               
                 Design_2-AAAA 
                 VH(CD3) 
                 HCJ2 
                 CBeta(S56C) (N69A) 
                 CJ′1G1 
                 FcG1 
               
               
                 (IgG1) HC 
               
               
                 SEQ ID NO: 15 
                 SEQ ID NO: 300 
                 SEQ ID NO: 50 
                 SEQ ID NO: 35 
                 SEQ ID NO: 53 
                 SEQ ID NO: 302 
               
               
                 Design_2-AAAA 
                 VL(CD3) 
                 LCJ2 
                 CAlpha(T49C) 
               
               
                 (IgG1) LC 
                   
                   
                 (N34A + N68A + N79A) 
               
               
                 SEQ ID NO: 14 
                 SEQ ID NO: 301 
                 SEQ ID NO: 52 
                 SEQ ID NO: 45 
               
               
                 Design_2-QSKE 
                 VH(CD3) 
                 HCJ2 
                 CBeta(S56C) (N69E) 
                 CJ′1G1 
                 FcG1 
               
               
                 (IgG1) HC 
               
               
                 SEQ ID NO: 17 
                 SEQ ID NO: 300 
                 SEQ ID NO: 50 
                 SEQ ID NO: 36 
                 SEQ ID NO: 53 
                 SEQ ID NO: 302 
               
               
                 Design_2-QSKE 
                 VL(CD3) 
                 LCJ2 
                 CAlpha(T49C) 
               
               
                 (IgG1) LC 
                   
                   
                 (N34Q + N68S + N79K) 
               
               
                 SEQ ID NO: 16 
                 SEQ ID NO: 301 
                 SEQ ID NO: 52 
                 SEQ ID NO: 46 
               
               
                 Design_2-ASKE 
                 VH(CD3) 
                 HCJ2 
                 CBeta(S56C) (N69E) 
                 CJ′1G1 
                 FcG1 
               
               
                 (IgG1) HC 
               
               
                 SEQ ID NO: 17 
                 SEQ ID NO: 300 
                 SEQ ID NO: 50 
                 SEQ ID NO: 36 
                 SEQ ID NO: 53 
                 SEQ ID NO: 302 
               
               
                 Design_2-ASKE 
                 VL(CD3) 
                 LCJ2 
                 CAlpha(T49C) 
               
               
                 (IgG1) LC 
                   
                   
                 (N34A + N68S + N79K) 
               
               
                 SEQ ID NO: 18 
                 SEQ ID NO: 301 
                 SEQ ID NO: 52 
                 SEQ ID NO: 47 
               
               
                 Design_2-QQQQQ 
                 VH(CD3) 
                 HCJ2 
                 CBeta(S56C) (N69Q) 
                 CJ′1G1 
                 FcG1 
               
               
                 (IgG1) HC 
               
               
                 SEQ ID NO: 20 
                 SEQ ID NO: 300 
                 SEQ ID NO: 50 
                 SEQ ID NO: 34 
                 SEQ ID NO: 53 
                 SEQ ID NO: 302 
               
               
                 Design_2-QQQQQ 
                 VL(CD3) 
                 LCJ2 
                 CAlpha(T49C) 
               
               
                 (IgG1) LC 
                   
                   
                 (N34Q + N68Q + 
               
               
                   
                   
                   
                 N79Q + N61Q) 
               
               
                 SEQ ID NO: 19 
                 SEQ ID NO: 301 
                 SEQ ID NO: 52 
                 SEQ ID NO: 48 
               
               
                   
               
            
           
         
       
     
     The expression quantities in supernatants were estimated by Q-ELISA shown in Table 25. Interestingly, only one of our de-glycosylation designs slightly decreased expression level. Simple mutations by Gln or Ala did not have any negative effects on the non-reduced gel ( FIG. 4 ), and 150 kd band was observed. On the reduced gel ( FIG. 4 ), the 25 kd band was observed. Both indicate the successful removal of glycosylation on light chain and heavy chain. The muteins with the removal of N-glycans on TCR constant region were tested on CD3-binding.  FIG. 5  showed the different muteins binding on CD3+ Jurkat cells. The curves of muteins only slightly shifted to the right compared to the wild type antibody T3, which might be due to detection antibody being more sensitive to human IgG than chimera. The maximum binding did not change. Overall, most of the de-glycosylation designs did not exhibit any obvious differences in either expression or binding. Design_2-QQQQ was chosen as the design for further studies. 
     In the similar study conducted by Wu et al., (Wu et al. 2015, supra), they performed de-glycosylation mutations on their “crossed orientation” formats, as their “normal orientation” formats did not express. 
     Example 4: Design of TCR Pre-Alpha/Beta Based WuXiBody 
     The pre-T-cell antigen receptor (pre-TCR), expressed by immature thymocytes, has a pivotal role in early T-cell development. Pre-TCR has a regular beta chain, but a special pre-alpha chain with only constant region available, whose sequence and structure are quite distinct from those of regular alpha chain. Since the constant region of regular TCR is compatible with antibody variable region, the Pre-TCR (see PDB 30F6, SEQ ID NO: 246) was expected to help design chimeric protein, too. The antibody designs were shown in Table 27. 
     
       
         
           
               
             
               
                 TABLE 27 
               
             
            
               
                   
               
               
                 Design of TCR pre-alpha/beta based chimeras for WuXiBody 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                 FG 
                 DE 
               
               
                   
                 Orientation 
                 Conjunction 
                 Conjunction′ 
                 loop 
                 loop 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 PreTCR_Design_A 
                 Normal 
                 Conjunction_A 
                 Conjunction′_1 
                 Native 
                 Native 
               
               
                   
                   
                   
                 (IgG1, IgG4) 
               
               
                 PreTCR_Design_B 
                 Normal 
                 Conjunction_B 
                 Conjunction′_1 
                 Native 
                 Native 
               
               
                   
                   
                   
                 (IgG1, IgG4) 
               
               
                 PreTCR_Design_C 
                 Cross 
                 Conjunction_C 
                 Conjunction′_2 
                 Native 
                 Native 
               
               
                   
                   
                   
                 (IgG1, IgG4) 
               
               
                 PreTCR_Design_D 
                 Cross 
                 Conjunction_D 
                 Conjunction′_2 
                 Native 
                 Native 
               
               
                   
                   
                   
                 (IgG1, IgG4) 
               
               
                   
               
            
           
         
       
     
     Total ten chimeric constructs were designed by combining these parameters, as listed in Table 28. 
     
       
         
           
               
             
               
                 TABLE 28 
               
             
            
               
                   
               
               
                 Correspondence of the design of chimeric Pre-TCR-antibody chimeric in IgG1 form 
               
            
           
           
               
               
               
            
               
                   
                   
                 SEQ ID NOs: 
               
               
                 Designs in Table 27 
                 Sequence file 
                 (HC/LC) 
               
               
                   
               
               
                 PreTCR_Design_B 
                 Design_1_Pre_TCR_Conjunction′1 
                 65/64 
               
               
                   
                 Design_2_Pre_TCR_Conjunction′_1_Cys10 
                 67/66 
               
               
                   
                 Design_3_Pre_TCR_Conjunction′_1_Cys11 
                 69/68 
               
               
                   
                 Design_4_Pre_TCR_Conjunction′_1_Cys12 
                 70/68 
               
               
                   
                 Design_5_Pre_TCR_Conjunction′_1_Cys13 
                 70/71 
               
               
                   
                 Design_6_Pre_TCR_Conjunction′_1_Cys14 
                 72/71 
               
               
                   
                 Design_7_Pre_TCR_Conjunction′_1_Cys15 
                 73/71 
               
               
                   
                 Design_8_Pre_TCR_Conjunction′_1_Cys1_4L4T_1 
                 75/74 
               
               
                   
                 Design_9_Pre_TCR_Conjunction′_1_Cys2_4L4T_2 
                 75/76 
               
               
                   
                 Design_10_Pre_TCR_Conjunction′_1_Cys4 
                 78/77 
               
               
                 PreTCR_Design_C 
                 PreTCR_Design_5_crossed_1 
                 137/136 
               
               
                   
                 PreTCR_Design_6_crossed_1 
                 137/139 
               
               
                 PreTCR_Design_D 
                 PreTCR_Design_5_crossed_2 
                 138/136 
               
               
                   
                 PreTCR_Design_6_crossed_2 
                 138/139 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 29 
               
               
                   
               
               
                 Components of the design of chimeric Pre-TCR-antibody chimeric in IgG1 form 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
            
               
                   
                 Domains from N-terminal to C-terminal and their SEQ ID NOs 
               
            
           
           
               
               
               
               
            
               
                   
                 Antibody Heavy 
                 First or Second 
                   
               
               
                   
                 Chain Variable 
                 Conjunction 
                 TCR Constant 
               
               
                 Complex name and 
                 Domain 
                 domain 
                 Domain 
               
               
                 chain SEQ ID NOs: 
                 (VH or VL) 
                 (CJ) 
                 (C1 or C2) 
               
               
                   
               
               
                 Design_1_Pre_TCR_Conjunction′1 HC 
                 VH(CD3) 
                 HCJB 
                 CBeta(N69Q) 
               
               
                 SEQ ID NO: 65 
                 SEQ ID NO: 300 
                 SEQ ID NO: 50 
                 SEQ ID NO: 84 
               
               
                 Design_1_Pre_TCR_Conjunction′1 LC 
                 VL(CD3) 
                 LCJB 
                 CPreAlpha(N50Q) 
               
               
                 SEQ ID NO: 64 
                 SEQ ID NO: 301 
                 SEQ ID NO: 309 
                 SEQ ID NO: 83 
               
               
                 Design_2_Pre_TCR_Conjunction′_1_Cys10 HC 
                 VH(CD3) 
                 HCJB 
                 Cbeta(S76C)(N69Q) 
               
               
                 SEQ ID NO: 67 
                 SEQ ID NO: 300 
                 SEQ ID NO: 50 
                 SEQ ID NO: 319 
               
               
                 Design_2_Pre_TCR_Conjunction′_1_Cys10 LC 
                 VL(CD3) 
                 LCJB 
                 CPreAlpha (Y59C)(N50Q) 
               
               
                 SEQ ID NO: 66 
                 SEQ ID NO: 301 
                 SEQ ID NO: 309 
                 SEQ ID NO: 311 
               
               
                 Design_3_Pre_TCR_Conjunction′_1_Cys11 HC 
                 VH(CD3) 
                 HCJB 
                 Cbeta(F13C)(N69Q) 
               
               
                 SEQ ID NO: 69 
                 SEQ ID NO: 300 
                 SEQ ID NO: 50 
                 SEQ ID NO: 320 
               
               
                 Design_3_Pre_TCR_Conjunction′_1_Cys11 LC 
                 VL(CD3) 
                 LCJB 
                 CPreAlpha (A13C)(N50Q) 
               
               
                 SEQ ID NO: 68 
                 SEQ ID NO: 301 
                 SEQ ID NO: 309 
                 SEQ ID NO: 312 
               
               
                 Design_4_Pre_TCR_Conjunction′_1_Cys12 HC 
                 VH(CD3) 
                 HCJB 
                 Cbeta(S16C)(N69Q) 
               
               
                 SEQ ID NO: 70 
                 SEQ ID NO: 300 
                 SEQ ID NO: 50 
                 SEQ ID NO: 321 
               
               
                 Design_4_Pre_TCR_Conjunction′_1_Cys12 LC 
                 VL(CD3) 
                 LCJB 
                 CPreAlpha (A13C)(N50Q) 
               
               
                 SEQ ID NO: 68 
                 SEQ ID NO: 301 
                 SEQ ID NO: 309 
                 SEQ ID NO: 312 
               
               
                 Design_5_Pre_TCR_Conjunction′_1_Cys13 HC 
                 VH(CD3) 
                 HCJB 
                 Cbeta(S16C)(N69Q) 
               
               
                 SEQ ID NO: 70 
                 SEQ ID NO: 300 
                 SEQ ID NO: 50 
                 SEQ ID NO: 321 
               
               
                 Design_5_Pre_TCR_Conjunction′_1_Cys13 LC 
                 VL(CD3) 
                 LCJB 
                 CPreAlpha (S11C)(N50Q) 
               
               
                 SEQ ID NO: 71 
                 SEQ ID NO: 301 
                 SEQ ID NO: 309 
                 SEQ ID NO: 313 
               
               
                 Design_6_Pre_TCR_Conjunction′_1_Cys14 HC 
                 VH(CD3) 
                 HCJB 
                 Cbeta(A18C)(N69Q) 
               
               
                 SEQ ID NO: 72 
                 SEQ ID NO: 300 
                 SEQ ID NO: 50 
                 SEQ ID NO: 322 
               
               
                 Design_6_Pre_TCR_Conjunction′_1_Cys14 LC 
                 VL(CD3) 
                 LCJB 
                 CPreAlpha (S11C)(N50Q) 
               
               
                 SEQ ID NO: 71 
                 SEQ ID NO: 301 
                 SEQ ID NO: 309 
                 SEQ ID NO: 313 
               
               
                 Design_7_Pre_TCR_Conjunction′_1_Cys15 HC 
                 VH(CD3) 
                 HCJB 
                 Cbeta(E19C)(N69Q) 
               
               
                 SEQ ID NO: 73 
                 SEQ ID NO: 300 
                 SEQ ID NO: 50 
                 SEQ ID NO: 323 
               
               
                 Design_7_Pre_TCR_Conjunction′_1_Cys15 LC 
                 VL(CD3) 
                 LCJB 
                 CPreAlpha (S11C)(N50Q) 
               
               
                 SEQ ID NO: 71 
                 SEQ ID NO: 301 
                 SEQ ID NO: 309 
                 SEQ ID NO: 313 
               
               
                 Design_8_Pre_TCR_Conjunction′_1_Cys1_4L4T_1 HC 
                 VH(CD3) 
                 HCJB 
                 Cbeta(S56C)(N69Q) 
               
               
                 SEQ ID NO: 75 
                 SEQ ID NO: 300 
                 SEQ ID NO: 50 
                 SEQ ID NO: 34 
               
               
                 Design_8_Pre_TCR_Conjunction′_1_Cys1_4L4T_1 LC 
                 VL(CD3) 
                 LCJB 
                 CPreAlpha (S62C)(N50Q) 
               
               
                 SEQ ID NO: 74 
                 SEQ ID NO: 301 
                 SEQ ID NO: 309 
                 SEQ ID NO: 314 
               
               
                 Design_9_Pre_TCR_Conjunction′_1_Cys2_4L4T_2 HC 
                 VH(CD3) 
                 HCJB 
                 Cbeta(S56C)(N69Q) 
               
               
                 SEQ ID NO: 75 
                 SEQ ID NO: 300 
                 SEQ ID NO: 50 
                 SEQ ID NO: 34 
               
               
                 Design_9_Pre_TCR_Conjunction′_1_Cys2_4L4T_2 LC 
                 VL(CD3) 
                 LCJB 
                 CPreAlpha (T65C)(N50Q) 
               
               
                 SEQ ID NO: 76 
                 SEQ ID NO: 301 
                 SEQ ID NO: 309 
                 SEQ ID NO: 315 
               
               
                 Design_10_Pre_TCR_Conjunction′_1_Cys4 HC 
                 VH(CD3) 
                 HCJB 
                 Cbeta(A11C)(N69Q) 
               
               
                 SEQ ID NO: 78 
                 SEQ ID NO: 300 
                 SEQ ID NO: 50 
                 SEQ ID NO: 324 
               
               
                 Design_10_Pre_TCR_Conjunction′_1_Cys4 LC 
                 VL(CD3) 
                 LCJB 
                 CPreAlpha (I16C)(N50Q) 
               
               
                 SEQ ID NO: 77 
                 SEQ ID NO: 301 
                 SEQ ID NO: 309 
                 SEQ ID NO: 316 
               
               
                 PreTCR_Design_5_crossed_1 HC 
                 VH(CD3) 
                 HCJC 
                 CPreAlpha (S11C)(N50Q) 
               
               
                 SEQ ID NO: 137 
                 SEQ ID NO: 300 
                 SEQ ID NO: 132 
                 SEQ ID NO: 313 
               
               
                 PreTCR_Design_5_crossed_1 LC 
                 VL(CD3) 
                 LCJC 
                 Cbeta (N69Q, S16C) 
               
               
                 SEQ ID NO: 136 
                 SEQ ID NO: 301 
                 SEQ ID NO: 50 
                 SEQ ID NO: 321 + 306 
               
               
                 PreTCR_Design_6_crossed_1 HC 
                 VH(CD3) 
                 HCJC 
                 CPreAlpha (S11C)(N50Q) 
               
               
                 SEQ ID NO: 137 
                 SEQ ID NO: 300 
                 SEQ ID NO: 132 
                 SEQ ID NO: 313 
               
               
                 PreTCR_Design_6_crossed_1 LC 
                 VL(CD3) 
                 LCJC 
                 Cbeta(N69Q, A18C) 
               
               
                 SEQ ID NO: 139 
                 SEQ ID NO: 301 
                 SEQ ID NO: 50 
                 SEQ ID NO: 322 + 306 
               
               
                 PreTCR_Design_5_crossed_2 HC 
                 VH(CD3) 
                 HCJD 
                 CPreAlpha (S11C)(N50Q) 
               
               
                 SEQ ID NO: 138 
                 SEQ ID NO: 300 
                 SEQ ID NO: 133 
                 SEQ ID NO: 313 
               
               
                 PreTCR_Design_5_crossed_2 LC 
                 VL(CD3) 
                 LCJC 
                 Cbeta (N69Q, S16C) 
               
               
                 SEQ ID NO: 136 
                 SEQ ID NO: 301 
                 SEQ ID NO: 50 
                 SEQ ID NO: 
               
               
                   
                   
                   
                 NO: 321 + 306 
               
               
                 PreTCR_Design_6_crossed_2 HC 
                 VH(CD3) 
                 HCJD 
                 CPreAlpha (S11C)(N50Q) 
               
               
                 SEQ ID NO: 138 
                 SEQ ID NO: 300 
                 SEQ ID NO: 133 
                 SEQ ID NO: 313 
               
               
                 PreTCR_Design_6_crossed_2 LC 
                 VL(CD3) 
                 LCJC 
                 Cbeta(N69Q, A18C) 
               
               
                 SEQ ID NO: 139 
                 SEQ ID NO: 301 
                 SEQ ID NO: 50 
                 SEQ ID NO: 322 + 306 
               
               
                   
               
            
           
           
               
               
            
               
                   
                 Domains from N-terminal to C-terminal and their SEQ ID NOs 
               
            
           
           
               
               
               
            
               
                   
                 Third Conjunction 
                 Dimerization 
               
               
                 Complex name and 
                 domain + Hinge 
                 Domain 
               
               
                 chain SEQ ID NOs: 
                 (CJ′) 
                 (D) 
               
               
                   
               
               
                 Design_1_Pre_TCR_Conjunction′1 HC 
                 CJ′1G1 
                 FcG1 
               
               
                 SEQ ID NO: 65 
                 SEQ ID NO: 53 
                 SEQ ID NO: 302 
               
               
                 Design_1_Pre_TCR_Conjunction′1 LC 
               
               
                 SEQ ID NO: 64 
               
               
                 Design_2_Pre_TCR_Conjunction′_1_Cys10 HC 
                 CJ′1G1 
                 FcG1 
               
               
                 SEQ ID NO: 67 
                 SEQ ID NO: 53 
                 SEQ ID NO: 302 
               
               
                 Design_2_Pre_TCR_Conjunction′_1_Cys10 LC 
               
               
                 SEQ ID NO: 66 
               
               
                 Design_3_Pre_TCR_Conjunction′_1_Cys11 HC 
                 CJ′1G1 
                 FcG1 
               
               
                 SEQ ID NO: 69 
                 SEQ ID NO: 53 
                 SEQ ID NO: 302 
               
               
                 Design_3_Pre_TCR_Conjunction′_1_Cys11 LC 
               
               
                 SEQ ID NO: 68 
               
               
                 Design_4_Pre_TCR_Conjunction′_1_Cys12 HC 
                 CJ′1G1 
                 FcG1 
               
               
                 SEQ ID NO: 70 
                 SEQ ID NO: 53 
                 SEQ ID NO: 302 
               
               
                 Design_4_Pre_TCR_Conjunction′_1_Cys12 LC 
               
               
                 SEQ ID NO: 68 
               
               
                 Design_5_Pre_TCR_Conjunction′_1_Cys13 HC 
                 CJ′1G1 
                 FcG1 
               
               
                 SEQ ID NO: 70 
                 SEQ ID NO: 53 
                 SEQ ID NO: 302 
               
               
                 Design_5_Pre_TCR_Conjunction′_1_Cys13 LC 
               
               
                 SEQ ID NO: 71 
               
               
                 Design_6_Pre_TCR_Conjunction′_1_Cys14 HC 
                 CJ′1G1 
                 FcG1 
               
               
                 SEQ ID NO: 72 
                 SEQ ID NO: 53 
                 SEQ ID NO: 302 
               
               
                 Design_6_Pre_TCR_Conjunction′_1_Cys14 LC 
               
               
                 SEQ ID NO: 71 
               
               
                 Design_7_Pre_TCR_Conjunction′_1_Cys15 HC 
                 CJ′1G1 
                 FcG1 
               
               
                 SEQ ID NO: 73 
                 SEQ ID NO: 53 
                 SEQ ID NO: 302 
               
               
                 Design_7_Pre_TCR_Conjunction′_1_Cys15 LC 
               
               
                 SEQ ID NO: 71 
               
               
                 Design_8_Pre_TCR_Conjunction′_1_Cys1_4L4T_1 HC 
                 CJ′1G1 
                 FcG1 
               
               
                 SEQ ID NO: 75 
                 SEQ ID NO: 53 
                 SEQ ID NO: 302 
               
               
                 Design_8_Pre_TCR_Conjunction′_1_Cys1_4L4T_1 LC 
               
               
                 SEQ ID NO: 74 
               
               
                 Design_9_Pre_TCR_Conjunction′_1_Cys2_4L4T_2 HC 
                 CJ′1G1 
                 FcG1 
               
               
                 SEQ ID NO: 75 
                 SEQ ID NO: 53 
                 SEQ ID NO: 302 
               
               
                 Design_9_Pre_TCR_Conjunction′_1_Cys2_4L4T_2 LC 
               
               
                 SEQ ID NO: 76 
               
               
                 Design_10_Pre_TCR_Conjunction′_1_Cys4 HC 
                 CJ′1G1 
                 FcG1 
               
               
                 SEQ ID NO: 78 
                 SEQ ID NO: 53 
                 SEQ ID NO: 302 
               
               
                 Design_10_Pre_TCR_Conjunction′_1_Cys4 LC 
               
               
                 SEQ ID NO: 77 
               
               
                 PreTCR_Design_5_crossed_1 HC 
                 CJ′2G1 
                 FcG1 
               
               
                 SEQ ID NO: 137 
                 SEQ ID NO: 134 
                 SEQ ID NO: 302 
               
               
                 PreTCR_Design_5_crossed_1 LC 
               
               
                 SEQ ID NO: 136 
               
               
                 PreTCR_Design_6_crossed_1 HC 
                 CJ′2G4 
                 FcG4 
               
               
                 SEQ ID NO: 137 
                 SEQ ID NO: 134 
                 SEQ ID NO: 303 
               
               
                 PreTCR_Design_6_crossed_1 LC 
               
               
                 SEQ ID NO: 139 
               
               
                 PreTCR_Design_5_crossed_2 HC 
                 CJ′2G1 
                 FcG1 
               
               
                 SEQ ID NO: 138 
                 SEQ ID NO: 134 
                 SEQ ID NO: 302 
               
               
                 PreTCR_Design_5_crossed_2 LC 
               
               
                 SEQ ID NO: 136 
               
               
                 PreTCR_Design_6_crossed_2 HC 
                 CJ′2G1 
                 FcG1 
               
               
                 SEQ ID NO: 138 
                 SEQ ID NO: 134 
                 SEQ ID NO: 302 
               
               
                 PreTCR_Design_6_crossed_2 LC 
               
               
                 SEQ ID NO: 139 
               
               
                   
               
            
           
         
       
     
     The experience learned in Examples 1-3 suggested that “normal orientation” and the conjunction domain with more TCR residues was more suitable in producing good chimeric proteins. Thus, the same strategy was adopted and the light chain and heavy chain conjunction domains as shown in Table 3 and Table 4 were designed. Different from regular alpha chain, there is only one glycosylation site (N50) in the TCR pre-alpha chain, which was mutated to Gln residue (see SEQ ID NO: 247). The entire heavy chain with beta constant region was the same as that of Design_2 in Table 22, with N-glycosylation site (N69) substituted to Gln residue (see SEQ ID NO: 244). 
     The third conjunction domains in normal orientation were designed identical to that in Table 7, and the third conjunction domains in cross orientation were designed identical to that in Table 8 (TCR alpha/beta based chimeric antibodies). 
     Pre-TCR does not have native interchain disulphide bond above the third conjunction domain. Similar to the engineering work conducted on regular TCR, we rationally introduced disulphide bond at the beta and pre-alpha interface in the constant region to improve the stability of the chimeric protein (see Table 11). All the interfacial residues on pre-TCR crystal structure (PDB 30F6) were inspected and the list of interchain pairs was obtained whose CAlpha and CBeta carbon atoms were within 7 Å and 5 Å, respectively (see Table 11). Each identified pair was then substituted to Cys residues and the mutein was expressed in Expi293 cells. 
     Example 5: Design of TCR Gamma/Delta Based Chimeric Antibodies 
     TCRs that are made up of gamma and delta chain are less common, but the heterodimeric nature of the protein could also help design new chimeric format. Following the same strategy and procedure that were validated in Example 1, we conducted new chimeric designs that used the constant region of delta-gamma TCR to replace the corresponding region of antibody. The structure of delta-gamma TCR (PDB 4LFH, see SEQ ID NO: 249 and 252) was used to facilitate the structure-guided sequence alignment between antibody and TCR. 
     Table 5 and Table 6 listed designed conjunction domains for “normal orientation” and “crossed orientation”, respectively. The corresponding IgG1 and IgG4 conjunction domains of different orientations were shown in Table 9 and Table 10. The structure of delta-gamma TCR is more similar to antibody, rather than that of alpha-beta TCR. No additional FG and DE loop designs were performed. N-glycosylation sites (N65 on gamma, and N16 and N79 on delta, see SEQ ID NO: 250) were all removed by Gln (Q) substitutions. The contact interface disulphide bond was designed based on the same strategy introduced in Example 4. 
     
       
         
           
               
             
               
                 TABLE 30 
               
             
            
               
                   
               
               
                 Design of chimeric TCR/antibody 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                 First and 
                   
                   
                   
               
               
                   
                   
                 second 
                 Third 
               
               
                   
                   
                 conjunction 
                 conjunction 
                 FG 
                 DE 
               
               
                   
                 Orientation 
                 domain 
                 domain 
                 loop 
                 loop 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 dg_Design_1 
                 Normal 
                 Conjunction_4 
                 Conjunction′_3 
                 Native 
                 Native 
               
               
                   
                   
                   
                 (IgG1, IgG4) 
               
               
                 dg_Design_2 
                 Normal 
                 Conjunction_5 
                 Conjunction′_3 
                 Native 
                 Native 
               
               
                   
                   
                   
                 (IgG1, IgG4) 
               
               
                 dg_Design_3 
                 Cross 
                 Conjunction_6 
                 Conjunction′_4 
                 Native 
                 Native 
               
               
                   
                   
                   
                 (IgG1, IgG4) 
               
               
                 dg_Design_4 
                 Cross 
                 Conjunction_7 
                 Conjunction′_4 
                 Native 
                 Native 
               
               
                   
                   
                   
                 (IgG1, IgG4) 
               
               
                   
               
            
           
         
       
     
     A total of thirteen chimeric constructs were designed by combining these parameters, as listed in Table 31. 
     
       
         
           
               
             
               
                 TABLE 31 
               
             
            
               
                   
               
               
                 Correspondence of the design of chimeric TCR/antibody for CGamma/CDelta 
               
            
           
           
               
               
               
            
               
                 Designs in Table 30 of IgG1 
                 Construct of Design 
                 SEQ ID NOs in HC/LC 
               
               
                   
               
               
                 dg_Design_1 
                 dg_Design_1 
                 108/107 
               
               
                 dg_Design_2 
                 dg_Design_2 
                 106/105 
               
               
                   
                 dg_Design_2_no_Glyco 
                 86/85 
               
               
                   
                 dg_Design_2_hypeCys1_no_Glyco 
                 90/89 
               
               
                   
                 dg_Design_2_hypeCys2_no_Glyco 
                 92/91 
               
               
                   
                 dg_Design_2_hypeCys3_no_Glyco 
                 94/93 
               
               
                   
                 dg_Design_2_Cys2_no_Glyco 
                 96/95 
               
               
                   
                 dg_Design_2_Cys1_no_Glyco 
                 98/97 
               
               
                   
                 dg_Design_2_Cys3_no_Glyco 
                 99/95 
               
               
                   
                 dg_Design_2_Cys4_no_Glyco 
                 101/100 
               
               
                   
                 dg_Design_2_Cys5_no_Glyco 
                 101/102 
               
               
                 dg_Design_3 
                 dg_crossed_Design_1 
                 110/109 
               
               
                 dg_Design_4 
                 dg_crossed_Deisgn_2 
                 112/111 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 32 
               
             
            
               
                   
               
               
                 Components of the design of chimeric TCR/antibody for CGamma/CDelta 
               
            
           
           
               
               
            
               
                   
                 Domains from N-terminal to C-terminal and their SEQ ID NOs 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 Antibody Heavy 
                 First or Second 
                   
                 Third 
                   
               
               
                   
                 Chain Variable 
                 Conjunction 
                 TCR Constant 
                 Conjunction 
                 Dimerization 
               
               
                 Complex name and 
                 Domain 
                 domain 
                 Domain 
                 domain + Hinge 
                 Domain 
               
               
                 chain SEQ ID NOs: 
                 (VH or VL) 
                 (CJ) 
                 (C1 or C2) 
                 (CJ′) 
                 (D) 
               
               
                   
               
               
                 dg_Design_1 HC 
                 VH(CD3) 
                 HCJ4 
                 CGamma 
                 CJ′3G1 
                 FcG1 
               
               
                 SEQ ID NO: 108 
                 SEQ ID NO: 300 
                 SEQ ID NO: 117 
                 SEQ ID NO: 113 
                 SEQ ID NO: 121 
                 SEQ ID NO: 302 
               
               
                 dg_Design_1 LC 
                 VL(CD3) 
                 LCJ4 
                 CDelta 
               
               
                 SEQ ID NO: 107 
                 SEQ ID NO: 301 
                 SEQ ID NO: 119 
                 SEQ ID NO: 310 
               
               
                 dg_Design_2 HC 
                 VH(CD3) 
                 HCJ5 
                 CGamma 
                 CJ′3G1 
                 FcG1 
               
               
                 SEQ ID NO: 106 
                 SEQ ID NO: 300 
                 SEQ ID NO: 118 
                 SEQ ID NO: 113 
                 SEQ ID NO: 121 
                 SEQ ID NO: 302 
               
               
                 dg_Design_2_LC 
                 VL(CD3) 
                 LCJ5 
                 CDelta 
               
               
                 SEQ ID NO: 105 
                 SEQ ID NO: 301 
                 SEQ ID NO: 120 
                 SEQ ID NO: 115 
               
               
                 dg_Design_2_no_Glyco HC 
                 VH(CD3) 
                 HCJ5 
                 CGamma (N65Q) 
                 CJ′3G1 
                 FcG1 
               
               
                 SEQ ID NO: 86 
                 SEQ ID NO: 300 
                 SEQ ID NO: 118 
                 SEQ ID NO: 114 
                 SEQ ID NO: 121 
                 SEQ ID NO: 302 
               
               
                 dg_Design_2_no_Glyco LC 
                 VL(CD3) 
                 LCJ5 
                 CDelta(N16Q + N79Q) 
               
               
                 SEQ ID NO: 85 
                 SEQ ID NO: 301 
                 SEQ ID NO: 120 
                 SEQ ID NO: 116 
               
               
                 dg_Design_2_hypeCys1_no_Glyco HC 
                 VH(CD3) 
                 HCJ5 
                 CGamma(T12C) (N65Q) 
                 CJ′3G1 
                 FcG1 
               
               
                 SEQ ID NO: 90 
                 SEQ ID NO: 300 
                 SEQ ID NO: 118 
                 SEQ ID NO: 333 
                 SEQ ID NO: 121 
                 SEQ ID NO: 302 
               
               
                 dg_Design_2_hypeCys1_no_Glyco LC 
                 VL(CD3) 
                 LCJ5 
                 CDelta (N16C) (N79Q) 
               
               
                 SEQ ID NO: 89 
                 SEQ ID NO: 301 
                 SEQ ID NO: 120 
                 SEQ ID NO: 325 
               
               
                 dg_Design_2_hypeCys2_no_Glyco HC 
                 VH(CD3) 
                 HCJ5 
                 CGamma (Q57C) (N65Q) 
                 CJ′3G1 
                 FcG1 
               
               
                 SEQ ID NO: 92 
                 SEQ ID NO: 300 
                 SEQ ID NO: 118 
                 SEQ ID NO: 334 
                 SEQ ID NO: 121 
                 SEQ ID NO: 302 
               
               
                 dg_Design_2_hypeCys2_no_Glyco LC 
                 VL(CD3) 
                 LCJ5 
                 CDelta (V50C) 
               
               
                   
                   
                   
                 (N16Q + N79Q) 
               
               
                 SEQ ID NO: 91 
                 SEQ ID NO: 301 
                 SEQ ID NO: 120 
                 SEQ ID NO: 326 
               
               
                 dg_Design_2_hypeCys3_no_Glyco HC 
                 VH(CD3) 
                 HCJ5 
                 CGamma (M62C) (N65Q) 
                 CJ′3G1 
                 FcG1 
               
               
                 SEQ ID NO: 94 
                 SEQ ID NO: 300 
                 SEQ ID NO: 118 
                 SEQ ID NO: 335 
                 SEQ ID NO: 121 
                 SEQ ID NO: 302 
               
               
                 dg_Design_2_hypeCys3_no_Glyco LC 
                 VL(CD3) 
                 LCJ5 
                 CDelta (D46C) 
               
               
                   
                   
                   
                 (N16Q + N79Q) 
               
               
                 SEQ ID NO: 93 
                 SEQ ID NO: 301 
                 SEQ ID NO: 120 
                 SEQ ID NO: 327 
               
               
                 dg_Design_2_Cys2_no_Glyco HC 
                 VH(CD3) 
                 HCJ5 
                 CGamma(S17C) (N65Q) 
                 CJ′3G1 
                 FcG1 
               
               
                 SEQ ID NO: 96 
                 SEQ ID NO: 300 
                 SEQ ID NO: 118 
                 SEQ ID NO: 336 
                 SEQ ID NO: 121 
                 SEQ ID NO: 302 
               
               
                 dg_Design_2_Cys2_no_Glyco LC 
                 VL(CD3) 
                 LCJ5 
                 CDelta (F12C) 
               
               
                   
                   
                   
                 (N16Q + N79Q) 
               
               
                 SEQ ID NO: 95 
                 SEQ ID NO: 301 
                 SEQ ID NO: 120 
                 SEQ ID NO: 328 
               
               
                 dg_Design_2_Cys1_no_Glyco HC 
                 VH(CD3) 
                 HCJ5 
                 CGamma(F14C) (N65Q) 
                 CJ′3G1 
                 FcG1 
               
               
                 SEQ ID NO: 98 
                 SEQ ID NO: 300 
                 SEQ ID NO: 118 
                 SEQ ID NO: 337 
                 SEQ ID NO: 121 
                 SEQ ID NO: 302 
               
               
                 dg_Design_2_Cys1_no_Glyco LC 
                 VL(CD3) 
                 LCJ5 
                 CDelta(M14C) 
               
               
                   
                   
                   
                 (N16Q + N79Q) 
               
               
                 SEQ ID NO: 97 
                 SEQ ID NO: 301 
                 SEQ ID NO: 120 
                 SEQ ID NO: 329 
               
               
                 dg_Design_2_Cys3_no_Glyco HC 
                 VH(CD3) 
                 HCJ5 
                 CGamma (E20C) (N65Q) 
                 CJ′3G1 
                 FcG1 
               
               
                 SEQ ID NO: 99 
                 SEQ ID NO: 300 
                 SEQ ID NO: 118 
                 SEQ ID NO: 338 
                 SEQ ID NO: 121 
                 SEQ ID NO: 302 
               
               
                 dg_Design_2_Cys3 no Glyco LC 
                 VL(CD3) 
                 LCJ5 
                 CDelta (F12C) 
               
               
                   
                   
                   
                 (N16Q + N79Q) 
               
               
                 SEQ ID NO: 95 
                 SEQ ID NO: 301 
                 SEQ ID NO: 120 
                 SEQ ID NO: 328 
               
               
                 dg_Design_2_Cys4_no_Glyco HC 
                 VH(CD3) 
                 HCJ5 
                 CGamma (A19C) (N65Q) 
                 CJ′3G1 
                 FcG1 
               
               
                 SEQ ID NO: 101 
                 SEQ ID NO: 300 
                 SEQ ID NO: 118 
                 SEQ ID NO: 339 
                 SEQ ID NO: 121 
                 SEQ ID NO: 302 
               
               
                 dg_Design_2_Cys4_no_Glyco LC 
                 VL(CD3) 
                 LCJ5 
                 CDelta(F87C) 
               
               
                   
                   
                   
                 (N16Q + N79Q) 
               
               
                 SEQ ID NO: 100 
                 SEQ ID NO: 301 
                 SEQ ID NO: 120 
                 SEQ ID NO: 330 
               
               
                 dg_Design_2_Cys5_no_Glyco HC 
                 VH(CD3) 
                 HCJ5 
                 CGamma (A19C) (N65Q) 
                 CJ′3G1 
                 FcG1 
               
               
                 SEQ ID NO: 101 
                 SEQ ID NO: 300 
                 SEQ ID NO: 118 
                 SEQ ID NO: 339 
                 SEQ ID NO: 121 
                 SEQ ID NO: 302 
               
               
                 dg_Design_2_Cys5_no_Glyco LC 
                   
                 LCJ5 
                 CDelta (E88C) 
               
               
                   
                   
                   
                 (N16Q + N79Q) 
               
               
                 SEQ ID NO: 102 
                   
                 SEQ ID NO: 120 
                 SEQ ID NO: 331 
               
               
                 dg_crossed_Design_1 HC 
                 VH(CD3) 
                 HCJ6 
                 CDelta 
                 CJ′4G1 
                 FcG1 
               
               
                 SEQ ID NO: 110 
                 SEQ ID NO: 300 
                 SEQ ID NO: 123 
                 SEQ ID NO: 332 
                 SEQ ID NO: 127 
                 SEQ ID NO: 302 
               
               
                 dg_crossed_Design_1 LC 
                 VL(CD3) 
                 LCJ6 
                 CGamma 
               
               
                 SEQ ID NO: 109 
                 SEQ ID NO: 301 
                 SEQ ID NO: 125 
                 SEQ ID NO: 340 
               
               
                 dg_crossed_Design_2 HC 
                 VH(CD3) 
                 HCJ7 
                 CDelta 
                 CJ′4G1 
                 FcG1 
               
               
                 SEQ ID NO: 112 
                 SEQ ID NO: 300 
                 SEQ ID NO: 124 
                 SEQ ID NO: 332 
                 SEQ ID NO: 127 
                 SEQ ID NO: 302 
               
               
                 dg_crossed_Design_2 LC 
                 VL(CD3) 
                 LCJ7 
                 CGamma 
               
               
                 SEQ ID NO: 111 
                 SEQ ID NO: 301 
                 SEQ ID NO: 126 
                 SEQ ID NO: 340 
               
               
                   
               
            
           
         
       
     
     Example 6: Antibody Heavy-Light Chain Mispairing Tests 
     One of the challenges in producing bispecific antibody in IgG-like format is the uncontrolled mispairing of light and heavy chains. We evaluated whether the TCR beta and alpha-replaced CH1 and CL domain can assemble with normal IgG heavy chain and light chain when they were co-expressed in a single host cell. 
     Besides the anti-CD3 antibody T3, we also developed a monoclonal antibody U4 that targets B-lymphocyte antigen CD19. In order to check how likely the light chains and heavy chains of two native antibodies can be mispaired, the light-heavy pairs of T3 and U4 were switched on purpose (T3_light-U4_heavy, T3_heavy-U4_light), and co-expressed in Expi293 cells. The same study using the TCR-modified T3 was also conducted as side-by-side comparison.  FIGS. 6A-6B  displayed SDS-PAGE data of the proteins in both IgG1 and IgG4. For the switched pairs using native antibodies, the 150 kd band in non-reduced page, and the 50 kd, 23 kd bands in reduced page, clearly confirmed the assembly of mispaired IgG protein. However, after introducing the TCR-modified T3, the 150 kd bands were not observed from the gel any more, indicating neither of the non-cognate pairs can assemble into antibody-like molecule. These data confirmed that our designed TCR-modified Fab can effectively prevent mispairing non-cognitive chains. 
     Example 7: Production and Characterization of Fab-TCR Chimera 
     To make sure that TCR-modified antibody Fab can be used to design bispecific antibody, Fab fragments truncated at two positions were constructed.  FIG. 8  shows that the TCR-modified T3 Fabs with N-glycan removed were successfully expressed and purified (T3-Fab-Design_2.his1 (SEQ ID NO: 30/12) and T3-Fab-Design_2.his2 (SEQ ID NO: 31/12)). Their binding capability to CD3 were also evaluated on CD3+ Jurkat cells, and compared to the monovalent form of wild type T3.  FIG. 9  showed that the chimeric Fab and monovalent T3 had qualitatively similar binding behaviors. The deviations might result from the difference in detection methods for proteins with His and Fc tag. 
     Example 8: Generation and Characterization of TCR-Based Knobs-into-Holes Bispecific Antibody 
     After successfully fusing the TCR constant domain into the monospecific antibody T3, and confirming that the new format can effectively prevent chain mispairing with antibody U4, we proceeded to construct bispecific formats. 
     TCR-modified T3 and wild type U4, with “knobs-into-holes” mutations employed in Fc CH3 domain, were co-expressed from Expi293 cells. The mutations for “knobs-into-holes” were made at S139C and T151W in the CH3 domain (SEQ ID NO: 295, knob) of T3 and Y134C, T151S, L153A and Y192V in the CH3 domain (SEQ ID NO: 296, hole) of U4 in the IgG1 isotype. Alternatively, the knobs-into-holes mutations were made at S136C and T148W in the CH3 domain (SEQ ID NO: 298, knob) of T3 and Y131C, T148S, L150A and Y189V in the CH3 domain (SEQ ID NO: 299, hole) of U4 in the IgG4 isotype.  FIGS. 7A-7B  show the SDS-PAGE data of the produced proteins in IgG1 and IgG4 after purifications. The yield after first-step protein A purification achieved 125 mg/L and 173.7 mg/L, for IgG1 and IgG4 respectively. The correct molecular weight, i.e. the bands around 150 kd in non-reduced gel as well as the bands around 50 and 25 kd in reduced gel, were all clearly observed. The purified samples were further inspected in SEC-HPLC. The purity of IgG1 and IgG4 achieved 98.63% and 100%. The data indicated that the IgG-like molecules, both IgG1 and IgG4 were well expressed and assembled. These TCR-involved new bispecific formats were referred as ‘E17-Design_2-QQQQ’ (SEQ ID NO: 22/12/24/23 for IgG1 and SEQ ID NO: 25/12/26/23 for IgG4). 
     Although the expected molecular weight was observed for the designed bispecific antibody, it was necessary to inspect whether each arm maintained their original binding capability to their individual cognate antigen. Since for each target, E17-Design_2 was monovalent binder, we also constructed the monovalent version of native T3 and native U4 to make the side-by-side comparisons.  FIG. 10A  and  FIG. 10B  show the FACS binding results of the designed bispecific antibody to CD3+ Jurkat cells and CD19+ Ramos cells, respectively. The arm of the TCR-modified T3 exhibited moderate binding loss compared to the wild type T3, but IgG4 was better than IgG1 and close to the native protein. The binding of the U4 arm was not reduced by the neighboring engineered T3 arm. It had binding similar to the original U4 antibody in monovalent form. But interestingly, this time IgG1 performed better than IgG4. It is unclear why isotype matters in maintaining the monovalent binding. Factors like stability of TCR constant region, selection of the third conjunction domain designs, or interactions between two Fab arms could result in observed phenomena. 
     The monovalent bindings of the TCR-modified bispecific format to CD3 and CD19 were both reduced compared with their bivalent parental antibodies. It is known that T cell activation via CD3 binding is quite sensitive. Strong stimulations to T cells may cause side effects. Therefore, the relatively weak CD3-binding was probably acceptable and even desired for safety reason. However, the weak CD19-binding might directly affect its capability in bispecific antibody directed B cell killing, and thus reduce the drug efficacy. To confirm the importance of CD19 binding, and to test the universality of our chimeric designs, we built another bispecific constructs named as “F16-Design_2-QQQQ” in IgG4, in which the designed T3 arm was still monovalent, but the U4 arm was bivalent. 
     The new construct was expressed and purified, and the binding experiment was directly carried out.  FIGS. 11A-11B  showed its FACS binding data compared to previously designed E17 and two parental antibodies T3 and U4. It is interesting that the F16-Design_2-QQQQ improved both CD3- and CD19-binding (SEQ ID NO: 25/12/27/23 in the order HC/LC (anti-CD3)/HC/LC (anti-CD19)). Its CD19-binding (SEQ ID NO: 27/23) was comparable to the wild type antibody U4. The data confirmed that our chimeric design on T3 can be applied to different bispecific formats. 
     Example 9: In Vitro Assay of Bispecific Antibody-Directed Tumor Cell Killing 
     In vitro functional assay was performed to check activity of the designed bispecific format in T cell engaged killing of malignant B cells. E17 construct was tested first. The parental monospecific antibodies T3 and U4 were used as negative controls.  FIG. 12  shows the dose-dependent cell killing function of this E17 bispecific format. E17-IgG4 (EC50=57 pM) was more potent than E17-IgG1 (EC50=624 pM). In order to improve the activity of cell killing, F16 format, which had two CD19-binding sites, was also compared with E17. As shown in  FIG. 13 , compared with E17 (EC50=17.7 pM), the potency of F16 (EC50=5.5 pM) was 3 times improved. The data confirmed the binding of CD19 affected the cell killing effect. An irrelevant human IgG4 antibody was used as negative control. 
     Example 10: Mass Spectrometry Characterization 
     To confirm that the produced bispecific antibody had the correct assembly, we characterized the molecule E17-Design_2-QQQQ in mass spectrometry. The differences of theoretical molecular weight between two heavy chains and two light chains are around 4000 Da and 500 Da, respectively.  FIG. 14A  showed the spectra of the protein in non-reduced condition. The peak at 148180 Da was the expected molecule weight of the correctly assembled bispecific antibody. No observed other peak indicates that the “knobs-into-holes” mutations in Fc region as well as our TCR-replaced CH1/CL region worked properly in pairing the desired four chains. It is noteworthy that the non-reduced mass spectra cannot help distinguish the correct assembled bispecific antibody from the IgG that has both light chains mispaired. However, Example 4 indicates that mis-paired heavy and light chains would not express or assemble, which eliminated the possibility of mis-pairing of both pairs of heavy and light chains. 
     In non-reduced condition (see  FIG. 14A ), there was a peak at 149128 Da, which is around 947 Da more than calculated molecule weight. A mass spectrometry analysis was also conducted using the protein in reduced condition.  FIG. 14B  showed that there was indeed a peak 948 Da away from the VL-CAlpha chimeric light chain, indicating O-glycan modifications (GlcNAc+Hex+2*NeuAc) on the light chain. 
     Example 11: Thermal Stability Tests 
     We further tested and compared the thermal stability of the designed bispecific antibodies in both IgG1 and IgG4 via measuring the protein melting temperature T m  using Differential Scanning Fluorimetry (DSF). The native monospecific T3 and the TCR-modified T3 (Design_2 and Design_2-QQQQ) were served as controls. 
     Table 33 listed measured T on , T m  values of the new constructs. Overall, all molecules displayed reasonable thermal stability. IgG1-like molecules were more stable than IgG4-like molecules. The T m  value of the native T3 antibody was 74° C. The TCR antibody chimeric proteins had a relatively lower T m  of around 60° C., suggesting that TCR CBeta-CAlpha might be less resistant to the elevated temperatures compared to the CH1-CL of normal antibody. This is consistent to what reported from Wu&#39;s study (Wu et al. 2015, supra), and the CAlpha domain was suggested to be less stable than CBeta (Toughiri et al.  mAbs,  862(July), pp. 1276-1285(2016)). 
     Mutations removing N-glycosylation on TCR constant region did not affect the thermal stability of the chimeric protein. Our bispecific antibody E17-Design_2-QQQQ had similar T m  to that of Design_2-QQQQ, and lower T m  than the native T3. 
     
       
         
           
               
             
               
                 TABLE 33 
               
             
            
               
                   
               
               
                 Thermostability of designed chimeric and bispecific antibody 
               
               
                 measured by Differential Scanning Fluorimetry (DSF) 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 Protein Name 
                   
                   
                   
                   
                   
                   
                   
               
               
                 HC/LC(anti- 
                   
                   
                   
                 T h  1 
               
               
                 CD3)/HC/LC(anti- 
                   
                 Concentration 
                 T on   
                 (T m ) 
                 T h  2 
               
               
                 CD19) 
                 Isotype 
                 (mg/ml) 
                 (° C.) 
                 (° C.) 
                 (° C.) 
                 pI 
                 Purity 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 T3 
                 IgG1 
                 2.7 
                 57 
                 74.2 
                 na 
                 8.31 
                 99.41% 
               
               
                 Design_2 ( SEQ ID 
                 IgG1 
                 1.6 
                 46 
                 59.3 
                 na 
                 6.07 
                 93.09% 
               
               
                 NO: 4/3/4/3) 
               
               
                 Design_2-QQQQ 
                 IgG1 
                 1.1 
                 45 
                 59.1 
                 na 
                 6.07 
                 99.03% 
               
               
                 (SEQ ID NO: 
               
               
                 13/12/13/12) 
               
               
                 E17-Design_2-QQQQ 
                 IgG1 
                 0.3 
                 49 
                 61.9 
                 76.2 
                 7.29 
                 98.63% 
               
               
                 (SEQ ID NO: 
               
               
                 22/12/24/23) 
               
               
                 T3 
                 IgG4 
                 1.4 
                 53 
                 65 
                 73.2 
                 8.24 
                 96.06% 
               
               
                 Design 2-QQQQ 
                 IgG4 
                 0.9 
                 45 
                 58.4 
                 na 
                 5.7 
                 96.05% 
               
               
                 (SEQ ID NO: 
               
               
                 21/12/21/12) 
               
               
                 E17-Design_2-QQQQ 
                 IgG4 
                 0.8 
                 47 
                 60.2 
                 72.7 
                 6.4 
                     100% 
               
               
                 (SEQ ID NO: 
               
               
                 25/12/26/23) 
               
               
                   
               
            
           
         
       
     
     Example 12: Materials and Methods 
     Antibody T3 Fv Homology Modeling 
     Antibody Fv structural model was built based on its Fv amino acid sequences using software Discovery Studio (BIOVIA). Both light and heavy chain sequences were firstly annotated in Kabat numbering to identify three CDRs as well as the framework of each chain. Each segment (either CDR or framework) was then BLAST-searched in the antibody database with sequences from all antibody structures in PDB. The structure of the most matched sequence, if having high resolution and low B factor, were used to build the homology model. All modeled segments were then assembled to construct the light and heavy chain structural model. The relative orientation between two modeled chains was predicted by taking the angle of the antibody structure that had the most similar overall sequence. All molecular visualization and analysis work was conducted using PyMOL software (Schrödinger). 
     Vector Constructions 
     The VL, VH, Ck, CH1 genes were amplified by PCR from existing in-house DNA templates. CAlpha and CBeta genes were synthesized by Genewiz Inc. Native or chimeric light chain genes were inserted into a linearized vector containing a CMV promoter and a kappa signal peptide. The DNA fragments of VH-CH1 or VH-CBeta were inserted into a linearized vector containing human IgG4/IgG1 constant region CH2-CH3. The vector contains a CMV promoter and a human antibody heavy chain signal peptide. Plasmid ligations, transformations, DNA preparations were performed using standard molecular biology protocols. The site-directed mutagenesis was conducted by PCR amplification using mutagenic primers and followed by DpnI digestion of template DNA. 
     Protein Expression 
     The constructed vectors of heavy chain and light chain were co-transfected into Expi293 cells (Thermofisher Scientific). The ratio of different vectors for co-transfection was adjusted according to the expected structure of the antibodies and the initial expression result shown on SDS-PAGE. Briefly, 40 μg plasmid and 108 μl of expifectamine were used to transfect 40 ml volume of 1.2×10 8  cells. Enhancer 1 and Enhancer 2 were added 20 hours after transfection. The transfected cells were cultured at 37° C. with 8% CO 2  on an orbital shaker, rotating at 120 rpm. Five days after transfection, the supernatants were harvested by centrifuge and cell fragments were removed by 0.22 m filtering. 
     Expression Detection by SDS-PAGE 
     Supernatant harvested on the 5th day was mixed with NuPAGE LDS Sample Buffer (4×), NuPAGE Sample Reducing Agent (10×) and H 2 O. The reduced samples were heated at 75° C. before loading on the gel. The gels were run using constant 200V for 35 minutes. Then the gels were stained with SimplyBlue™ SafeStain (Invitrogen, LC6065), and microwaved for 5 minutes. Distaining was conducted by incubating with water and microwaved for 7 minutes. The images of the gels were taken using Universal HoodIII (Bio-Rad). 
     Purification 
     Protein A Chromatography Purification 
     MabSelect™ SuRe™ (MSS) Protein A resins were acquired from GE Healthcare and packed into glass columns (BioRad). Purification by Protein A chromatography was performed at room temperature using peristaltic pump as power at a flow rate of 0.2 ml/min. After samples were loaded, 10 column volume of 100 mM Glycine, pH3.5 was used for elution, and different fractions were collected. The protein concentration in different fractions was measured using a NanoDrop™ 2000 (Thermo Fisher Scientific). The protein purity was detected by SDS-PAGE and SEC-HPLC. 
     Ion-Exchange Chromatography (IEC) 
     The IEC chromatographic experiments were performed using a Hi trap SP HP 1 ml column from GE Healthcare life sciences with an ÄKTA Pure system (GE Healthcare). The programmed method settings were: wash the column 10 CV with wash buffer A (10 mM NaH 2 PO 4 , pH 6.0); apply the sample using sample inlet; equilibrate the column 10 CV with wash buffer A (10 mM NaH 2 PO 4 , pH 6.0); elute column with wash buffer A and wash buffer B (10 mM NaH 2 PO 4 , 1 M NaCl, pH 6.0). A gradient elution condition was applied as liner step for 50 CV with 30% wash buffer B, liner step for 5 CV with 100% wash buffer B and a step with fill for 10 CV with 100% wash buffer B. The fractions was collected as 0.5 ml per tube according to the UV absorbance value (collection threshold was set as 5 mAU). 
     Size Exclusion Chromatography (SEC) 
     The chromatographic experiments were performed using a Superdex™ 200 increase 10/300 GL column and an ÄKTA system from GE Healthcare life sciences. The experiment was run using PBS (137 mM NaCl, 2.68 mM KCl, 1.76 mM KH 2 PO 4 , 10 Mm Na2HPO4, pH 7.0) at 0.5 ml/min. The fractions were collected using automated collection program (collect value was set as 5 mAU of UV absorbance) with 0.5 ml of each fractions. 
     Ni Sepharose™ Excel Chromatography Purification 
     Purification of 6×His-tagged protein using Ni Sepharose™ Excel Chromatography Ni Sepharose™ excel resins were purchased from GE Healthcare. The resin was packed into glass columns (BioRad). After the column was washed with 10 column volume (CV) ddH 2 O to removal the resin storage buffer, it was used for purification of 6×His tagged proteins. Briefly, purification by Ni column was performed at room temperature using peristaltic pump at a flow rate of 0.2 ml/min. After sample loading, 10 CV PBS (50 mM phosphate, 150 mM NaCl, pH 7.0) was used for wash, followed by 5 CV elution buffer1 (50 mM phosphate, 150 mM NaCl, 20 mM imidazole, pH 7.0) to remove weakly bound protein. 10 CV elution buffer2 (50 mM phosphate, 150 mM NaCl, 500 mM imidazole, pH 7.0) was used to elute bound protein. After elution, collected protein was measured using a NanoDrop™ 2000 (Thermo Fisher Scientific). The purity of eluted protein was detected by SDS-PAGE and SEC-HPLC. The column was regenerated using 10 CV ddH 2 O, 10 CV stripping buffer (50 mM Tris, 500 mM NaCl, 50 mM EDTA, pH 7.4) for sanitation, 10 CV 6 M Guanidine hydrochloride, pH 7.4 and 10 CV 0.1 M Nickel sulfate. The regenerated column was filled with 20% ethanol and stored in 4° C. 
     Size Exclusion-High Performance Liquid Chromatography (SEC-HPLC) 
     Purity of the samples was analyzed using a TSK-GEL G3000SWXL column (7.8 mm×300 mm) from Tosoh Bioscience and an Agilent 1200 HPLC system (Agilent Technologies). The column was equilibrated at a flow rate of 1.0 ml/min with phosphate buffer (50 mM sodium phosphate, 150 mM NaCl, pH 7.0). After protein sample of 50 μl was filtered and injected, UV absorbance at 280 nm was monitored. The purity was estimated by integrating the chromatograms. 
     Measurement of Antibody Concentration by ELISA 
     ELISA plates were coated with 200 ng/ml (Fab) 2  form of goat anti-human IgG-Fc in coating buffer (200 mM Na 2 CO 3 /NaHCO 3 , pH 9.2). After incubation over night at 4° C., the plates were washed once with PBS buffer using a deep well washer machine (Biotek ELx405). Then the plates were blocked with 2% BSA in PBS buffer and incubated at room temperature for 1 hour. The plates were washed 3 times with washing buffer, and the positive control antibody and the diluted samples were added. After 2-hour incubation, the plates were washed 6 times with 300 μl washing buffer, and biotinylated goat anti-human Ig-Fc (Bethyl, 100 μl/well, 1:5000 dilution in 2% BSA) was added as detection antibody. After incubation and wash steps, SA-HRP (Invitrogen, 1:8000 dilution in 2% BSA) was added. Then the plates were incubated at room temperature for another 1 hour. The plates were washed 6 times with 300 μl/well washing buffer. Substrate TMB was added and developed for 10 minutes. Stop solution (2 M HCl, 100 μl/well) was added to stop further color developing and the absorbance was read at 450 nm using a plate reader (Molecular Device SpectraMax®MV5e). 
     Target-Binding Assays 
     The binding ability of designed molecules was evaluated using CD3+ Jurkat and CD19+ Ramos cell lines, respectively. Both cell lines were obtained from American Type Culture Collection (ATCC), and were maintained in RPMI 1640 medium (Invitrogen, Cat. No. 22400105), supplemented with 10% fetal bovine serum (FBS, Corning, Cat. No. 35-076-CV). 
     Aliquots of 10 5  cells per well were collected and washed with 1% bovine serum albumin (BSA, BovoGen-BSAS), followed by the incubation with serial-diluted studied antibodies in 96-well round-bottom plate (Corning, Cat. No. 3799) at 4° C. for 1 hour. After being washed twice with 1% BSA, the plates were further incubated with PE-conjugated goat anti-human IgG Fc antibodies (Jackson Immuno Research Laboratories, Cat. No 109-115-098) at 4° C. for 30 minutes. After the plates were washed twice again, the cells were analyzed by flow cytometry using a FACSCanto II cytometer (BD Biosciences) and associated fluorescence intensity was quantified using the FlowJo software. Four-parameter non-linear regression analysis was used to obtain EC50 values in Prism software (GraphPad Software, Inc). 
     Bispecific Antibodies-Directed Tumor Cell Killing 
     In order to obtain human T cells, peripheral blood mononuclear cells (PBMCs) from healthy donors were freshly isolated by Ficoll-Paque PLUS (GE Healthcare-17-1440-03) density centrifugation from heparinized venous blood. After being cultured in RPMI 1640 medium supplemented with 10% FBS, 1% Penicillin/Streptomycin Solution (ScienCell, Cat. No.: 0503), 50 units per mL of human IL-2 ligand protein and 10 ng/mL OKT3 antibody (EBioscience, Cat. No.: 16-0037-85) for 6 days, the PBMCs were passed through EasySep (Stemcell, Cat. No.: 19053) columns for the enrichment of CD8+ T cells. The CD8+ T cells from the negative selection columns were used as effector cells. 
     In the cytotoxicity assay, CD19+ Raji cells as target cells were pre-labeled with 20 nM CellTrace Far Red (Invitrogen, Cat. No. C34564) at 37° C. for 30 minutes. The cell pellets were then washed twice with phenol-free RPMI 1640 medium (Invitrogen, Cat. No. 11835030) supplemented with 10% FBS. In 96-well round-bottom plate (Corning, Cat. No. 3799), Far Red-stained Raji B cells (20,000 cells/well) were incubated with isolated CD8+ T cells (target: effector cell ratio 1:5) and serial-diluted bispecific antibodies at 37° C. for 4 hours. After incubation, 3 μM propidium iodide (PI, Invitrogen, Cat. No. P3566) was added and mixed thoroughly to identify dead cells. After 15 minutes, cells were analyzed by flow cytometry using a FACSCanto II cytometer. The bispecific antibody-mediated cytotoxicity can be defined as the percentage of PI-positive target cells in Far Red-positive target cells. EC50 of T cell engaged cytotoxicity were determined using Prism software (GraphPad Software, Inc.). 
     Mass Spectrometry Characterization 
     The protein was diluted to 0.4 mg/mL and de-glycosylated by incubation with 1 μL of PNGase F (Glyko, GKE-5006D) (protein to enzyme ratio 40:1) in 100 μL of 20 mM Tris buffer (pH 8.0) at 37° C. for at least 4 hours. A aliquot of de-glycosylated bispecific antibodies were partially reduced by addition of 2 μL 1M DTT to final concentration of 20 mM at room temperature for 15 minutes. Each sample at 2 μg was injected onto a Acquity UPLC BEH300 C4 column (2.1×100 mm, 1.7 μm) at 0.4 mL/min. Mobile phase A was 0.1% Formic Acid (FA) in HPLC grade water. Mobile phase B was 0.1% FA in acetonitrile. For both non-reduced and reduced conditions, an efficient elution gradient of 24% B to 34% B from 3.0 to 15.0 minutes was used. After separation by RP UPLC, the mass of the bispecific protein in both non-reduced and reduced conditions were detected by Waters Xevo G2 Q-TOF. The MS signals were deconvoluted using BiophamaLynx 1.3 software. Theoretical mass-averaged molecular weights of the light chain and heavy chain components were determined using the GPMaw program (v. 6.00). 
     Thermostability Test by DSF 
     A DSF assay was performed using 7500 Fast Real-Time PCR system (Applied Biosystems). Briefly, 19 μL of antibody solution was mixed with 1 μL of 62.5×SYPRO Orange solution (Invitrogen) and added to a 96 well plate (Biosystems). The plate was heated from 26° C. to 95° C. at a rate of 2° C./min, and the resulting fluorescence data were collected. The negative derivatives of the fluorescence changes with respect to different temperatures were calculated, and the maximal value was defined as melting temperature T h . If a protein has multiple unfolding transitions, the first two T h  were reported, named as T h1  and T h2 . T h1  is always interpreted as the formal melting temperature T m  to facilitate comparisons between different proteins. Data collection and T h  calculation were conducted automatically by its operation software. Once the plot of negative derivatives of different temperatures was reported by the software, the point in the plot where the curve starts to decrease from a pre-transition baseline could be roughly estimated as the onset temperature T on . 
     Example 13: O-Glycan Identification 
     Previous mass spectrometry data discovered O-glycans on the TCR-modified T3 light chain. Unlike N-glycosylation sites, which can be located based on amino acid sequence patterns, O-glycosylation sites are difficult to predict from the sequence. This T3 TCR-chimeric light chain was composed of the V region of the T3 parental antibody as well as the constant region of TCR alpha chain. Both regions could potentially have O-glycosylation sites. Mass spectrometry analysis was conducted again on the original T3 monoclonal antibody and it was found that this parental antibody was free of O-glycans, which indicated that the O-glycans were located in the TCR alpha constant region. 
     It is known that O-glycosylation mostly happens on Ser or Thr residues, and there are 21 Ser/Thr residues in the sequence of the TCR alpha constant region (shown in bold in the sequence below). To locate the exact position of the O-glycosylation sites, Ala scanning was carried out to substitute each individual Ser/Thr with Ala, and 21 TCR-modified monospecific T3 molecules were constructed. The potential O-glycans on each mutant were released from the protein, labeled with 2-amino benzoic acid and quantified by HPLC coupled with Fluorescence Detector. The loss of O-glycan signal could guide us the location of O-glycosylation site. 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 411) 
               
               
                   
                 1          11         21         31          
               
               
                   
                 PDIQNPDPAV YQLRD S K SS D K S VCLF T DFD  S Q T QV S Q S KD 
               
               
                   
                   
               
               
                   
                 41         51         61         71          
               
               
                   
                   S DVYI T DKCV LDMR S MDFK S  N S AVAW S QK S  DFACANAFQN 
               
               
                   
                   
               
               
                   
                 81         91 
               
               
                   
                   S IIPED T FFP  S PE SS   
               
            
           
         
       
     
     In order to identify and quantify the amount of O-glycan, an acidic hydrolysis and HPLC based method was developed. The sample was hydrolyzed by 2M TFA (Trifluoroacetic Acid) and the monosaccharide of the O-glycans was released. The released GalN (Galactosamine) from GalNAc (N-Acetyl-D-galactosamine) of the O-glycan and Gal (Galactose) was labeled with 2-amino benzoic acid and analyzed by HPLC coupled with FLD (Fluorescence Detector) detector and quantified by an external calibration curve. The released GalN content was directly correlated to the amount of O-glycan as it is the specific monosaccharide to the O-glycans. The results reported the amount of mol GalN per mol protein which stands for one mol protein contains the amount of mol O-glycan. 
     Table 34 shows the quantified O-glycan levels on all mutants. Bispecific molecule E17-Design_2-QQQQ was used as a control protein. The data showed that there were 0.24 mol O-glycans available on each mole of E17-Design_2-QQQQ protein. Since this is a bispecific antibody having only one TCR-modified T3 light chain, the total O-glycan level of two chains should be doubled, i.e. at around 0.48 mol/mol. Among all the 21 mutants, most of them kept the expected O-glycan quantity. Samples #3, #8, #10 and #20 had slight signal decrease. Sample #19 exhibited obvious O-glycan loss. The signal was even lower than that from the control protein. Therefore position S91 was identified as the major O-glycosylation site. S19, S36, S41 and S94 were identified as possible O-glycosylation sites. 
     
       
         
           
               
             
               
                 TABLE 34 
               
             
            
               
                   
               
               
                 Quantified O-glycans on various single Ala mutants (residue numbering was listed in FIG. 19A) 
               
            
           
           
               
               
               
               
            
               
                   
                 WBP3438 
                 Analytical NO. 
                 AS1803474 
               
               
                 Project NO. 
                 Monosaccharide Analysis 
                 SOP NO. 
                 PD-PAS-LAB-090-02 
               
            
           
           
               
               
            
               
                 Test Item 
                 Test Result(mol/mol protein) 
               
            
           
           
               
               
               
            
               
                 Sample No. 
                 Sample ID 
                 Galactosamine 
               
               
                   
               
            
           
           
               
               
               
            
               
                 196388 
                 E17-Design_2-QQQQ 
                 0.24 (x2) 
               
               
                 1 
                 T3.uIgG4.SP(S16A) 
                 0.41 
               
               
                 2 
                 T3.uIgG4.SP(S18A) 
                 0.43 
               
               
                 3 
                 T3.uIgG4.SP(S19A) 
                 0.38 
               
               
                 4 
                 T3.uIgG4.SP(S22A) 
                 0.45 
               
               
                 5 
                 T3.uIgG4.SP(T27A) 
                 0.57 
               
               
                 6 
                 T3.uIgG4.SP(S31A) 
                 0.44 
               
               
                 7 
                 T3.uIgG4.SP(T33A) 
                 0.42 
               
               
                 8 
                 T3.uIgG4.SP(S36A) 
                 0.36 
               
               
                 9 
                 T3.uIgG4.SP(S38A) 
                 0.47 
               
               
                 10 
                 T3.uIgG4.SP(S41A) 
                 0.37 
               
               
                 11 
                 T3.uIgG4.SP(T46A) 
                 0.50 
               
               
                 12 
                 T3.uIgG4.SP(S55A) 
                 0.43 
               
               
                 13 
                 T3.uIgG4.SP(S60A) 
                 0.53 
               
               
                 14 
                 T3.uIgG4.SP(S62A) 
                 0.56 
               
               
                 15 
                 T3.uIgG4.SP(S67A) 
                 0.57 
               
               
                 16 
                 T3.uIgG4.SP(S70A) 
                 0.52 
               
               
                 17 
                 T3.uIgG4.SP(S81A) 
                 0.53 
               
               
                 18 
                 T3.uIgG4.SP(T87A) 
                 0.55 
               
               
                 19 
                 T3.uIgG4.SP(S91A) 
                 0.12 
               
               
                 20 
                 T3.uIgG4.SP(S94A) 
                 0.32 
               
               
                 21 
                 T3.uIgG4.SP(S95A) 
                 0.61 
               
               
                   
               
            
           
         
       
     
     Example 14: Binding to Fcγ Receptor, C1q, and FcRn 
     Methods 
     Fcγ Receptor Binding Affinity by SPR 
     Antibody binding affinity to FcγRs was detected using Biacore T200 (or Biacore 8K). Each receptor was captured on an anti-his antibody immobilized CM5 sensor chip (GE). Antibodies at different concentrations were injected over the sensor chip at a flow rate of 30 uL/min for an association phase of 60 s, followed by 60 s dissociation. The chip was then regenerated by 10 mM glycine (pH 1.5) after each binding cycle. 
     The sensorgrams of blank surface and buffer channel were subtracted from the test sensorgrams. The experimental data was fitted by 1:1 model using Langmiur analysis (for FcγRI) or steady state model (for other receptors). Molecular weight of 150 KDa was used to calculate the molar concentration of antibodies. 
     C1q Binding by ELISA 
     ELISA Plates (Nunc) were coated with antibody samples at 3 μg/mL overnight at 4° C. After blocking and washing, C1q was gradient diluted starting from 600 μg/mL and incubated at room temperature for 2 hr. The plates were then washed and subsequently incubated with sheep anti-human C1q Ab-HRP for 1 hr. After washing, TMB substrate was added and the interaction was stopped by 2 M HCl. The absorbance at 450 nm was read using a microplate reader (Molecular Device). 
     FcRn Binding Affinity by SPR 
     Antibody binding affinity to FcRn was detected using Biacore T200 (or Biacore 8K). Each antibody was immobilized on CM5 sensor chip (GE). FcRn at different concentrations in running buffer (50 mM Na2HPO4/NaH2PO4, 150 mM NaCl, 0.05% Tween20, pH 6.0) were injected over the sensor chip at a flow rate of 30 uL/min for an association phase of 60 s, followed by 60 s dissociation. The chip was then regenerated by 1×PBS (pH 7.4) after each binding cycle. 
     The sensorgrams of blank surface and buffer channel were subtracted from the test sensorgrams. The experimental data was fitted by steady state model. A molecular weight of 45 KDa was used to calculate the molar concentration of FcRn. 
     Results 
     As all the IgG1s mentioned above were IgG1 with LALA mutation, the binding activity of E17-Design_2-QQQQ in both IgG4 and wild type IgG1 (T3U4.E17-2.(2).uIgG1 (wild type IgG1 with knobs-into-holes)) to FcγRI, FcγRIIa (H167), FcγRIIa (R167), FcγRIIb, FcγRIIIa (F176), FcγRIIIa (V176) and FcγRIIIb were investigated by SPR. 
     Relevant sequences of the T3U4.E17-2.(2).uIgG1 construct are provided below. 
     
       
         
           
               
               
               
             
               
                   
               
             
            
               
                 T3U4.E17- 
                 U4-LC 
                 DIQLTQSPSFLSASVGDRVTITCSASSTVNYMHWYQQKPGKAPKLLIYS 
               
               
                 2.(2).uIgG1 
                 (SEQ ID 
                 TSNLASGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCHQWSSYPYTFGQ 
               
               
                   
                 NO: 371) 
                 GTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWK 
               
               
                   
                   
                 VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT 
               
               
                   
                   
                 HQGLSSPVTKSFNRGEC 
               
               
                   
               
               
                   
                 U4-HC 
                 QMQLVQSGPEVKKPGTSVKVSCKASGYAFTSYNMYWVRQARGQRLE 
               
               
                   
                 (SEQ ID 
                 WIGYIDPYNGDTTYNQKFKGRVTITRDMSTSTAYMELSSLRSEDTAVY 
               
               
                   
                 NO: 372) 
                 YCLTTAYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALG 
               
               
                   
                   
                 CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS 
               
               
                   
                   
                 LGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVF 
               
               
                   
                   
                 LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT 
               
               
                   
                   
                 KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS 
               
               
                   
                   
                 KAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNG 
               
               
                   
                   
                 QPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALH 
               
               
                   
                   
                 NHYTQKSLSLSPGK 
               
               
                   
               
               
                   
                 T3-LC 
                 DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQQKPGQ 
               
               
                   
                 (SEQ ID 
                 PPKLLIYWASTRQSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTQS 
               
               
                   
                 NO: 373) 
                 HTLRTFGGGTKVEIKPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTQ 
               
               
                   
                   
                 VSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSQKSDFACANAFQNS 
               
               
                   
                   
                 IIPEDTFFPSPESS 
               
               
                   
               
               
                   
                 T3-HC 
                 QVQLVQSGAEVKKPGSSVKVSCKASGFAFTDYYIHWVRQAPGQGLEW 
               
               
                   
                 (SEQ ID 
                 MGWISPGNVNTKYNENFKGRVTITADKSTSTAYMELSSLRSEDTAVYY 
               
               
                   
                 NO: 374) 
                 CARDGYSLYYFDYWGQGTLVTVLEDLKNVFPPEVAVFEPSEAEISHTQ 
               
               
                   
                   
                 KATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALQDS 
               
               
                   
                   
                 RYALSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQI 
               
               
                   
                   
                 VSAEAWGRASDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEV 
               
               
                   
                   
                 TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL 
               
               
                   
                   
                 TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPC 
               
               
                   
                   
                 REEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG 
               
               
                   
                   
                 SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 
               
               
                   
               
            
           
         
       
     
     The affinities were summarized in Table 35 (IgG4) and Table 36 (wild type IgG1). Both molecules showed typical human IgG4 and wild type IgG1 binding affinity to all the Fcγ receptors. 
     
       
         
           
               
             
               
                 TABLE 35 
               
             
            
               
                   
               
               
                 IgG4 Affinity to Fc receptor by SPR 
               
            
           
           
               
               
               
            
               
                   
                 Fc receptor 
                 K D  (M) 
               
               
                   
                   
               
               
                   
                 FcγRI 
                 9.79E−09 
               
               
                   
                 FcγRIIa (H167) 
                 2.05E−05 
               
               
                   
                 FcγRIIa (R167) 
                 1.58E−05 
               
               
                   
                 FcγRIIb 
                 2.41E−05 
               
               
                   
                 FcγRIIIa (F176) 
                 2.93E−05 
               
               
                   
                 FcγRIIIa (V176) 
                 1.40E−05 
               
               
                   
                 FcγRIIIb 
                 &gt;4.10E−05  
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 36 
               
             
            
               
                   
               
               
                 Wild type IgG1 Affinity to Fc receptor by SPR 
               
            
           
           
               
               
               
            
               
                   
                 Fc receptor 
                 K D  (M) 
               
               
                   
                   
               
               
                   
                 FcγRI 
                 1.30E−09 
               
               
                   
                 FcγRIIa (H167) 
                 3.58E−06 
               
               
                   
                 FcγRIIa (R167) 
                 4.83E−06 
               
               
                   
                 FcγRIIb 
                 8.07E−06 
               
               
                   
                 FcγRIIIa (F176) 
                 2.08E−06 
               
               
                   
                 FcγRIIIa (V176) 
                 6.44E−07 
               
               
                   
                 FcγRIIIb 
                 5.16E−06 
               
               
                   
                   
               
            
           
         
       
     
     The binding activity of antibodies to C1Q was tested by ELISA ( FIGS. 21A-21B ). E17-Design_2-QQQQ in IgG4 showed no binding signal in ELISA ( FIG. 21A ), while the E17-Design_2-QQQQ in wild type IgG1 and the control human IgG1 antibody showed normal binding signal ( FIG. 21B ). 
     Example 15: Symmetric Formats G19, G19R, G25, G25R 
     Antibody Therapeutic targets like CD3×CD19 benefit from a bispecific antibody with monovalent CD3 binding, due to safety concerns. With this in mind, asymmetric bispecific formats E17 and F16 via integrating the WuXiBody Fab as well as the knobs-into-holes techniques were designed and successfully generated. Some bispecific targets like CTLA-4×PD-1, however, benefit from a symmetric format, which can assemble two different antibodies while keeping their original valances (i.e. tetravalent in total) to achieve desired synergetic effects. The core unit of WuXiBody is a chimeric Fab, which can be easily incorporated into both asymmetric and symmetric formats to assure the correct pairing of cognate light-heavy chains. Four WuXiBodybased symmetric formats, named G19, G19R, G25 and G25R were designed. 
       FIG. 22  provides a schematic description of four symmetric formats. In G19 and G25, two WuXiBody chimeric Fabs were grafted at the c-terminus and n-terminus of a normal antibody, respectively. The difference between G19 and G19R, or between G25 and G25R, is the reversed locations of the normal and chimeric Fab in each individual format. The heavy parts of two Fabs as well as the IgG-Fc were integrated into one chain, while both light chains were free to fold and assemble independently. When three vectors were co-transfected into host cells, heavy-heavy association were expected to take place like normal antibodies during expression process, while each light chain was expected to self-assemble to its own cognate partner on the heavy chain. 
     The bispecific CTLA-4×PD-1 antibodies in symmetric WuXiBody format were designed. A novel anti-PD-1 antibody W3055_1.153.7 (named as U6) and a commercial anti-CTLA-4 antibody ipilimumab (named as T1) were adopted to plug in the new formats. IgG4 isotype was chosen to assure the depletion of ADCC and CDC effect on the molecule. Because both U6 and T1 could be put on the top or bottom side of the format (named as U6T1 and T1U6, respectively), single format G19 was firstly used to investigate both cases. 
     Relevant sequences of the tested WuXiBody are provided below: 
     
       
         
           
               
               
               
             
               
                   
               
               
                   
                 Plasmid 
                   
               
               
                 Samples 
                 No 
                 Sequences 
               
               
                   
               
             
            
               
                 T1U6.G1 
                 T1-LC 
                 EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKPGQAPRLLIYGAFSR 
               
               
                 9.IgG4 
                 (SEQ ID 
                 ATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIKRT 
               
               
                   
                 NO: 375) 
                 VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES 
               
               
                   
                   
                 VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 
               
               
                   
               
               
                   
                 T1-U6-HC 
                 QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKGLEWVTFIS 
               
               
                   
                 (SEQ ID 
                 YDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGWLGP 
               
               
                   
                 NO: 376) 
                 FDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVS 
               
               
                   
                   
                 WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDRKPSNTK 
               
               
                   
                   
                 VDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQ 
               
               
                   
                   
                 EDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEY 
               
               
                   
                   
                 KCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFY 
               
               
                   
                   
                 PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCS 
               
               
                   
                   
                 VMHEALHNHYTQKSLSLSLGGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLV 
               
               
                   
                   
                 QPGGSLRLSCAASGFTFSSHAMSWVRQAPGKGLEWVSTITGGGGSIYYADSVK 
               
               
                   
                   
                 GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKNRAGEGYFDYWGQGTLVTV 
               
               
                   
                   
                 LEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEV 
               
               
                   
                   
                 HSGV C TDPQPLKEQPAL Q DSRYALSSRLRVSATFWQNPRNHFRCQVQFYGLSE 
               
               
                   
                   
                 NDEWTQDRAKPVTQIVSAEAWGR 
               
               
                   
               
               
                   
                 U6-LC 
                 SYELTQPLSVSVALGQTARITCGGDNIGNKDVHWYQQKPGQAPVLVIYRDSNR 
               
               
                   
                 (SEQ ID 
                 PSGIPEGFSGSNSGNTATLTISRAQAGDEADYYCQVWDSIWVFGGGTKLTVLPD 
               
               
                   
                 NO: 377) 
                 IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQT Q VSQSKDSDVYITDK C VLDMRS 
               
               
                   
                   
                 MDFKSNSAVAWS Q KSDFACANAF Q NSIIPEDTFFPSPESS 
               
               
                   
               
               
                 U6T1.G1 
                 U6-LC 
                 SYELTQPLSVSVALGQTARITCGGDNIGNKDVHWYQQKPGQAPVLVIYRDSNR 
               
               
                 9.IgG4 
                 (SEQ ID 
                 PSGIPEGFSGSNSGNTATLTISRAQAGDEADYYCQVWDSIWVFGGGTKLTVLG 
               
               
                   
                 NO: 378) 
                 QPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVET 
               
               
                   
                   
                 TTPSKQSNNKYAASSYLSLTPEQWKSHKSYSCQVTHEGSTVEKTVAPTECS 
               
               
                   
               
               
                   
                 U6-T1-HC 
                 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSHAMSWVRQAPGKGLEWVSTITG 
               
               
                   
                 (SEQ ID 
                 GGGSIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKNRAGEGY 
               
               
                   
                 NO: 379) 
                 FDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVS 
               
               
                   
                   
                 WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDRKPSNTK 
               
               
                   
                   
                 VDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQ 
               
               
                   
                   
                 EDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEY 
               
               
                   
                   
                 KCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFY 
               
               
                   
                   
                 PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCS 
               
               
                   
                   
                 VMHEALHNHYTQKSLSLSLGGGGGSGGGGSGGGGSGGGGSQVQLVESGGGV 
               
               
                   
                   
                 VQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKGLEWVTFISYDGNNKYYADS 
               
               
                   
                   
                 VKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGWLGPFDYWGQGTLVT 
               
               
                   
                   
                 VLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKE 
               
               
                   
                   
                 VHSGV C TDPQPLKEQPAL Q DSRYALSSRLRVSATFWQNPRNHFRCQVQFYGLS 
               
               
                   
                   
                 ENDEWTQDRAKPVTQIVSAEAWGR 
               
               
                   
               
               
                   
                 T1-LC 
                 EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKPGQAPRLLIYGAFSR 
               
               
                   
                 (SEQ ID 
                 ATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIKPD 
               
               
                   
                 NO: 380) 
                 IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQT Q VSQSKDSDVYITDK C VLDMRS 
               
               
                   
                   
                 MDFKSNSAVAWS Q KSDFACANAF Q NSIIPEDTFFPSPESS 
               
               
                   
               
               
                 U6T1.G1 
                 U6-LC 
                 SYELTQPLSVSVALGQTARITCGGDNIGNKDVHWYQQKPGQAPVLVIYRDSNR 
               
               
                 9R.IgG4 
                 (SEQ ID 
                 PSGIPEGFSGSNSGNTATLTISRAQAGDEADYYCQVWDSIWVFGGGTKLTVLPD 
               
               
                   
                 NO: 381) 
                 IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQT Q VSQSKDSDVYITDK C VLDMRS 
               
               
                   
                   
                 MDFKSNSAVAWS Q KSDFACANAF Q NSIIPEDTFFPSPESS 
               
               
                   
               
               
                   
                 U6-T1-HC 
                 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSHAMSWVRQAPGKGLEWVSTITG 
               
               
                   
                 (SEQ ID 
                 GGGSIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKNRAGEGY 
               
               
                   
                 NO: 382) 
                 FDYWGQGTLVTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDH 
               
               
                   
                   
                 VELSWWVNGKEVHSGV C TDPQPLKEQPAL Q DSRYALSSRLRVSATFWQNPRN 
               
               
                   
                   
                 HFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRYGPPCPPCPAPEFLGGP 
               
               
                   
                   
                 SVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKP 
               
               
                   
                   
                 REEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPR 
               
               
                   
                   
                 EPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV 
               
               
                   
                   
                 LDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGG 
               
               
                   
                   
                 GSGGGGSGGGGSGGGGSQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMH 
               
               
                   
                   
                 WVRQAPGKGLEWVTFISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLR 
               
               
                   
                   
                 AEDTAIYYCARTGWLGPFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTA 
               
               
                   
                   
                 ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG 
               
               
                   
                   
                 TKTYTCNVDHKPSNTKVDKRV 
               
               
                   
               
               
                   
                 T1-LC 
                 EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKPGQAPRLLIYGAFSR 
               
               
                   
                 (SEQ ID 
                 ATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIKRT 
               
               
                   
                 NO: 383) 
                 VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES 
               
               
                   
                   
                 VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 
               
               
                   
               
               
                 U6T1.G2 
                 U6-LC 
                 SYELTQPLSVSVALGQTARITCGGDNIGNKDVHWYQQKPGQAPVLVIYRDSNR 
               
               
                 5.IgG4 
                 (SEQ ID 
                 PSGIPEGFSGSNSGNTATLTISRAQAGDEADYYCQVWDSIWVFGGGTKLTVLPD 
               
               
                   
                 NO: 384) 
                 IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQT Q VSQSKDSDVYITDK C VLDMRS 
               
               
                   
                   
                 MDFKSNSAVAWS Q KSDFACANAF Q NSIIPEDTFFPSPESS 
               
               
                   
               
               
                   
                 U6-T1-HC 
                 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSHAMSWVRQAPGKGLEWVSTITG 
               
               
                   
                 (SEQ ID 
                 GGGSIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKNRAGEGY 
               
               
                   
                 NO: 385) 
                 FDYWGQGTLVTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDH 
               
               
                   
                   
                 VELSWWVNGKEVHSGV C TDPQPLKEQPAL Q DSRYALSSRLRVSATFWQNPRN 
               
               
                   
                   
                 HFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRGGGGSGGGGSQVQLV 
               
               
                   
                   
                 ESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKGLEWVTFISYDGNN 
               
               
                   
                   
                 KYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGWLGPFDYWG 
               
               
                   
                   
                 QGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGA 
               
               
                   
                   
                 LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVE 
               
               
                   
                   
                 SKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQ 
               
               
                   
                   
                 FNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSN 
               
               
                   
                   
                 KGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVE 
               
               
                   
                   
                 WESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEAL 
               
               
                   
                   
                 HNHYTQKSLSLSLGK 
               
               
                   
               
               
                   
                 T1-LC 
                 EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKPGQAPRLLIYGAFSR 
               
               
                   
                 (SEQ ID 
                 ATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIKRT 
               
               
                   
                 NO: 386) 
                 VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES 
               
               
                   
                   
                 VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 
               
               
                   
               
               
                 U6T1.G2 
                 U6-LC 
                 SYELTQPLSVSVALGQTARITCGGDNIGNKDVHWYQQKPGQAPVLVIYRDSNR 
               
               
                 5R.IgG4 
                 (SEQ ID 
                 PSGIPEGFSGSNSGNTATLTISRAQAGDEADYYCQVWDSIWVFGGGTKLTVLG 
               
               
                   
                 NO: 387) 
                 QPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVET 
               
               
                   
                   
                 TTPSKQSNNKYAASSYLSLTPEQWKSHKSYSCQVTHEGSTVEKTVAPTECS 
               
               
                   
               
               
                   
                 U6-T1-HC 
                 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSHAMSWVRQAPGKGLEWVSTITG 
               
               
                   
                 (SEQ ID 
                 GGGSIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKNRAGEGY 
               
               
                   
                 NO: 388) 
                 FDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVS 
               
               
                   
                   
                 WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTK 
               
               
                   
                   
                 VDKRVGGGGSGGGGSQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWV 
               
               
                   
                   
                 RQAPGKGLEWVTFISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAE 
               
               
                   
                   
                 DTAIYYCARTGWLGPFDYWGQGTLVTVLEDLKNVFPPEVAVFEPSEAEISHTQ 
               
               
                   
                   
                 KATLVCLATGFYPDHVELSWWVNGKEVHSGV C TDPQPLKEQPAL Q DSRYALS 
               
               
                   
                   
                 SRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRY 
               
               
                   
                   
                 GPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNW 
               
               
                   
                   
                 YVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGL 
               
               
                   
                   
                 PSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWES 
               
               
                   
                   
                 NGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNH 
               
               
                   
                   
                 YTQKSLSLSLGK 
               
               
                   
               
               
                   
                 T1-LC 
                 EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKPGQAPRLLIYGAFSR 
               
               
                   
                 (SEQ ID 
                 ATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIKPD 
               
               
                   
                 NO: 389) 
                 IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQT Q VSQSKDSDVYITDK C VLDMRS 
               
               
                   
                   
                 MDFKSNSAVAWS Q KSDFACANAF Q NSIIPEDTFFPSPESS 
               
               
                   
               
               
                 U6T4.G2 
                 U6-LC 
                 SYELTQPLSVSVALGQTARITCGGDNIGNKDVHWYQQKPGQAPVLVIYRDSNR 
               
               
                 6.IgG4 
                 (SEQ ID 
                 PSGIPEGFSGSNSGNTATLTISRAQAGDEADYYCQVWDSIWVFGGGTKLTVLPD 
               
               
                   
                 NO: 390) 
                 IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQT Q VSQSKDSDVYITDK C VLDMRS 
               
               
                   
                   
                 MDFKSNSAVAWS Q KSDFACANAF Q NSIIPEDTFFPSPESS 
               
               
                   
               
               
                   
                 U6-HC 
                 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSHAMSWVRQAPGKGLEWVSTITG 
               
               
                   
                 T4-LC 
                 GGGSIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKNRAGEGY 
               
               
                   
                 (SEQ ID 
                 FDYWGQGTLVTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDH 
               
               
                   
                 NO: 391) 
                 VELSWWVNGKEVHSGV C TDPQPLKEQPAL Q DSRYALSSRLRVSATFWQNPRN 
               
               
                   
                   
                 HFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRGGGGSGGGGSDIVMTQ 
               
               
                   
                   
                 TPLSLSVTPGQPASISCRSSQSLLNSDGNTYLYWYLQKPGQSPQLLIYLVSKLGS 
               
               
                   
                   
                 GVPNRFSGSGSGTDFTLKISRVEAEDVGVYYCVQGTHDPWTFGGGTKVEIKRT 
               
               
                   
                   
                 VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES 
               
               
                   
                   
                 VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 
               
               
                   
               
               
                   
                 T4-HC 
                 QVQLQESGPGLVKPSETLSLTCSVTYHTITSGYDWTWIRKPPGKGMEWIGYISY 
               
               
                   
                 (SEQ ID 
                 SGNTNYNPSLKSRVTISRDTSKNQFFLKLSSVTAADTAVYYCASMMVPHYYV 
               
               
                   
                 NO: 392) 
                 MDAWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVS 
               
               
                   
                   
                 WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTK 
               
               
                   
                   
                 VDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQ 
               
               
                   
                   
                 EDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEY 
               
               
                   
                   
                 KCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFY 
               
               
                   
                   
                 PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCS 
               
               
                   
                   
                 VMHEALHNHYTQKSLSLSLGK 
               
               
                   
               
               
                 U6T5.G1 
                 U6-LC 
                 SYELTQPLSVSVALGQTARITCGGDNIGNKDVHWYQQKPGQAPVLVIYRDSNR 
               
               
                 9.IgG4 
                 (SEQ ID 
                 PSGIPEGFSGSNSGNTATLTISRAQAGDEADYYCQVWDSIWVFGGGTKLTVLG 
               
               
                   
                 NO: 393) 
                 QPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVET 
               
               
                   
                   
                 TTPSKQSNNKYAASSYLSLTPEQWKSHKSYSCQVTHEGSTVEKTVAPTECS 
               
               
                   
               
               
                   
                 U6-T5-HC 
                 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSHAMSWVRQAPGKGLEWVSTITG 
               
               
                   
                 (SEQ ID 
                 GGGSIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKNRAGEGY 
               
               
                   
                 NO: 394) 
                 FDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVS 
               
               
                   
                   
                 WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTK 
               
               
                   
                   
                 VDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQ 
               
               
                   
                   
                 EDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEY 
               
               
                   
                   
                 KCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFY 
               
               
                   
                   
                 PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCS 
               
               
                   
                   
                 VMHEALHNHYTQKSLSLSLGGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEV 
               
               
                   
                   
                 KKPGSSVKVSCKASGYTFTNYFMNWVRQAPGQGLEWMGRVDPEQGRADYAE 
               
               
                   
                   
                 KFKKRVTITADKSTSTAYMELSSLRSEDTAVYYCARRAMDNYGFAYWGQGTL 
               
               
                   
                   
                 VTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNG 
               
               
                   
                   
                 KEVHSGV C TDPQPLKEQPAL Q DSRYALSSRLRVSATFWQNPRNHFRCQVQFY 
               
               
                   
                   
                 GLSENDEWTQDRAKPVTQIVSAEAWGR 
               
               
                   
               
               
                   
                 T5-LC 
                 EIVLTQSPDFQSVTPKEKVTITCSANSALSYMYWYQQKPDQSPKLWVHGTSNL 
               
               
                   
                 (SEQ ID 
                 ASGVPSRFSGSGSGTDFTLTINSLEAEDAATYYCHHWSNTQWTFGGGTKVEIKP 
               
               
                   
                 NO: 395) 
                 DIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQT Q VSQSKDSDVYITDK C VLDMR 
               
               
                   
                   
                 SMDFKSNSAVAWS Q KSDFACANAF Q NSIIPEDTFFPSPESS 
               
               
                   
               
               
                 U6T5.G1 
                 U6-LC 
                 SYELTQPLSVSVALGQTARITCGGDNIGNKDVHWYQQKPGQAPVLVIYRDSNR 
               
               
                 9R.IgG4 
                 (SEQ ID 
                 PSGIPEGFSGSNSGNTATLTISRAQAGDEADYYCQVWDSIWVFGGGTKLTVLPD 
               
               
                   
                 NO: 396) 
                 IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQT Q VSQSKDSDVYITDK C VLDMRS 
               
               
                   
                   
                 MDFKSNSAVAWS Q KSDFACANAF Q NSIIPEDTFFPSPESS 
               
               
                   
               
               
                   
                 U6-T5-HC 
                 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSHAMSWVRQAPGKGLEWVSTITG 
               
               
                   
                 (SEQ ID 
                 GGGSIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKNRAGEGY 
               
               
                   
                 NO: 397) 
                 FDYWGQGTLVTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDH 
               
               
                   
                   
                 VELSWWVNGKEVHSGV C TDPQPLKEQPAL Q DSRYALSSRLRVSATFWQNPRN 
               
               
                   
                   
                 HFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRYGPPCPPCPAPEFLGGP 
               
               
                   
                   
                 SVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKP 
               
               
                   
                   
                 REEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPR 
               
               
                   
                   
                 EPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV 
               
               
                   
                   
                 LDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGG 
               
               
                   
                   
                 GSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTNYFMN 
               
               
                   
                   
                 WVRQAPGQGLEWMGRVDPEQGRADYAEKFKKRVTITADKSTSTAYMELSSL 
               
               
                   
                   
                 RSEDTAVYYCARRAMDNYGFAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSE 
               
               
                   
                   
                 STAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS 
               
               
                   
                   
                 SLGTKTYTCNVDHKPSNTKVDKRV 
               
               
                   
               
               
                   
                 T5-LC 
                 EIVLTQSPDFQSVTPKEKVTITCSANSALSYMYWYQQKPDQSPKLWVHGTSNL 
               
               
                   
                 (SEQ ID 
                 ASGVPSRFSGSGSGTDFTLTINSLEAEDAATYYCHHWSNTQWTFGGGTKVEIK 
               
               
                   
                 NO: 398) 
                 RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ 
               
               
                   
                   
                 ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 
               
               
                   
               
               
                 U6T5.G2 
                 U6-LC 
                 SYELTQPLSVSVALGQTARITCGGDNIGNKDVHWYQQKPGQAPVLVIYRDSNR 
               
               
                 5.IgG4 
                 (SEQ ID 
                 PSGIPEGFSGSNSGNTATLTISRAQAGDEADYYCQVWDSIWVFGGGTKLTVLPD 
               
               
                   
                 NO: 399) 
                 IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQT Q VSQSKDSDVYITDK C VLDMRS 
               
               
                   
                   
                 MDFKSNSAVAWS Q KSDFACANAF Q NSIIPEDTFFPSPESS 
               
               
                   
               
               
                   
                 U6-T5-HC 
                 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSHAMSWVRQAPGKGLEWVSTITG 
               
               
                   
                 (SEQ ID 
                 GGGSIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKNRAGEGY 
               
               
                   
                 NO: 400) 
                 FDYWGQGTLVTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDH 
               
               
                   
                   
                 VELSWWVNGKEVHSGV C TDPQPLKEQPAL Q DSRYALSSRLRVSATFWQNPRN 
               
               
                   
                   
                 HFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRGGGGSGGGGSQVQLV 
               
               
                   
                   
                 QSGAEVKKPGSSVKVSCKASGYTFTNYFMNWVRQAPGQGLEWMGRVDPEQG 
               
               
                   
                   
                 RADYAEKFKKRVTITADKSTSTAYMELSSLRSEDTAVYYCARRAMDNYGFAY 
               
               
                   
                   
                 WGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNS 
               
               
                   
                   
                 GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKR 
               
               
                   
                   
                 VESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPE 
               
               
                   
                   
                 VQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKV 
               
               
                   
                   
                 SNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIA 
               
               
                   
                   
                 VEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHE 
               
               
                   
                   
                 ALHNHYTQKSLSLSLGK 
               
               
                   
               
               
                   
                 T5-LC 
                 EIVLTQSPDFQSVTPKEKVTITCSANSALSYMYWYQQKPDQSPKLWVHGTSNL 
               
               
                   
                 (SEQ ID 
                 ASGVPSRFSGSGSGTDFTLTINSLEAEDAATYYCHHWSNTQWTFGGGTKVEIK 
               
               
                   
                 NO: 401) 
                 RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ 
               
               
                   
                   
                 ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 
               
               
                   
               
               
                 U6T5.G2 
                 U6-LC 
                 SYELTQPLSVSVALGQTARITCGGDNIGNKDVHWYQQKPGQAPVLVIYRDSNR 
               
               
                 5R.IgG4 
                 (SEQ ID 
                 PSGIPEGFSGSNSGNTATLTISRAQAGDEADYYCQVWDSIWVFGGGTKLTVLG 
               
               
                   
                 NO: 402) 
                 QPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVET 
               
               
                   
                   
                 TTPSKQSNNKYAASSYLSLTPEQWKSHKSYSCQVTHEGSTVEKTVAPTECS 
               
               
                   
               
               
                   
                 U6-T5-HC 
                 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSHAMSWVRQAPGKGLEWVSTITG 
               
               
                   
                 (SEQ ID 
                 GGGSIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKNRAGEGY 
               
               
                   
                 NO: 403) 
                 FDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVS 
               
               
                   
                   
                 WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTK 
               
               
                   
                   
                 VDKRVGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTNYFMNW 
               
               
                   
                   
                 VRQAPGQGLEWMGRVDPEQGRADYAEKFKKRVTITADKSTSTAYMELSSLRS 
               
               
                   
                   
                 EDTAVYYCARRAMDNYGFAYWGQGTLVTVLEDLKNVFPPEVAVFEPSEAEIS 
               
               
                   
                   
                 HTQKATLVCLATGFYPDHVELSWWVNGKEVHSGV C TDPQPLKEQPAL Q DSR 
               
               
                   
                   
                 YALSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEA 
               
               
                   
                   
                 WGRYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPE 
               
               
                   
                   
                 VQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKV 
               
               
                   
                   
                 SNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIA 
               
               
                   
                   
                 VEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHE 
               
               
                   
                   
                 ALHNHYTQKSLSLSLGK 
               
               
                   
               
               
                   
                 T5-LC 
                 EIVLTQSPDFQSVTPKEKVTITCSANSALSYMYWYQQKPDQSPKLWVHGTSNL 
               
               
                   
                 (SEQ ID 
                 ASGVPSRFSGSGSGTDFTLTINSLEAEDAATYYCHHWSNTQWTFGGGTKVEIKP 
               
               
                   
                 NO: 404) 
                 DIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQT Q VSQSKDSDVYITDK C VLDMR 
               
               
                   
                   
                 SMDFKSNSAVAWS Q KSDFACANAF Q NSIIPEDTFFPSPESS 
               
               
                   
               
               
                 T4U6.G2 
                 T4-HC 
                 QVQLQESGPGLVKPSETLSLTCSVTYHTITSGYDWTWIRKPPGKGMEWIGYISY 
               
               
                 7.IgG4 
                 U6-LC 
                 SGNTNYNPSLKSRVTISRDTSKNQFFLKLSSVTAADTAVYYCASMMVPHYYV 
               
               
                   
                 (SEQ ID 
                 MDAWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVS 
               
               
                   
                 NO: 405) 
                 WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTK 
               
               
                   
                   
                 VDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQ 
               
               
                   
                   
                 EDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEY 
               
               
                   
                   
                 KCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFY 
               
               
                   
                   
                 PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCS 
               
               
                   
                   
                 VMHEALHNHYTQKSLSLSLGKGGGGSGGGGSGGGGSGGGGSSYELTQPLSVS 
               
               
                   
                   
                 VALGQTARITCGGDNIGNKDVHWYQQKPGQAPVLVIYRDSNRPSGIPEGFSGS 
               
               
                   
                   
                 NSGNTATLTISRAQAGDEADYYCQVWDSIWVFGGGTKLTVLPDIQNPDPAVY 
               
               
                   
                   
                 QLRDSKSSDKSVCLFTDFDSQT Q VSQSKDSDVYITDK C VLDMRSMDFKSNSAV 
               
               
                   
                   
                 AWS Q KSDFACANAF Q NSIIPEDTFFPSPESS 
               
               
                   
               
               
                   
                 U6-HC 
                 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSHAMSWVRQAPGKGLEWVSTITG 
               
               
                   
                 (SEQ ID 
                 GGGSIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKNRAGEGY 
               
               
                   
                 NO: 406) 
                 FDYWGQGTLVTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDH 
               
               
                   
                   
                 VELSWWVNGKEVHSGV C TDPQPLKEQPAL Q DSRYALSSRLRVSATFWQNPRN 
               
               
                   
                   
                 HFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGR 
               
               
                   
               
               
                   
                 T4-LC 
                 DIVMTQTPLSLSVTPGQPASISCRSSQSLLNSDGNTYLYWYLQKPGQSPQLLIYL 
               
               
                   
                 (SEQ ID 
                 VSKLGSGVPNRFSGSGSGTDFTLKISRVEAEDVGVYYCVQGTHDPWTFGGGTK 
               
               
                   
                 NO: 407) 
                 VEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS 
               
               
                   
                   
                 GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN 
               
               
                   
                   
                 RGEC 
               
               
                   
               
               
                   
                 T4-HC 
                 QVQLQESGPGLVKPSETLSLTCSVTYHTITSGYDWTWIRKPPGKGMEWIGYISY 
               
               
                   
                 U6-LC 
                 SGNTNYNPSLKSRVTISRDTSKNQFFLKLSSVTAADTAVYYCASMMVPHYYV 
               
               
                   
                 (SEQ ID 
                 MDAWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVS 
               
               
                   
                 NO: 408) 
                 WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTK 
               
               
                   
                   
                 VDKRVGGGGSGGGGSSYELTQPLSVSVALGQTARITCGGDNIGNKDVHWYQQ 
               
               
                   
                   
                 KPGQAPVLVIYRDSNRPSGIPEGFSGSNSGNTATLTISRAQAGDEADYYCQVWD 
               
               
                   
                   
                 SIWVFGGGTKLTVLPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTQVSQSKD 
               
               
                   
                   
                 SDVYITDKCVLDMRSMDFKSNSAVAWSQKSDFACANAFQNSIIPEDTFFPSPES 
               
               
                   
                   
                 S 
               
               
                 T4U6.G2 
                 U6-HC 
                 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSHAMSWVRQAPGKGLEWVSTITG 
               
               
                 6R.IgG4 
                 (SEQ ID 
                 GGGSIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKNRAGEGY 
               
               
                   
                 NO: 409) 
                 FDYWGQGTLVTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDH 
               
               
                   
                   
                 VELSWWVNGKEVHSGV C TDPQPLKEQPAL Q DSRYALSSRLRVSATFWQNPRN 
               
               
                   
                   
                 HFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRYGPPCPPCPAPEFLGGP 
               
               
                   
                   
                 SVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKP 
               
               
                   
                   
                 REEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPR 
               
               
                   
                   
                 EPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV 
               
               
                   
                   
                 LDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 
               
               
                   
               
               
                   
                 T4-LC 
                 DIVMTQTPLSLSVTPGQPASISCRSSQSLLNSDGNTYLYWYLQKPGQSPQLLIYL 
               
               
                   
                 (SEQ ID 
                 VSKLGSGVPNRFSGSGSGTDFTLKISRVEAEDVGVYYCVQGTHDPWTFGGGTK 
               
               
                   
                 NO: 410) 
                 VEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS 
               
               
                   
                   
                 GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN 
               
               
                   
                   
                 RGEC 
               
               
                   
               
            
           
         
       
     
     Both U6T1 and T1U6 constructs were expressed normally in Expi293 system, and the expressed protein achieved around 90% purity after one-step purification of protein A chromatography.  FIGS. 23A-23B  showed the SDS-PAGE and the SEC-HPLC characterizations of the purified proteins. To inspect their binding capability, cell-based binding assays to both PD-1 and CTLA-4 targets were conducted afterwards.  FIGS. 24A-24B  showed that both U6 and T1 had reduced binding if located at the bottom side of the format. Considering that the function of PD-1 has relatively higher importance than that of CTLA-4 (CTLA-4 antibodies are known to have more severe side effect), the PD-1 binding side was put on the top to maximize the U6 binding (i.e. U6T1, rather than T1U6), and to test how to optimize the CTLA-4 binding that is located at the bottom side. 
     The other three WuXiBody formats G19R, G25, and G25R (shown in  FIG. 22 ) were further investigated. In addition, a benchmark antibody AK-104 (Akeso Biopharma, Inc), which is a PD-1/CTLA-4 bispecific antibody used in clinical trial was obtained and used as a control for direct comparison. 
     Due to the importance of PD-1 function, U6 was kept on the top side of all the formats to maximize the PD-1 binding, while T1 was kept at the bottom to realize the decent CTLA-4 binding. All the constructed molecules were well expressed in Expi293, and easily achieved &gt;90% purity after one-step purification of protein A chromatography.  FIGS. 25A-25B  showed that purified proteins characterized by SDS-PAGE as well as SEC-HPLC. 
     The cell-based binding assays to both PD-1 and CTLA-4 were then conducted to check the binding capability of all the new-built molecules.  FIGS. 26A-26B  showed the binding curve comparisons between the designed constructs and the benchmark antibody. The data showed that all of the proteins had very similar PD-1 binding to the benchmark antibody. In addition, the CTLA-4 binding significantly improved in G25 and G25R formats and achieved comparable performance to the benchmark antibody (&lt;=2 fold). The G19R format, however, still did not work well. It is likely that G19 and G19R shared the same issue that prevented the effective binding of T1. 
     The functions of the molecules were further characterized by inspecting their competition capabilities to each ligand of two targets, PD-L1 and CD80.  FIGS. 27A-27B  confirmed that these molecules have comparable performance to the benchmark in competing with PD-L1. For the CTLA-4 side, the format G25R exhibited similar capability to the benchmark in competing with CD80. The other two formats had relatively worse results. The difference between G25 and G25R is the location of TCR constant region. It seems that the conversion of T1 into WuXiBody format facilitated the activity of T1, although T1 was still beneath U6. This provided a good example demonstrating that functional leads could be effectively screened out by scanning over limited number of WuXiBody derived formats. 
     Accordingly, a functional PD-1/CTLA-4 bispecific antibody similar to the benchmark antibody was obtained. WuXiBody formats are very universal, i.e., any new antibodies can fit into these formats and play its function. If a good parental antibody is available, it could be used to create a molecule superior to the benchmark antibody. 
     To prove the concept, another anti-CTLA-4 antibody W3162_1.154.8-z35 (named T5), which has much stronger affinity than Ipilimumab, was developed and implemented in all the four formats G19, G19R, G25, and G25R shown in  FIG. 22 . Again, all the new constructs were well expressed in Expi293 cells, and easily purified by one-step protein A chromatography. The purities of the proteins were shown in  FIGS. 28A-28B . 
     The bindings of all the U6T5 molecules, the previously identified U6T1.G25R molecule, as well as the benchmark antibody were all conducted and compared. Results were listed in  FIGS. 29A-29B . The PD-1 side kept the original binding behaviors as observed before, because no PD-1 antibodies were replaced in any of the formats. However, for the CTLA-4 side, all the U6T5 constructs (even G19 and G19R formats) exhibited obvious superior bindings than U6T1.G25R as well as the benchmark molecule. U6T5.G25 was the strongest one among all the new proteins, which has 1.6× improved EC 50  and &gt;3× improved top values compared to the benchmark antibody. This molecule was further characterized in the ELISA dual binding assay and FACS competition assays.  FIG. 30  proved the effective dual bindings of the molecules to both targets simultaneously. The data in  FIGS. 31A-31B  confirmed that U6T5.G25 had significantly improved competition capability with CD80 to CTLA-4. This proved that WuXiBody formats were sufficiently flexible to handle different parental antibodies. The superior part of a parental antibody could be well conserved and reflected when the molecule is plugged into WuXiBody formats. 
     The thermal stability of the molecules that covered all the four symmetric formats was characterised. Most of the molecules showed the melting temperature around 60° C. (shown in Table 37), which is consistent with the asymmetric format shown above. 
     
       
         
           
               
             
               
                 TABLE 37 
               
             
            
               
                   
               
               
                 Melting Temperatures of Representative Antibodies in WuXiBody Formats 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                 Concentration 
                 T h 1 
                 T h 2 
               
               
                 Protein Name 
                 Isotype 
                 pI 
                 Buffer 
                 (mg/ml) 
                 (° C.) 
                 (° C.) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 T1U6.G19.IgG4 
                 IgG4, kappa, lamda 
                 5.92 
                 PBS 
                 1.3 
                 60.8 
                 69.9 
               
               
                 U6T1.G19.IgG4 
                 IgG4, kappa, lamda 
                 5.92 
                 PBS 
                 0.9 
                 59.1 
                 72.8 
               
               
                 U6T1.G25R.IgG4 
                 IgG4, kappa, lamda 
                 6.06 
                 PBS 
                 1.385 
                 60.8 
                 73.9 
               
               
                 U6T5.G19.IgG4 
                 IgG4, kappa, lamda 
                 5.93 
                 PBS 
                 0.6 
                 56.2 
                 74.1 
               
               
                 U6T5.G19R.IgG4 
                 IgG4, kappa, lamda 
                 5.87 
                 PBS 
                 1.2 
                 63.4 
                 — 
               
               
                 U6T5.G25.IgG4 
                 IgG4, kappa, lamda 
                 5.93 
                 PBS 
                 0.7 
                 63.4 
                 — 
               
               
                 U6T5.G25R.uIgG4 
                 IgG4, kappa, lamda 
                 5.99 
                 PBS 
                 0.5 
                 57.2 
                 74.1 
               
               
                   
               
            
           
         
       
     
     Example 16: Light-Heavy Switched Chimeric Fab 
     In total, 111 potential WuXiBody based formats were successfully designed. Besides E17, F16, G19, G19R, G25 and G25R shown above, a few formats with light-heavy crossed TCR-chimeric Fab were also designed. These were named G26, G26R, and G27, shown in  FIG. 32 . This time, antibody pair U6 and T4 was used, where T4 was an anti-CTLA-4 antibody WBP3162-1.146.19-z12. The T4U6 pair was developed on format G27 and G26R.  FIGS. 33A-33B  showed the purified protein characterized by SDS-PAGE and SEC-HPLC. Although both proteins were expressed, T4U6.G27.IgG4 had low purity, but T4U6.G26R.IgG4 had correct molecular weight and high purity. The binding capability of the later molecule was characterized in FACS binding.  FIGS. 34A-34B  showed that the PD-1 binding, since located in the bottom side, was affected, while the CTLA binding side showed full recovery as it was put at the top side of the format. 
     The U6T4 pair was tried on format G26. The expression and purification steps both worked well as shown in  FIGS. 35A-35B . The ELISA and FACS binding were both conducted and the data were shown in  FIGS. 36A-36D . These data proved that the light-heavy crossed chimeric Fab can still work well. The melting temperature of this molecule is around 63.4, as shown in Table 38. 
     
       
         
           
               
             
               
                 TABLE 38 
               
             
            
               
                   
               
               
                 Melting Temperature of U6T4.G26.IgG4 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                 Concentration 
                 T h 1 
                 T h 2 
               
               
                 Protein Name 
                 Isotype 
                 pI 
                 Buffer 
                 (mg/ml) 
                 (° C.) 
                 (° C.) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 U6T4.G26.IgG4 
                 IgG4, kappa, lamda 
                 5.93 
                 PBS 
                 0.8 
                 63.4 
                 — 
               
               
                   
               
            
           
         
       
     
     Example 17: Bispecific Anti-CD13×CD19 WuXiBody 
     Background 
     Target Biology 
     The human CD19 is a type I transmembrane protein belonging to the immunoglobulin superfamily (Carter et al., Curr Dir Autoimmun, 2004, 7:4-32). It is expressed on most B cells, but not detected on plasma cells, stem cells, or on normal myeloid lineage (Tedder, Nat Rev Rheumatol, 2009, 5(10):572-577). CD19 is critically involved in establishing intrinsic B cell signaling thresholds through modulating both B cell receptor (BCR)-dependent and independent signaling (Wang et al., Experimental Hematology &amp; Oncology, 2012, 1:36). CD19 has broader expression than CD20. The pattern of CD19 expression is maintained in B-cell malignancies, covering all subtypes of B-cell lymphoma, from indolent to aggressive forms, as well as B-cell chronic lymphocytic leukemia and non-T acute lymphoblastic leukemia, and allows the targeting of tumor indications of early B cells, such as acute lymphoblastic leukemia (ALL), which cannot be targeted by Rituximab. Several CD19 monoclonal antibody have been explored for lymphoma therapy (U.S. Patent Application Publication No. 20140072587 A1, U.S. Pat. No. 8,242,252 B2, and U.S. Pat. No. 8,097,703 B2). 
     The CD3 T-cell co-receptor is a protein complex composed of four distinct chains, a CD3gamma chain, a CD3delta chain, and two CD3epsilon chains. The four chains associate with a molecule known as T-cell receptor (TCR) and the zeta-chain to generate activation signal in T lymphocytes. The TCR, zetachain, and CD3 molecules compose the TCR complex, in which TCR as a subunit recognizes and binds to antigen, and CD3 as a subunit transfers and conveys the antigen stimulation to signaling pathway, and ultimately regulates T-cell activity. The CD3 protein is present in virtually all T cells. The CD3-TCR complex modulates T cell functions in both innate and adoptive immune response, as well as cellular and humoral immune functions. These include eliminating pathogenic organisms and controlling tumor growth by broad range of cytotoxic effects. Mouse monoclonal antibodies specific for human CD3, such as OKT3 (Kung et al., Science, 1979, 206: 347-9), were the first generation CD3 antibodies developed for treatment. Although OKT3 has strong immunosuppressive potency, its clinical use was hampered by serious side effects linked to its immunogenic and mitogenic potentials (Chatenoud, Nature Reviews, 2003, 3:123-132). OKT3 induced an anti-globulin response, promoting its own rapid clearance and neutralization (Chatenoud et al., Eur. J. Immunol., 1982, 137:830-8). In addition, OKT3 induced T-cell proliferation and cytokine production in vitro, and led to a large scale release of cytokine in vivo (Hirsch et al., J. Immunol, 1989, 142: 737-43). Such serious side effects limited the more widespread use of OKT3 in transplantation as well as the extension of its use to other clinical fields such as autoimmunity (Id.). 
     A bispecific antibody targeting CD3 and CD19 can bind to T cells and B cells simultaneously. Once the bispecific antibody binds to a CD3-positive T cell and a CD19-positive B cell, a cytolytic synapse is formed. Cytotoxicity is then induced by the release of perforin and granzymes from granules in the cytotoxic T cell, the latter inducing apoptosis and lysis of the malignant B cell. 
     The activity of blinatumomab has been proved to be independent of antigen presentation by class I MHC and TCR recognition. Therefore, it can circumvent a variety of tumor-mediated immune escape mechanisms, such as impairment of antigen presentation machinery and activation of negative costimulatory signals in the tumor microenvironment. 
     Unmet Medical Needs 
     The treatment of acute lymphoblastic leukemia (ALL) in adults remains challenging and novel therapies are needed. With the current therapies, the response rates range from 30 to 50% depending on the duration of the initial remission, age and cytogenetics. The overall response rates for a subset of non-Hodgkin lymphoma (NHL) are now greater than 90% under regimens employing the first generation of anti-CD20 antibodies. However, several NHL subtypes are not as responsive to these therapies, and the majority of patients with responsive NHL eventually relapse after the standard combined immunotherapy/chemotherapy regimen. Thus, both new first-line therapies and new salvage regimens are required for these unmet needs. 
     Materials and Methods 
     Generation of Cynomolgus Monkey CD19 Expressing Cell Line 
     The gene of full length human or cynomolgus monkey CD19 was cloned into pcDNA3.3 vector. Each expression vector was then transfected into CHO-K1 cells respectively using Lipofectamine 2000. The cells were cultured in F12-K with 10% FBS. Blasticidin was added 24-48 hours after transfection. After two to three passages of selection, the cells were enriched by PE conjugated anti-CD19 antibody and Anti-PE Microbeads (Miltenyi-013-048-801). Stable single cell clones were isolated by limiting dilution and screened by FACS using anti-CD19 antibody. 
     Target-Expressing Tumor Lines 
     Raji and Jurkat cells were from ATCC. Ramos cell was from ECACC. All the tumor cells were cultured in RPMI1640/10% FBS. 
     Construction of WuXiBody W3438-T3U4.E17-1.uIgG4.SP and W3438-T3U4.F16-1.uIgG4.SP 
     The VL, VH, Ck, CH1 genes were amplified by PCR from existing in-house DNA templates. CAlpha and CBeta genes were synthesized by Genewiz Inc. Anti CD19 Native or Anti CD3 chimeric light chain genes were inserted into a linearized vector containing a CMV promoter and a kappa signal peptide. The DNA fragments of Anti CD3 VH-CBeta were inserted into a linearized vector containing human IgG4S228P constant region CH2-CH3 with a knob mutation. The DNA fragments of Anti CD19 VH-CH1 were inserted into a linearized vector containing human IgG4S228P constant region CH2-CH3 with a hole mutation. The vector contains a CMV promoter and a human antibody heavy chain signal peptide. 
     Expression and Purification of W3438-T3U4.E17-1.uIgG4.SP and W3438-T3U4.F16-1.uIgG4.SP 
     Heavy chain and light chain expression plasmids were co-transfected into Expi293 cells using Expi293 expression system kit (ThermoFisher-A14635) according to the manufacturer&#39;s instructions. Five days after transfection, the supernatants were collected and the protein was purified using Protein A column (GE Healthcare-17543802) and further size exclusion column (GE Healthcare-17104301). Antibody concentration was measured by Nano Drop. The purity of proteins was evaluated by SDS-PAGE and HPLC-SEC. 
     Target Binding by FACS 
     The binding of bi-specific antibodies to CD3- and CD19-expressing cells was evaluated using Jurkat and Ramos, respectively. A non-relevant antibody was used as an isotype control. Cells were spread in 96-well plates (Corning-3799) at a density of 10 5  cells/well and washed with PBS/1% BSA. The antibodies were serial-diluted and incubated with cells at 4° C. for 1 hr. PE-conjugated goat anti-human IgG Fc Antibody (Jackson-109-115-098) was used for detection. After washing and resuspending, cells were analyzed by flow cytometry (Canto II, BD Biosciences). Data were analyzed using FlowJo software. Four-parameter non-linear regression analysis was used to calculate EC 50  values using Prism GraphPad Software. 
     Binding to Cynomolgus CD3 
     The binding of the CD3×CD19 bispecific antibody to Cynomolgus CD3 were tested by protein binding ELISA. 96-well high protein binding ELISA plates (Nunc MaxiSorp, ThermoFisher, Thermo-442404) were coated overnight at 4° C. with 100 ul of 1 μg/ml Cynomolgus CD3 epsilon protein (Acro, # CDE-C5226) in Carbonate-bicarbonate buffer (20 mM Na2CO3, 180 mM NaHCO3, PH9.2). All wells were washed one time with 300 μL per well of PBS/0.5‰ Tween-20 (v/v). The wells were then blocked for one hour at room temperature with 200 μL per well of PBS/2% BSA (BOVOGEN, # BSAS) and washed three times with 300 μL per well of PBS/0.5‰ Tween-20 (v/v). For the primary antibody binding, CD3×CD19 bispecific antibody serially diluted in PBS/2% BSA were added to the relevant wells and incubated at room temperature for two hours. Plates were washed three times like before prior to the addition of 100 ul of 100 ng/ml secondary antibody Goat-anti-human IgG Fc-HRP (Bethyl, # A80-304P). Plates were incubated at room temperature for one hour, followed by six washes as described above. For the binding detection, 100 ul Tetramethylbenzidine (TMB) Substrate solution (Sigma-860336) was added to all wells for 10 minutes at room temperature in the dark before stopping the reaction with 100 ul 2M HCl. The extent of bispecific antibody binding to Cynomolgus CD3 was determined by measuring the OD450 absorbance using the SpectraMax® M5e microplate reader. Wherever appropriate, binding EC50 values were obtained by the four-parameter non-linear regression analysis using GraphPad Prism5 software. 
     Binding to Cynomoglus CD19 
     Binding of the CD3×CD19 bispecific antibody to Cynomoglus CD19 target protein expressed on CHOK1 cells was determined by flow cytometry analysis. In brief, cynomoglus CD19 over-expressed stable cell line (WBP701.CHOK1.cPro1.C9, WuXi Biologics) were harvested with trypsin and diluted to 1×10 6  cells/ml in 1% BSA/1×PBS. 1×10 5  cells/well (100 ul) were added to each well of a 96-well U-plate (Corning, #3799) and centrifuged at 1500 rpm (Eppendorf, #5810R) for 5 minutes before removing the supernatant. Antibodies serially diluted in 1% BSA/1×PBS were added at 100 ul/well to the pelleted cells and incubated at 4° C. for 1 hour. A non-related hIgG4 antibody was used as an isotype control. Cells were washed two times with 180 ul/well of 1% BSA/1×PBS by centrifugation at 1500 rpm for 5 minutes at 4° C. Pelleted cells were resuspended in 100 ul/well Fluorescence-labeled anti-human IgG Fc antibody (Jackson, #109-115-098) 1:150 diluted in 1% BSA/1×PBS for 30 minutes at 4° C. in the dark. Cells were then washed two times as described above. After the final wash, cells were resuspended in 80 ul 1% BSA/1×PBS and fluorescence values were measured with a FACS Canto II cytometer (BD Biosciences). The amount of cell surface bound anti-CD19&amp;CD3 bispecific antibody was assessed by measuring the mean fluorescence (MFI). The FACS raw data were analyzed by FlowJo software, wells containing no antibody or secondary antibody only were used to establish background fluorescence. Binding EC50 values were obtained by the four-parameter non-linear regression analysis using GraphPad Prism 5 software. 
     Affinity by FACS 
     Binding affinity to CD3 and CD19 was determined by flow cytometry using Jurkat and Ramos cells, respectively. The cells were transferred in to 96-well U-bottom plates (BD) at a density of 5×10 4  cells/well. Antibodies to be tested were 1:2-fold serially diluted in 1% 1×PBS/1% BSA and incubated with cells at 4° C. for 1 hr. Then, the plates were centrifuged at 1500 rpm for 4 mins and the supernatant discarded. The secondary antibody, Alexa647 conjugated goat anti-human IgG Fc (Jackson, Cat #109-605-098) or FITC conjugated goat anti-His (Bethyl, Cat # A190-113F) was added to re-suspended cells and incubated at 4° C. in the dark for 30 min. The cells were washed once and re-suspended in 100 μL 1×PBS/1% BSA. Fluorescence intensity was measured by flow cytometry (BD Canto II) and analyzed by FlowJo. Fluorescence intensity was converted to bound molecules/cell based on the quantitative beads (Quantum™ MESF Kits, Bangs Laboratories). K D  was calculated by Graphpad PrismS. 
     Dual-Binding on Target Cells 
     The ability of bispecific antibodies to bridge CD3 T cells and CD19 B cells was tested by FACS. Jurkat cells and Raji cells were pre-labeled separately with 20 nM CellTrace Far Red (Invitrogen-C34564) and 50 nM Calcein-AM (Invitrogen-C3099) at 37° C. for 30 min, at a density of 1*10 6  cells/ml. The pre-labeled cell pellets were washed twice with PBS/1% BSA, then mixed 1:1 to a final density of 1*10 6  cells/ml. The cell mixture was centrifuged and resuspended with 10 nM antibody followed by 1 hr incubation. The cell mixture was analyzed by flow cytometry immediately after incubation. Bridging percentage was calculated as the percentage of events that are simultaneously labeled Far-Red and Calcein. 
     Cytotoxicity Assay 
     Peripheral Blood Mononuclear Cells (PBMCs) were freshly isolated by Ficoll-Paque PLUS (GE Healthcare-17-1440-03) density centrifugation from heparinized venous blood. Then obtained PBMCs were passed through EasySep (Stemcell-19053) columns for the enrichment of CD8+ T cells, which were used as effector cells. The efficacy of the antibodies to mediate tumor cell lysis by CD8+ T cells was determined by flow cytometry. In the cyotoxicity assay, Raji CD19 B cells as target cells were pre-labeled with 20 nM CellTrace Far Red (Invitrogen-C34564) at 37° C. for 30 min followed by washing the cell pellets twice with phenol-free RPMI 1640 (Invitrogen-11835030) supplemented with 1% FBS. Far Red-stained Raji (20000 cells per well) was incubated in 96-well round-bottom plate (Corning-3799) with isolated CD8+ T cells (effector/target cells ratio 5:1) and serial-diluted antibodies at 37° C. for 4 h. Following incubation, 3 μM Propidium Iodide (PI, Invitrogen-P3566) was mixed thoroughly for identifying dead cells. After 15 min, cells were analyzed by flow cytometry using a FACSCanto II cytometer. The Ab-mediated cytotoxicity can be defined as the PI-positive target cells percentage in Far Red-positive target cells. EC50 of the cytotoxicity was determined using Prism. 
     T Cell Activation Assay 
     Secreted Cytokine TNFα and IFNγ 
     Whether T cells were activated was reflected by the quantity of TNFα and IFNγ secreted to supernatant. The isolation procedure of CD4 and CD8 positive T cells was described in Section “T Cell Activation (Intracellular Cytokine TNFα &amp; IFNγ Staining)”. The mixture of Raji human B cells (2*10 4  cells/well), CD4 or CD8 T cells (1*10 5  cells/well), and antibodies was co-incubated at 37° C. for 24 h. The supernatant was collected followed by centrifuging the reaction mixture at 1500 rpm for 5 min. The quantity of TNFα and IFNγ in the supernatant was determined by Human TNF ELISA Set (R&amp;D-DY210) and Human IFNγ ELISA Set (Capture Ab: Thermo Fisher-M700A, Detection Ab: Thermo Fisher-M701B, Standard substance: PEROTECH-300-02) respectively. 
     The procedure of sandwich ELISA was as follows. 96-well high protein binding ELISA plates (ThermoFisher-442404) were coated overnight at 4° C. or room temperature with 50 μl/well capture antibody in Carbonate-bicarbonate buffer (20 mM Na2CO3, 180 mM NaHCO3, pH 9.2) according to the kit specifications. All wells were washed three times with 300 μl per well of PBS/0.5% Tween-20 (v/v) and all the following wash steps in the assay were performed the same. The wells were then blocked for one hour with PBS/2% BSA (BovoGen Biologicals-BSAS) for TNFα and 100% casein (Pierce-37528) for IFNγ then washed three times, followed by binding of collected supernatant above or standard substance (50 μl/well) for 1 hour at room temperature and three washes afterwards. For the detection antibody binding, corresponding antibodies diluted in PBS/2% BSA for TNFα and 50% casein for IFNγ were added to the relevant wells and incubated at room temperature for two hours. Plates were washed three times prior to the addition of 50 μl of secondary antibody SA-HRP. Plates were incubated at room temperature for one hour, followed by six washes as described above. For the binding detection, 50 μl Tetramethylbenzidine (TMB) Substrate solution (Sigma-860336) was added to all wells for 10 minutes before stopping the reaction with 50 μl 2M HCl. The quantity of TNFα and IFNγ was determined by measuring the OD450 absorbance using the SpectraMax® M5e microplate reader. 
     T Cell Activation Assay-Surface Marker CD25 and CD69 Expression 
     Whether T cells were activated was reflected by staining signals of surface receptors CD25 and CD69. The isolation procedure of CD4 and CD8 positive T cells was described in Section “T Cell Activation (Intracellular Cytokine TNFα &amp; IFNγ Staining)”. The mixture of Raji human B cells (2*10 4  cells/well), CD4 or CD8 T cells (1*10 5  cells/well), and antibodies was co-incubated at 37° C. for 24 h. Following washing once with 1% BSA, the cell pellets were resuspended with staining buffer containing FITC Mouse Anti-human CD4 (BD-550628) or PerCpCy5.5 Mouse Anti-human CD8 (BD-560662), PE Mouse Anti-human CD69 (BD-560968) and APC Mouse Anti-human CD25 (BD-555434), followed by a 30 min incubation at 4° C. After washing cells twice, the percentage of PE and APC positive cells in FITC or PerCpCy5.5 positive cells was determined by flow cytometry. 
     Thermal Stability (DSF) 
     Melting temperature (Tm) of antibodies was investigated using QuantStudio™ 7 Flex Real-Time PCR system (Applied Biosystems). 19 μL of antibody solution was mixed with 1 μL of 62.5×SYPRO Orange solution (Invitrogen) and transferred to a 96 well plate (Biosystems). The plate was heated from 26° C. to 95° C. at a rate of 0.9° C./min, and the resulting fluorescence data was collected. The negative derivatives of the fluorescence changes with respect to different temperatures were calculated, and the maximal value was defined as melting temperature Tm. If a protein had multiple unfolding transitions, the first two Tm were reported, named as Tm1 and Tm2. Data collection and Tm calculation were conducted automatically by the operation software. 
     Serum Stability 
     Human blood was freshly collected from selected donors to polystyrene tubes without anticoagulant. Following 30 min&#39;s standing at room temperature, the human blood was centrifuged at 4000 rpm for 10 min to collect the serum layer. The centrifugation step was repeated until the serum was clarifying. Antibodies were mixed under detection with collected serum at the ratio of 1:9, and aliquots were drawn at 37° C. for the indicated times: 0 day, 1 day, 4 days, 7 days and 14 days. The samples were quick-frozen at different time points in liquid nitrogen and stored at −80° C. until use. The samples were analyzed by FACS to assess the binding ability on Jurkat CD3 T cells and Ramos CD19 B cells by comparison with that of corresponding antibodies without serum treatment. 
     Fcγ Receptor Binding Affinity by SPR 
     Antibody binding affinity to FcγRs was detected using Biacore T200 (or Biacore 8K). Each receptor was captured on an anti-his antibody immobilized CM5 sensor chip (GE). Antibodies at different concentrations were injected over the sensor chip at a flow rate of 30 uL/min for an association phase of 60 s, followed by 60 s dissociation. The chip was then regenerated by 10 mM glycine (pH 1.5) after each binding cycle. 
     The sensorgrams of blank surface and buffer channel were subtracted from the test sensorgrams. The experimental data was fitted by 1:1 model using Langmiur analysis (for FcγRI) or steady state model (for other receptors). A molecular weight of 150 KDa was used to calculate the molar concentration of antibodies. 
     C1q Binding by ELISA 
     ELISA Plates (Nunc) were coated with antibody samples at 3 μg/mL overnight at 4° C. After blocking and washing, C1q was gradient diluted starting from 600 μg/mL and incubated at room temperature for 2 hr. The plates were then washed and subsequently incubated with sheep anti-human C1q Ab-HRP for 1 hr. After washing, TMB substrate was added and the interaction was stopped by 2 M HCl. The absorbance at 450 nm was read using a microplate reader (Molecular Device). 
     FcRn Binding Affinity by SPR 
     Antibody binding affinity to FcRn was detected using Biacore T200 (or Biacore 8K). Each antibody was immobilized on CM5 sensor chip (GE). FcRn at different concentrations in running buffer (50 mM Na2HPO4/NaH2PO4, 150 mM NaCl, 0.05% Tween20, pH 6.0) were injected over the sensor chip at a flow rate of 30 uL/min for an association phase of 60 s, followed by 60 s dissociation. The chip was then regenerated by 1×PBS (pH 7.4) after each binding cycle. 
     The sensorgrams of blank surface and buffer channel were subtracted from the test sensorgrams. The experimental data was fitted by steady state model. Molecular weight of 45 KDa was used to calculate the molar concentration of FcRn. 
     Efficacy Study in Murine Raji/PBMC Model 
     The Raji tumor cells (ATCC® CCL-86™) were maintained in vitro as a monolayer culture in RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin at 37° C. in an atmosphere of 5% CO 2  in air. The tumor cells were routinely subcultured twice weekly. The cells growing in an exponential growth phase were harvested and counted for tumor inoculation. 
     Human PBMCs were isolated from heparin whole blood by using Ficoll-Paque Plus per manufacturer&#39;s instructions. 
     Each mouse was co-inoculated subcutaneously at the right flank with Raji tumor cells mixed with Matrigel and fresh PBMC in 0.2 ml of PBS on D0. Antibodies injection was conducted from D3 (i.v. BIW×4 times). 
     Testing Article Preparation 
     
       
         
           
               
               
               
               
             
               
                   
               
               
                 Compounds 
                 Package 
                 Preparation 
                 Conc. mg/ml 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
            
               
                 Isotype control 
                 9.38 mg/ml 
                 0.031 ml solution + 1.908 ml PBS 
                 1.5 
               
               
                 W3438-T3U4.E17-1.uIgG4.SP 
                  2.6 mg/ml 
                 B: 0.138 ml solution + 2.254 ml PBS 
                 1.5 
               
               
                 W3438-T3U4.E17-1.uIgG4.SP 
                   
                 B1: 0.450 ml B + 1.800 ml PBS 
                 0.3 
               
               
                 W3438-T3U4.E17-1.uIgG4.SP 
                   
                 B2: 0.450 ml B1 + 1.800 ml PBS 
                 0.06 
               
               
                   
               
            
           
         
       
     
     Tumor Measurements and Endpoints 
     The major endpoint was to see if the tumor growth could be delayed or mice could be cured. Tumor size was measured twice weekly in two dimensions using a caliper, and the volume was expressed in mm 3  using the formula: V=0.5 a×b 2  where a and b are the long and short diameters of the tumor, respectively. The T/C value (in percent) is an indication of antitumor effectiveness. 
     TGI was calculated for each group using the formula: TGI (%)=[1−(Ti−T0)/(Vi−V0)]×100; Ti is the average tumor volume of a treatment group on a given day, T0 is the average tumor volume of the treatment group on the day of treatment start, Vi is the average tumor volume of the vehicle control group on the same day with Ti, and V0 is the average tumor volume of the vehicle group on the day of treatment start. 
     Cynomolgus Monkey PK, Toxicity and Immunogenicity 
     One male and one female cynomolgus monkeys were were administered with WBP3438 at 1 mg/kg once by intravenous bolus administration. The formulations were formulated in 20 mM NaAc-HAc, 7.0% (w/w) Sucrose, 0.02% (w/v) PS80, pH5.0. PK blood samples were collected at pre-dose (Day-1), 0.25 h, 0.5 h, 1 h, 4 h, 8 h, 24 h, Day 3, Day 7, Day 14, Day 21 and Day 28. Antidrug antibody (ADA) samples were collected at 3 d, 14 d (312 h) and 28 d (480 h). 
     Serum concentrations of WBP3438 and ADA in serum samples were determined by ELISA. The serum concentration of WBP3438 in monkeys was subjected to a non-compartmental pharmacokinetic analysis by using the Phoenix WinNonlin software (version 6.3, Pharsight, Mountain View, Calif.). The linear/log trapezoidal rule was applied in obtaining the PK parameters. 
     Cage-side observations for general health and appearance, especially skin irritation was observed. Whole blood sample analysis for hematology (CBC) and serum analysis for chemistry detection were determined by hematology analyzer (ADVIA2120) and chemistry (HITACHI 7180), respectively. 
     Results 
     Generation of Cynomolgus CD19 Expressing Cell Line 
     The expression of cynomolgus CD19 expressing cell line WBP701.CHO-K1.cpro1.FL.C9 was detected using anti-CD19 antibody by flow cytometry. WBP701.CHO-K1.cpro1.FL.C9 showed high expression of monkey CD19 ( FIG. 37 ). 
     WuXiBody Generation and Optimization 
       FIG. 1  presents schematic representations of studied antibodies and formats. Both anti-CD3 antibody T3 and anti-CD19 antibody U4 were developed. The constant region (CL and CH1) of T3 was replaced by the constant domain of TCR to design unique light-heavy chain interface that is orthogonal to regular antibody. The TCR-modified T3 and native U4 in conjunction with “knobs-into-holes” mutations in Fc domain were used to design bispecific antibody format E17 and F16. 
     Variable heavy chain and light chain sequences of anti-CD3 and anti-CD19 binding moieties from W3438-T3U4.E17-1.uIgG4.SP and W3438-T3U4.F16-1.uIgG4.SP are provided below: 
     
       
         
           
               
               
               
               
             
               
                   
               
             
            
               
                 W3438- 
                 anti-CD3 
                 DNA sequence 
                 CAGGTGCAGCTTGTGCAGTCTGGGGCAGAAGTG 
               
               
                 T3U4.E17- 
                 antibody 
                 (SEQ ID NO: 353) 
                 AAGAAGCCTGGGTCTAGTGTCAAGGTGTCATGC 
               
               
                 1.uIgG4.SP 
                 VH 
                   
                 AAGGCTAGCGGGTTCGCCTTTACTGACTACTACA 
               
               
                 &amp; W3438- 
                   
                   
                 TCCACTGGGTGCGGCAGGCTCCCGGACAAGGGT 
               
               
                 T3U4.F16- 
                   
                   
                 TGGAGTGGATGGGATGGATCTCCCCAGGCAATG 
               
               
                 1.uIgG4.SP 
                   
                   
                 TCAACACAAAGTACAACGAGAACTTCAAAGGC 
               
               
                   
                   
                   
                 CGCGTCACCATTACCGCCGACAAGAGCACCTCC 
               
               
                   
                   
                   
                 ACAGCCTACATGGAGCTGTCCAGCCTCAGAAGC 
               
               
                   
                   
                   
                 GAGGACACTGCCGTCTACTACTGTGCCAGGGAT 
               
               
                   
                   
                   
                 GGGTACTCCCTGTATTACTTTGATTACTGGGGCC 
               
               
                   
                   
                   
                 AGGGCACACTGGTGACAGTGAGCTCC 
               
               
                   
               
               
                   
                   
                 Amino acid sequence 
                 QVQL V QSG A E VK KPGSSV KV SCKAS GFAFTDYYI   
               
               
                   
                   
                 (SEQ ID NO: 352) 
                   H W VR Q A PGQGLEW M G WISPGNVNTKYNENFKG   
               
               
                   
                   
                   
                 R V T I TAD K STSTAYM E LSSL R SED T AVY Y CAR DG   
               
               
                   
                   
                   
                   YSLYYFDY WGQGT LV TVSS 
               
               
                   
               
               
                   
                 anti-CD3 
                 DNA sequence 
                 GATATCGTGATGACCCAGAGCCCAGACTCCCTTG 
               
               
                   
                 antibody 
                 (SEQ ID NO: 355) 
                 CTGTCTCCCTCGGCGAAAGAGCAACCATCAACT 
               
               
                   
                 VL 
                   
                 GCAAGAGCTCCCAAAGCCTGCTGAACTCCAGG 
               
               
                   
                   
                   
                 ACCAGGAAGAATTACCTGGCCTGGTATCAGCAG 
               
               
                   
                   
                   
                 AAGCCCGGCCAGCCTCCTAAGCTGCTCATCTACT 
               
               
                   
                   
                   
                 GGGCCTCCACCCGGCAGTCTGGGGTGCCCGATC 
               
               
                   
                   
                   
                 GGTTTAGTGGATCTGGGAGCGGGACAGACTTCA 
               
               
                   
                   
                   
                 CATTGACAATTAGCTCACTGCAGGCCGAGGACG 
               
               
                   
                   
                   
                 TGGCCGTCTACTACTGTACTCAGAGCCACACTCT 
               
               
                   
                   
                   
                 CCGCACATTCGGCGGAGGGACTAAAGTGGAGAT 
               
               
                   
                   
                   
                 TAAG 
               
               
                   
               
               
                   
                   
                 Amino acid sequence 
                 DIVM T QSP D SL A VS L GE RA T IN C KSSQSLLNSRTR   
               
               
                   
                   
                 (SEQ ID NO: 354) 
                   KNYLA WYQQKPGQ P PKLLIY WASTRQS GVPDRFS 
               
               
                   
                   
                   
                 GSGSGT D FTLTIS SL QAED V AVY Y C TQSHTLRT FG 
               
               
                   
                   
                   
                 GGTK V EIK 
               
               
                   
               
               
                   
                 anti-CD19 
                 DNA sequence 
                 CAAATGCAGCTCGTCCAGTCTGGACCTGAAGTG 
               
               
                   
                 antibody 
                 (SEQ ID NO: 368) 
                 AAGAAGCCCGGGACATCCGTCAAGGTCTCATGT 
               
               
                   
                 VH 
                   
                 AAGGCTAGCGGGTACGCATTCACTTCCTACAAC 
               
               
                   
                   
                   
                 ATGTACTGGGTGCGCCAGGCCAGAGGACAGAG 
               
               
                   
                   
                   
                 GTTGGAGTGGATCGGCTACATCGACCCATACAA 
               
               
                   
                   
                   
                 CGCCGATACTACCTACAATCAGAAGTTTAAAGG 
               
               
                   
                   
                   
                 GCGGGTGACCATTACCCGGGATATGTCCACCTCC 
               
               
                   
                   
                   
                 ACCGCCTACATGGAGCTGAGCAGCCTGAGGAGC 
               
               
                   
                   
                   
                 GAGGACACAGCCGTGTACTACTGCCTGACAACA 
               
               
                   
                   
                   
                 GCCTATGCCATGGACTATTGGGGCCAGGGCACA 
               
               
                   
                   
                   
                 CTTGTGACTGTGAGCAGT 
               
               
                   
               
               
                   
                   
                 Amino acid sequence 
                 QMQLVQSGPEVKKPGTSVKVSCKAS GYAFTSYN   
               
               
                   
                   
                 (SEQ ID NO: 367) 
                   MY WVRQARGQRLEWIG YIDPYNADTTYNQKFKG   
               
               
                   
                   
                   
                 RVTITRDMSTSTAYMELSSLRSEDTAVYYCLT TAYA   
               
               
                   
                   
                   
                   MDY WGQGTLVTVSS 
               
               
                   
               
               
                   
                 anti-CD19 
                 DNA sequence 
                 GACATCCAGCTCACCCAATCCCCTTCTTTCCTCT 
               
               
                   
                 antibody 
                 (SEQ ID NO: 370) 
                 CCGCAAGTGTCGGAGATAGGGTGACTATCACCT 
               
               
                   
                 VL 
                   
                 GCTCAGCTTCTTCAACCGTGAACTACATGCATTG 
               
               
                   
                   
                   
                 GTACCAGCAGAAGCCCGGGAAAGCCCCAAAGC 
               
               
                   
                   
                   
                 TGCTGATCTACAGCACCTCCAATCTGGCCAGTGG 
               
               
                   
                   
                   
                 AGTGCCAAGCCGGTTTAGCGGGAGCGGCTCCGG 
               
               
                   
                   
                   
                 CACTGAATTCACTTTGACAATTAGCAGCCTTCAG 
               
               
                   
                   
                   
                 CCTGAGGACTTTGCCACATATTACTGTCACCAGT 
               
               
                   
                   
                   
                 GGTCCAGCTACCCCTACACATTCGGGCAGGGCA 
               
               
                   
                   
                   
                 CAAAGCTGGAGATTAAG 
               
               
                   
               
               
                   
                   
                 Amino acid sequence 
                 DIQLTQSPSFLSASVGDRVTITC SASSTVNYMH WY 
               
               
                   
                   
                 (SEQ ID NO: 369) 
                 QQKPGKAPKLLIY STSNLAS GVPSRFSGSGSGTEF 
               
               
                   
                   
                   
                 TLTISSLQPEDFATYYC HQWSSYPYT FGQGTKLEIK 
               
               
                   
               
            
           
         
       
     
     Full-length W3438-T3U4.E17-1.uIgG4.SP and W3438-T3U4.F16-1.uIgG4.SP sequences are provided below: 
     
       
         
           
               
               
               
             
               
                   
               
               
                 Antibody 
                 Chain 
                 Sequences 
               
               
                   
               
             
            
               
                 W3438- 
                 T3-LC 
                 DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQQKPGQP 
               
               
                 T3U4.E17- 
                 (SEQ ID 
                 PKLLIYWASTRQSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTQSHT 
               
               
                 1.uIgG4.SP 
                 NO: 12) 
                 LRTFGGGTKVEIKPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQT Q VS 
               
               
                   
                   
                 QSKDSDVYITDK C VLDMRSMDFKSNSAVAWS Q KSDFACANAF Q NSIIP 
               
               
                   
                   
                 EDTFFPSPESS 
               
               
                   
               
               
                   
                 T3-HC 
                 QVQLVQSGAEVKKPGSSVKVSCKASGFAFTDYYIHWVRQAPGQGLEW 
               
               
                   
                 (SEQ ID 
                 MGWISPGNVNTKYNENFKGRVTITADKSTSTAYMELSSLRSEDTAVYY 
               
               
                   
                 NO: 25) 
                 CARDGYSLYYFDYWGQGTLVTVLEDLKNVFPPEVAVFEPSEAEISHTQ 
               
               
                   
                   
                 KATLVCLATGFYPDHVELSWWVNGKEVHSGV C TDPQPLKEQPAL Q DS 
               
               
                   
                   
                 RYALSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQI 
               
               
                   
                   
                 VSAEAWGRYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVV 
               
               
                   
                   
                 VDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLH 
               
               
                   
                   
                 QDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPCQEEM 
               
               
                   
                   
                 TKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL 
               
               
                   
                   
                 YSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 
               
               
                   
               
               
                   
                 U4-LC 
                 DIQLTQSPSFLSASVGDRVTITCSASSTVNYMHWYQQKPGKAPKLLIYS 
               
               
                   
                 (SEQ ID 
                 TSNLASGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCHQWSSYPYTFGQ 
               
               
                   
                 NO: 23) 
                 GTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWK 
               
               
                   
                   
                 VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT 
               
               
                   
                   
                 HQGLSSPVTKSFNRGEC 
               
               
                   
               
               
                   
                 U4-HC 
                 QMQLVQSGPEVKKPGTSVKVSCKASGYAFTSYNMYWVRQARGQRLE 
               
               
                   
                 (SEQ ID 
                 WIGYIDPYNGDTTYNQKFKGRVTITRDMSTSTAYMELSSLRSEDTAVYY 
               
               
                   
                 NO: 26) 
                 CLTTAYAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCL 
               
               
                   
                   
                 VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG 
               
               
                   
                   
                 TKTYTCNVDRKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPK 
               
               
                   
                   
                 PKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPRE 
               
               
                   
                   
                 EQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKG 
               
               
                   
                   
                 QPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPEN 
               
               
                   
                   
                 NYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALHNHYT 
               
               
                   
                   
                 QKSLSLSLGK 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
             
               
                   
               
               
                 Antibody 
                 Chain 
                 Sequences 
               
               
                   
               
             
            
               
                 W3438- 
                 T3-LC 
                 DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQQKPGQ 
               
               
                 T3U4.F16- 
                 (SEQ ID 
                 PPKLLIYWASTRQSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTQS 
               
               
                 1.uIgG4.SP 
                 NO: 12) 
                 HTLRTFGGGTKVEIKPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQT 
               
               
                   
                   
                   Q VSQSKDSDVYITDK C VLDMRSMDFKSNSAVAWS Q KSDFACANAF Q   
               
               
                   
                   
                 NSIIPEDTFFPSPESS 
               
               
                   
               
               
                   
                 T3-HC 
                 QVQLVQSGAEVKKPGSSVKVSCKASGFAFTDYYIHWVRQAPGQGLE 
               
               
                   
                 (SEQ ID 
                 WMGWISPGNVNTKYNENFKGRVTITADKSTSTAYMELSSLRSEDTAV 
               
               
                   
                 NO: 25) 
                 YYCARDGYSLYYFDYWGQGTLVTVLEDLKNVFPPEVAVFEPSEAEISH 
               
               
                   
                   
                 TQKATLVCLATGFYPDHVELSWWVNGKEVHSGV C TDPQPLKEQPAL 
               
               
                   
                   
                   Q DSRYALSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKP 
               
               
                   
                   
                 VTQIVSAEAWGRYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPE 
               
               
                   
                   
                 VTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVS 
               
               
                   
                   
                 VLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTL 
               
               
                   
                   
                 PPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL 
               
               
                   
                   
                 DSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSL 
               
               
                   
                   
                 GK 
               
               
                   
               
               
                   
                 U4-LC 
                 DIQLTQSPSFLSASVGDRVTITCSASSTVNYMHWYQQKPGKAPKLLIY 
               
               
                   
                 (SEQ ID 
                 STSNLASGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCHQWSSYPYTF 
               
               
                   
                 NO: 23) 
                 GQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ 
               
               
                   
                   
                 WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYAC 
               
               
                   
                   
                 EVTHQGLSSPVTKSFNRGEC 
               
               
                   
               
               
                   
                 U4-HC 
                 QMQLVQSGPEVKKPGTSVKVSCKASGYAFTSYNMYWVRQARGQRL 
               
               
                   
                 (SEQ ID 
                 EWIGYIDPYNGDTTYNQKFKGRVTITRDMSTSTAYMELSSLRSEDTAV 
               
               
                   
                 NO: 27) 
                 YYCLTTAYAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAAL 
               
               
                   
                   
                 GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS 
               
               
                   
                   
                 SLGTKTYTCNVDHKPSNTKVDKRVGGGGSGGGGSQMQLVQSGPEVK 
               
               
                   
                   
                 KPGTSVKVSCKASGYAFTSYNMYWVRQARGQRLEWIGYIDPYNGDT 
               
               
                   
                   
                 TYNQKFKGRVTITRDMSTSTAYMELSSLRSEDTAVYYCLTTAYAMDY 
               
               
                   
                   
                 WGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPV 
               
               
                   
                   
                 TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNV 
               
               
                   
                   
                 DHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMIS 
               
               
                   
                   
                 RTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTY 
               
               
                   
                   
                 RVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQ 
               
               
                   
                   
                 VCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTP 
               
               
                   
                   
                 PVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLS 
               
               
                   
                   
                 LSLGK 
               
               
                   
               
            
           
         
       
     
     Production of W3438-T3U4.F16-1.uIgG4.SP 
     The expression titer of antibody W3438-T3U4.F16-1.uIgG4.SP is higher than 90 mg/L through transient expression. After 2-step purification, the purity of W3438-T3U4.F16-1.uIgG4.SP reaches 97.5% (SEC-HPLC,  FIG. 39 ). W3438-T3U4.F16-1.uIgG4.SP migrates with the apparent molecular mass of 75 kDa, 55 kDa and 25 kDa on SDS-PAGE under reducing conditions, corresponding to the two heavy chains and two light chains. The two light chains may overlap due to similar molecular weights. The antibody migrates with the apparent molecular mass of 200 kDa under non-reducing condition indicating the intact bispecific molecule ( FIG. 38 ). 
     Production of W3438-T3U4.E17-1.uIgG4.SP 
     The expression titer of antibody W3438-T3U4.E17-1.uIgG4.SP is higher than 100 mg/L through transient expression. After 2-step purification, the purity of W3438-T3U4.E17-1.uIgG4.SP reaches 95% (SEC-HPLC,  FIG. 41 ). W3438-T3U4.E17-1.uIgG4.SP migrates with the apparent molecular mass of 54 kDa, 56 kDa and 25 kDa on SDS-PAGE under reducing conditions, corresponding to the two heavy chains and two light chains. The two light chains may overlap due to similar molecular weights. The antibody migrates with the apparent molecular mass of 150 kDa under non-reducing condition indicating the intact bispecific molecule ( FIG. 40 ). 
     Target Binding 
     The binding of W3438-T3U4.E17-1.uIgG4.SP to CD19 and CD3 was tested on Ramos and Jurkat cells by flow cytometry ( FIGS. 42A-42B ). The antibody W3438-T3U4.E17-1.uIgG4.SP showed strong binding activities to Ramos and Jurkat cells, with EC 50  values of 15.6 nM and 47 nM respectively. 
     The binding of W3438-T3U4.F16-1.uIgG4.SP to CD19 and CD3 was tested on Ramos and Jurkat cells by flow cytometry ( FIGS. 43A-43B ). The antibody W3438-T3U4.F16-1.uIgG4.SP showed strong binding activities to Ramos and Jurkat cells, with EC 50  values of 1.8 nM and 19.3 nM respectively. 
     Cross Species Binding 
     The binding of W3438-T3U4.E17-1.uIgG4.SP to cynomolgus CD19 was tested on WBP701.CHO-K1.cpro1.FL.C9 cell (CD19-expressing cell) by flow cytometry ( FIG. 44 ). The binding EC 50  was 26 nM. The binding of W3438-T3U4.E17-1.uIgG4.SP to cynomolgus CD3 was tested using W331-cynoPro1.ECD.His (Cynomolgus CD3 epsilon protein) by ELISA ( FIG. 45 ). The binding EC 50  was 0.04 nM. 
     Affinity to Target Cells 
     The binding affinity of W3438-T3U4.E17-1.uIgG4.SP to human CD19 and CD3 was tested on Ramos and Jurkat cells by flow cytometry. The bound IgG/free IgG versus bound IgG was plotted in  FIGS. 46A and 46B . The fitted K D  values of binding to CD19 and CD3 were 23 nM and 9.0 nM, respectively. 
     Dual Binding on Target Cells 
     The activity of W3438-T3U4.E17-1.uIgG4.SP to bridge CD3 T cell and CD19 B cell was tested using pre-labeled Jurkat and Raji cells by flow cytometry ( FIGS. 47A-47B ). Q2 shows the population of bridged Jurkat and Raji cells. Compared with the negative control, roughly 18% of cells were bridged through bispecific antibody W3438-T3U4.E17-1.uIgG4.SP. 
     Cytotoxicity Assay 
     The cytotoxic activity of W3438-T3U4.E17-1.uIgG4.SP was evaluated using CD8+ T cell and raji cell. W3438-T3U4.E17-1.uIgG4.SP induced rapid and efficacious cell lysis after 4 hours incubation ( FIG. 48A ) with an EC 50  value of 15 nM. The maximum cell killing percentage was 90%. 
     The cytotoxic activity of W3438-T3U4.F16-1.uIgG4.SP was evaluated using CD8+ T cell and raji cell. W3438-T3U4.F16-1.uIgG4.SP induced rapid and efficacious cell lysis after 4 hours incubation ( FIG. 48B ) with an EC 50  value of 3.2 nM. The maximum cell killing percentage was 90%. 
     Target Specific T Cell Activation 
     W3438-T3U4.E17-1.uIgG4.SP was investigated in assays that indicate T cell activation through activation markers CD69 and CD25 in the presence or absence of CD19+ target cells. The results demonstrated that W3438-T3U4.E17-1.uIgG4.SP induces the expression of the T cell activation markers CD25 and CD69 in a dose-dependent manner only in the presence of CD19+ target cells ( FIGS. 49A-49D ). When the B cell is absent, no expression of CD25 and CD69 was observed in both CD4+ and CD8+ T cell subsets. 
     W3438-T3U4.E17-1.uIgG4.SP was also investigated in T cell activation assays of cytokine release in the presence or absence of CD19+ target cells. The results demonstrated that W3438-T3U4.E17-1.uIgG4.SP induces IFN-γ and TNF-α release in a dose-dependent manner only in the presence of CD19+ target cells ( FIGS. 50A-50D ). When the B cell is absent, no IFN-γ and TNF-α was detected in both CD4+ and CD8+ T cell subsets. 
     Thermal Stability 
     The thermal stability of W3438-T3U4.E17-1.uIgG4.SP was investigated using Real-Time PCR. T m 1 and T m 2 of W3438-T3U4.E17-1.uIgG4.SP are 60.2° C. and 72.7° C. 
     Serum Stability 
     W3438-T3U4.E17-1.uIgG4.SP was incubated in serum at 37° C. for 14 days. The binding activity of the antibody incubated for 0, 1, 4, 7 and 14 days was detected by flow cytometry. The results showed that the binding activity of W3438-T3U4.E17-1.uIgG4.SP to both CD3 and CD19 cells was unchanged after incubating in human serum for 14 days ( FIGS. 51A-51B ). 
     Fcγ Receptor Binding 
     The binding activity of W3438-T3U4.E17-1.uIgG4.SP to FcγRI, FcγRIIa (H167), FcγRIIa (R167), FcγRIIb, FcγRIIIa (F176), FcγRIIIa (V176) and FcγRIIIb were investigated by SPR. The affinities were summarized in Table 39. W3438-T3U4.E17-1.uIgG4.SP showed typical human IgG4 binding affinity to all the Fcγ receptors. 
     
       
         
           
               
             
               
                 TABLE 39 
               
             
            
               
                   
               
               
                 Affinity of W3438-T3U4.E17-1.uIgG4.SP to Fc Receptor by SPR 
               
            
           
           
               
               
               
            
               
                   
                 Fc receptor 
                 K D  (M) 
               
               
                   
                   
               
               
                   
                 FcγRI 
                 9.79E−09 
               
               
                   
                 FcγRIIa (H167) 
                 2.05E−05 
               
               
                   
                 FcγRIIa (R167) 
                 1.58E−05 
               
               
                   
                 FcγRIIb 
                 2.41E−05 
               
               
                   
                 FcγRIIIa (F176) 
                 2.93E−05 
               
               
                   
                 FcγRIIIa (V176) 
                 1.40E−05 
               
               
                   
                 FcγRIIIb 
                 &gt;4.10E−05  
               
               
                   
                   
               
            
           
         
       
     
     The binding activity of antibodies to C1Q was tested by ELISA. W3438-T3U4.E17-1.uIgG4.SP showed no binding signal in ELISA ( FIG. 52 ), and the control human IgG1 antibody showed normal binding signal. 
     FcRn Binding 
     The binding of W3438-T3U4.E17-1.uIgG4.SP to FcRn was tested by SPR at pH 6.0. The affinity was fitted as 2.58 μM which is a typical affinity of human IgG4 to FcRn. 
     Efficacy Study in the PBMC/Raji Xenograft Model 
     In this study, anti-tumor efficacy of W3438-T3U4.E17-1.uIgG4.SP in the admixed PBMC humanized model bearing Raji cell in NOG mice was investigated. The tumor growth curve is shown in  FIG. 53 . 
     At D14, the mean tumor size of the isotype control treatment group reached 342 mm 3 . The treatment with 1.5 mg/kg and 0.5 mg/kg of W3438-T3U4.E17-1.uIG4.SP produced a significant antitumor activity. The mean tumor size was respectively 78 mm 3  (T/C=23.0%, TGI=93.9%, p=0.016) and 75 mm 3  (T/C=22.0%, TGI=95.3%, p=0.014), and the tumor of one animal in high dosing level group was eradicated. W3438-T3U4.E17-1.uIgG4.SP at very low dose (0.06 mg/kg) did not show any antitumor activity. 
     Pharmacokinetics of WuXiBody in Cynomolgus Monkey 
     The concentration of W3438-T3U4.E17-1.uIgG4.SP in cynomolgus serum was tested by ELISA ( FIG. 54 ). The calculated PK parameters were listed in Table 40. The half life of W3438-T3U4.E17-1.uIgG4.SP for once single IV injection at 1 mg/kg was 152 hours. W3438-T3U4.E17-1.uIgG4.SP showed much longer half life in monkey than blinatumomab which has a very short half-life (1.5-2.6) hours in chimpanzees (European Medicines Agency assessment report EMA/CHMP/469312/2015). 
     
       
         
           
               
             
               
                 TABLE 40 
               
             
            
               
                   
               
               
                 Cynomolgus PK of W3438-T3U4.E17-1.uIgG4.SP 
               
            
           
           
               
               
               
            
               
                   
                 PK parameter 
                 W3438-T3U4.E17-1.uIgG4.SP 
               
               
                   
                   
               
            
           
           
               
               
               
            
               
                   
                 C 0  (μg/mL) 
                 60.4 
               
               
                   
                 T 1/2  (h) 
                 152 
               
               
                   
                 Vd ss  (L/kg) 
                 0.0513 
               
               
                   
                 Cl (mL/min/kg) 
                 0.00462 
               
               
                   
                 AUC 0-last  (h*μg/mL) 
                 3552 
               
               
                   
                 AUC 0-inf  (h*μg/mL) 
                 3708 
               
               
                   
                 MRT 0-last  (h) 
                 157 
               
               
                   
                 MRT 0-inf  (h) 
                 187 
               
               
                   
                   
               
            
           
         
       
     
     Toxicity 
     All monkeys tolerated the drug well during the entire course of the study. No adverse effects were observed during the in-life phase of the study. There was no obvious change in food consumption and weight. The parameters for Hematology and Clinical Chemistry, including AST, ALT, WBC, HGB and HCT were generally within the reference range. 
     Immunogenicity 
     The immunogenicity test results of W3438-T3U4.E17-1.uIgG4.SP are shown in  FIGS. 55A-55B . The titers of anti-drug antibody (ADA) against W3438-T3U4.E17-1.uIgG4.SP in monkey serum of 3, 14 and 28 days post dose showed no significant difference from predose. Therefore, the single IV injection of W3438-T3U4.E17-1.uIgG4.SP at 1 mg/kg appeared not immunogenic in monkeys. 
     Example 18: Bispecific Anti-CTLA-4×PD-1 WuXiBody 
     Background 
     Cancer immunotherapy has become a hot research area for treating cancer. Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) is one of the validated targets of immune checkpoints. After T cell activation, CTLA-4 quickly expresses on those T cells, generally within one hour of antigen engagement with TCR. CTLA-4 can inhibit T cell signaling through competition with CD28, which mediates a well characterized T cell co-stimulatory signal. CD28 binding to its ligands CD80 (B7-1) and CD86 (B7-2) on antigen presenting cells leads to T cell proliferation by inducing production of interleukin-2 and anti-apoptotic factors. Due to much higher affinity binding of CTLA-4 to CD80 and CD86 than that of CD28, CTLA-4 can out-compete with CD28 binding on CD80 and CD86, leading to suppression of T cell activation. In addition to induced expression on activated T cells, CTLA-4 is constitutively expressed on the surface of regulatory T cells (Treg), suggesting that CTLA-4 may be required for contact-mediated suppression and associated with Treg production of immunosuppressive cytokines such as transforming growth factor beta and interleukin-10. 
     CTLA-4 blockade can induce tumor regression, as demonstrated in a number of preclinical and clinical studies. Two antibodies against CTLA-4 are in clinical development. Ipilimumab (MDX-010, BMS-734016), a fully human anti-CTLA-4 monoclonal antibody of IgG1-kappa isotype, is an immunomodulatory agent that has been approved as monotherapy for treatment of advanced melanoma. The proposed mechanism of action for Ipilimumab is interference in the interaction of CTLA-4, which is expressed on a subset of activated T cells, with CD80/CD86 molecules on professional antigen presenting cells. This results in T-cell potentiation due to blockade of the inhibitory modulation of T-cell activation promoted by the CTLA-4 and CD80/CD86 interaction. The resulting T-cell activation, proliferation and lymphocyte infiltration into tumors, leads to tumor cell death. The commercial dosage form is a 5 mg/ml concentrate for solution for infusion. Ipilimumab is also under clinical investigation for other tumor types, including prostate and lung cancers. The second anti-CTLA-4 antibody in clinical development, Tremelimumab, was evaluated as monotherapy in melanoma and malignant mesothelioma. 
     Programmed Death-1 (PD-1, CD279) is a member of CD28 family expressed on activated T cells and other immune cells. Engagement of PD-1 inhibits function in these immune cells. PD-1 has two known ligands, PD-L1 (B7-H1, CD274) and PD-L2 (B7-DC, CD273), both belong to B7 family. PD-L1 expression is inducible on a variety of cell types in lymphoid and peripheral tissues, whereas PD-L2 is more restricted to myeloid cells including dendritic cells. The major role of PD-1 pathway is to reduce inflammatory immune response in tissues and organs. 
     Immunotherapy with the combination of monoclonal antibodies (mAbs) that block CTLA-4 (Ipilimumab) and PD-1 (Nivolumab) has shown clinical benefit beyond that observed with either mAb alone. Bispecific anti-CTLA-4×PD-1 WuXiBody were developed to induce antitumor immunity through simultaneous blockade of both of the checkpoint molecules. 
     Materials and Methods 
     General Materials 
     General research materials and their sources are listed in Table below. 
     
       
         
           
               
               
               
             
               
                   
               
               
                 Materials 
                 Vendor 
                 Cat. 
               
               
                   
               
             
            
               
                 Expi293F ™ Cells 
                 Thermo Fisher 
                 Cat. A14527 
               
               
                 ExpiFectamine293 transfection kit 
                 Thermo Fisher 
                 Cat. A14524 
               
               
                 Expi293F ™ expression medium 
                 Thermo Fisher 
                 Cat. A1435101 
               
               
                 Lipofectamine ™ 2000 Transfection Reagent 
                 Thermo Fisher 
                 Cat. 11668019 
               
               
                 FreeStyle ™ 293-F Cells 
                 Thermo Fisher 
                 Cat. R79007 
               
               
                 FreeStyle ™ 293 Expression Medium 
                 Thermo Fisher 
                 Cat. 12338002 
               
               
                 CHO-S Cells 
                 Thermo Fisher 
                 Cat. A1155701 
               
               
                 FreeStyle ™ CHO Expression Medium 
                 GIBCO 
                 Cat. 12651014 
               
               
                 Fetal bovine serum (FBS) 
                 Corning 
                 Cat. 35-076-CV 
               
               
                 Opti-MEM 
                 Thermo Fisher 
                 Cat. 31985070 
               
               
                 Ni column 
                 GE healthcare 
                 Cat. 17-5247-01 
               
               
                 Protein A column 
                 GE healthcare 
                 Cat. 17-5438-02 
               
               
                 Superdex200 prep grade 
                 GE Healthcare 
                 Cat. 17-1043-01 
               
               
                 HPLC-SEC 
                 TOSOH 
                 Cat. 0008541 
               
               
                 NuPAGE4%-12% Bis-Tris Gel 
                 Thermo Fisher 
                 Cat. NP0322BOX 
               
               
                 Human CTLA-4: W316-hPro1.ECD.His 
                 Sino Biological 
                 Cat. 11159-H08H 
               
               
                 Cynomolgus CTLA-4: W316-cpro1.ECD.his 
                 Sino Biological 
                 Cat. 90213-C08H 
               
               
                 Human PD-1: W305-hPro1.ECD.His 
                 In house 
               
               
                 Cynomolgus PD-1: W305-cynoPro1.ECD.His 
                 R&amp;D 
                 Cat.R&amp;D-8509-PD-050 
               
               
                 Coating 96-well plates for ELISA 
                 Nunc MaxiSorp, ThermoFisher 
               
               
                 U-bottom 96-well plates for FACS 
                 Coming-COSTAR 
                 3799 
               
               
                 Human PD-1+ cell line: W305-CHO-S.hPro1.C6 
                 In house 
               
               
                 Cynomolgus PD-1+ cells: W305-293F.cPro1.FL.Pool 
                 In house 
               
               
                 Human CTLA-4+ cell line: W316-293F.hPro1.FL 
                 In house 
               
               
                 Cynomolgus CTLA-4+ cell line W316-293F.cPro1.FL.Pool 
                 In house 
               
               
                 Human CD80+ cell line: W316-CHO-K1.hPro1L1.B9B11 
                 In house 
               
               
                 Human CD86+ cell line W316-CHO-K1.hPro1L2.A4A7 
                 In house 
               
               
                 Human PD-1: W305-hPro1.ECD.mFc 
                 In house 
               
               
                 Human PD-1: W305-hPro1.ECD.hFc 
                 In house 
               
               
                 Human PD-1: W305-hPro1.ECD.His 
                 In house 
               
               
                 Cynomolgus PD-1: W305-cPro1.ECD.His 
                 In house 
               
               
                 CynoPD-1.hFc protein 
                 SinoBiological 
                 90311C02H 
               
               
                 Human CTLA-4: W316-hPro1.ECD.mFc 
                 In house 
               
               
                 Human CTLA-4: W316-hPro1.ECD.hFc 
                 In house 
               
               
                 Human CTLA-4: W316-hPro1.ECD.His 
                 In house 
               
               
                 Cynomolgus CTLA-4: W316-cPro1.ECD.His 
                 In house 
               
               
                 Human PDL1: W315-hPro1.ECD.mFc 
                 In house 
               
               
                 CynoPD-L1.hFc-Biotin 
                 In house 
               
               
                 Biotin-labeled W316.hPro1.ECD.hFc 
                 In house 
               
               
                 Human CD80: W316-hPro1L1.ECD.His 
                 In house 
               
               
                 WBP316-BMK1 (Ipilimumab) 
                 In house 
               
               
                 WBP305 BMK1 (nivolumab) 
                 In house 
               
               
                 WBP324-BMK1.IgG1.KDL 
                 In house 
               
               
                 Isotye control: WBP332-1.80.12.xAb.hIgG4 
                 In house 
               
               
                 HRP-labeled goat anti-human IgG Fc 
                 Bethyl Laboratories 
                 A80-304P 
               
               
                 HRP-labeled mouse anti-Human IgG Fc (CH2) 
                 Thermo 
                 MA5-16859 
               
               
                 HRP-labeled goat anti-mouse IgG Fc 
                 Bethyl Laboratories 
                 A90-231P 
               
               
                 HRP-labeled Streptavidin 
                 Lifetechnologies 
                 SNN1004 
               
               
                 Biotin-labeled anti-His mAb 
                 GenScript 
                 A00613 
               
               
                 FITC-labeled goat anti-human IgG 
                 Jackson 
                 109-095-008 
               
               
                 PE-labeled goat anti-human IgG 
                 Jackson 
                 109-115-098 
               
               
                 FITC-labeled goat anti-Mouse IgG 
                 Abcam 
                 98716 
               
               
                 PE-labeled Streptavidin 
                 BD 
                 554061 
               
               
                 Human Ficoll-Paque 
                 Stemcell 
                 07861 
               
               
                 Monocyte enrichment kit 
                 Miltenyi 
                 Biotec-130-050-201 
               
               
                 CD4 +  T cell enrichment kit 
                 Stemcell 
                 19052 
               
               
                 CD4 + CD25 +  T cell enrichment kit 
                 Miltenyi 
                 130-093-631 
               
               
                 Recombinant human GM-CSF 
                 R&amp;D 
                 215-GM 
               
               
                 Recombinant human IL-2 
                 SLPHARM 
               
               
                 Recombinant human IL-4; anti-IL-2 Ab 
                 R&amp;D 
                 AG1815401; MAB602 
               
               
                 Recombinant human IFN-γ standard 
                 Peprotech 
                 300-02 
               
               
                 Anti-IFN-γ antibodies 
                 life technology 
                 M700A; M7001B 
               
               
                 H3-thymidine and MicroScint 
                 Perkin Elmer 
                 NET027001MC 
               
               
                 Fetal bovine serum (FBS) 
                 GIBCO 
                 10100147 
               
               
                 RPMI 1640 medium 
                 GIBCO 
                 22400089 
               
               
                 DPBS 
                 Corning 
                 21-031-CVR 
               
               
                 DELFIA ® EuTDA Cytotoxicity Reagents 
                 PerkinElmer 
                 AD0116 
               
               
                 CellTiter-Glo Luminescent Cell Viability Assay Kit 
                 Promega 
                 G7573 
               
               
                 Calcein-AM 
                 Corning-354216 
                 354216 
               
               
                 Far red 
                 Invitrogen 
                 C34572 
               
               
                   
               
            
           
         
       
     
     Generation of Soluble Antigens 
     DNA sequences encoding the extracellular domain sequence of human PD-1 (Uniport No.: Q15116) were synthesized in Sangon Biotech (Shanghai, China), and then subcloned into modified pcDNA3.3 expression vectors with 6×his in C-terminal. Protein of human, cynomolgus and mouse CTLA-4 and mouse and cynomolgus PD-1 were purchased from Sino Biological. 
     Expi293 cells (Invitrogen-A14527) were transfected with the purified expression vector pcDNA3.3. Cells were cultured for 5 days and supernatant was collected for protein purification using Ni-NTA column (GE Healthcare, 175248). The obtained human PD-1 was QC&#39;ed by SDS-PAGE and SEC, and then stored at −80° C. 
     Generation of Reference Antibodies 
     DNA sequence encoding the variable region of anti-CTLA-4 antibody (WBP316-BMK1), anti-PD-1 antibody (WBP305-BMK1) was synthesized in Sangon Biothech (Shanghai, China), and then subcloned into modified pcDNA3.4 expression vectors with constant region of human IgG1 or human IgG4 (S228P). Anti-PD-1 WBP3055-1.153.7.uIgG4k and WBP3055-1.103.11.uIgG4k antibodies were generated after immunizing rats with human PD-1 and mouse PD-1, and were converted to IgG4 (S228P) format. DNA sequence encoding a benchmark bispecific anti-CTLA-4×PD-1 antibody (WBP324-BMK1.IgG1.KDL) was synthesized. 
     The plasmids containing the VH and VL genes were co-transfected into Expi293 cells. Cells were cultured for 5 days and supernatant was collected for protein purification using Protein A column (GE Healthcare, 175438) or Protein G column (GE Healthcare, 170618). The obtained antibodies were tested by SDS-PAGE and SEC, and then stored at −80° C. 
     Generation of Target-Expressing Cell Lines 
     Using Lipofectamine 2000, CHO-S or 293F cells were transfected with the expression vectors containing the genes encoding full length human PD-1 or mouse PD-1. The cells were cultured in medium containing proper selection markers. The human PD-1 high expression stable cell line (WBP305.CHO-S.hPro1.C6) and mouse PD-1 high expression stable cell line (WBP305.293F.mPro1.B4) were obtained by limiting dilution. 
     Generation of Bispecific Anti-CTLA-4/PD-1 Bispecific Antibodies 
     Construction of W3248-U6T1.G25R-1.uIgG4.SP: DNA sequence encoding anti-PD-1 heavy chain variable region, constant region 1, anti-CTLA-4 heavy chain variable region, TCR beta constant region, and IgG4 (S228P) constant region 2 and 3, linked from 5′ end to 3′ end, were cloned into a modified pcDNA3.3 expression vector. DNA sequence encoding anti-CTLA-4 antibody light chain variable region on the 5′ of TCR alpha constant region was cloned into another modified pcDNA3.3 expression vector. Anti-PD-1 light chain was cloned into the third modified pcDNA3.3 expression vector. 
     Construction of W3248-U6T5.G25-1.uIgG4.SP: DNA sequence encoding anti-PD-1 heavy chain variable region, constant region of TCR beta chain, anti-CTLA-4 heavy chain variable region and IgG4 (S228P) constant region, linked from 5′ end to 3′ end, were cloned into a modified pcDNA3.3 expression vector. DNA sequence encoding anti-PD-1 antibody light chain variable region on the 5′ of TCR alpha constant region was cloned into another modified pcDNA3.3 expression vector. Anti-CTLA-4 light chain was cloned into the third modified pcDNA3.3 expression vector. 
     
       
         
           
               
               
               
             
               
                   
               
             
            
               
                 LC1 
                 Amino acid sequence 
                 SYELTQPLSVSVALGQTARITCGGDNIGNKDVHWYQQKPGQ 
               
               
                   
                 (SEQ ID NO: 412) 
                 APVLVIYRDSNRPSGIPEGFSGSNSGNTATLTISRAQAGDEAD 
               
               
                   
                   
                 YYCQVWDSIWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQA 
               
               
                   
                   
                 NKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSN 
               
               
                   
                   
                 NKYAASSYLSLTPEQWKSHKSYSCQVTHEGSTVEKTVAPTEC 
               
               
                   
                   
                 S 
               
               
                   
               
               
                 LC2 
                 Amino acid sequence 
                 EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKPGQ 
               
               
                   
                 (SEQ ID NO: 413) 
                 APRLLIYGAFSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVY 
               
               
                   
                   
                 YCQQYGSSPWTFGQGTKVEIKPDIQNPDPAVYQLRDSKSSDK 
               
               
                   
                   
                 SVCLFTDFDSQTQVSQSKDSDVYITDKCVLDMRSMDFKSNSA 
               
               
                   
                   
                 VAWSQKSDFACANAFQNSIIPEDTFFPSPESS 
               
               
                   
               
               
                 HC 
                 Amino acid sequence 
                 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSHAMSWVRQAPG 
               
               
                   
                 (SEQ ID NO: 414) 
                 KGLEWVSTITGGGGSIYYADSVKGRFTISRDNSKNTLYLQMN 
               
               
                   
                   
                 SLRAEDTAVYYCAKNRAGEGYFDYWGQGTLVTVSSASTKGP 
               
               
                   
                   
                 SVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSG 
               
               
                   
                   
                 VHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNT 
               
               
                   
                   
                 KVDKRVGGGGSGGGGSQVQLVESGGGVVQPGRSLRLSCAAS 
               
               
                   
                   
                 GFTFSSYTMHWVRQAPGKGLEWVTFISYDGNNKYYADSVKG 
               
               
                   
                   
                 RFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGWLGPFDYW 
               
               
                   
                   
                 GQGTLVTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLAT 
               
               
                   
                   
                 GFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALQDSRYA 
               
               
                   
                   
                 LSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKP 
               
               
                   
                   
                 VTQIVSAEAWGRYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMI 
               
               
                   
                   
                 SRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREE 
               
               
                   
                   
                 QFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTI 
               
               
                   
                   
                 SKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAV 
               
               
                   
                   
                 EWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGN 
               
               
                   
                   
                 VFSCSVMHEALHNHYTQKSLSLSLG 
               
               
                   
               
            
           
         
       
     
     Relevant sequences of W3248-U6T1.G25R-1.uIgG4.SP are provided below: 
     
       
         
           
               
               
               
             
               
                   
               
             
            
               
                 LC1 
                 Amino acid sequence 
                 SYELTQPLSVSVALGQTARITCGGDNIGNKDVHWYQQKPGQA 
               
               
                   
                 (SEQ ID NO: 415)  
                 PVLVIYRDSNRPSGIPEGFSGSNSGNTATLTISRAQAGDEADYY 
               
               
                   
                   
                 CQVWDSIWVFGGGTKLTVLPDIQNPDPAVYQLRDSKSSDKSV 
               
               
                   
                   
                 CLFTDFDSQTQVSQSKDSDVYITDKCVLDMRSMDFKSNSAVA 
               
               
                   
                   
                 WSQKSDFACANAFQNSIIPEDTFFPSPESS 
               
               
                   
               
               
                 LC2 
                 Amino acid sequence 
                 EIVLTQSPDFQSVTPKEKVTITCSANSALSYMYWYQQKPDQSP 
               
               
                   
                 (SEQ ID NO: 416) 
                 KLWVHGTSNLASGVPSRFSGSGSGTDFTLTINSLEAEDAATYY 
               
               
                   
                   
                 CHHWSNTQWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTA 
               
               
                   
                   
                 SVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDS 
               
               
                   
                   
                 TYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 
               
               
                   
               
               
                 HC 
                 Amino acid sequence 
                 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSHAMSWVRQAPG 
               
               
                   
                 (SEQ ID NO: 417) 
                 KGLEWVSTITGGGGSIYYADSVKGRFTISRDNSKNTLYLQMNS 
               
               
                   
                   
                 LRAEDTAVYYCAKNRAGEGYFDYWGQGTLVTVLEDLKNVFP 
               
               
                   
                   
                 PEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGK 
               
               
                   
                   
                 EVHSGVCTDPQPLKEQPALQDSRYALSSRLRVSATFWQNPRN 
               
               
                   
                   
                 HFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRGGGGS 
               
               
                   
                   
                 GGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTNYFMNW 
               
               
                   
                   
                 VRQAPGQGLEWMGRVDPEQGRADYAEKFKKRVTITADKSTS 
               
               
                   
                   
                 TAYMELSSLRSEDTAVYYCARRAMDNYGFAYWGQGTLVTVS 
               
               
                   
                   
                 SASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN 
               
               
                   
                   
                 SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNV 
               
               
                   
                   
                 DHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPK 
               
               
                   
                   
                 DTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKT 
               
               
                   
                   
                 KPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSS 
               
               
                   
                   
                 IEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYP 
               
               
                   
                   
                 SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSR 
               
               
                   
                   
                 WQEGNVFSCSVMHEALHNHYTQKSLSLSLG 
               
               
                   
               
            
           
         
       
     
     For both bispecific antibodies, one heavy chain expression vector and two light chain expression vectors were co-transfected into Expi293 cells (ThermoFisher-A14527) according to the manufacturer&#39;s instructions. Five days after transfection, the supernatants were harvested and purified using Protein A column (GE Healthcare-17543802) and further size-exclusion chromatography (GE Healthcare-17104301). Antibody concentration was measured by Nano Drop. The low endotoxin level was confirmed by using endotoxin detection kit (GenScript-L00350), and the endotoxin level of two bispecific antibodies was less than 10 EU/mg. The purity of proteins was evaluated by SDS-PAGE and HPLC-SEC. 
     In Vitro Charactrization 
     Differential Scanning Fluorimetry (DSF) 
     A DSF assay was performed using 7500 Fast Real-Time PCR system (Applied Biosystems). Briefly, 19 μL of bispecific antibody solution was mixed with 1 μL of 62.5×SYPRO Orange solution (TheromFisher-S6650) and added to a 96 well plate. The plate was heated from 26° C. to 95° C. at a rate of 2° C./min and the resulting fluorescence data was collected. The data was analyzed automatically by its operation software and Th was calculated by taking the maximal value of negative derivative of the resulting fluorescence data with respect to temperature. Ton can be roughly determined as the temperature of negative derivative plot beginning to decrease from a pre-transition baseline. 
     Human PD-1-Binding by FACS 
     Engineered human PD-1 expressing cells W305-CHO-S.hPro1.C6 were seeded at 1×10 5  cells/well in U-bottom 96-well plates (COSTAR 3799). Antibodies with 3.16-fold titration in 1% BSA DPBS from 200 nM to 0.002 nM were added to the cells. The plates were incubated at 4° C. for 1 hour. After wash, 100 μL 1:125 diluted PE-labeled goat anti-human antibody (Jackson 109-115-098) was added to each well and the plates were incubated at 4° C. for 1 hour. The binding of the antibodies onto the cells was tested by flow cytometry and the mean fluorescence intensity (MFI) was analyzed by FlowJo. 
     Cynomolgus PD-1-Binding by FACS 
     Engineered cynomolgus PD-1 expressing cells W305-293F.cynoPro1.FL.pool were seeded at 1×10 5  cells/well in U-bottom 96-well plates (COSTAR 3799). 4.0-fold titrated Abs with 1% BSA DPBS from 40 μg/ml to 0.0001526 μg/ml were added to the cells. Plates were incubated at 4° C. for 1 hour. After wash, 100 μL 1:150 diluted PE-labeled goat anti-human antibody (Jackson 109-115-098) was added to each well and the plates were incubated at 4° C. for 1 hour. The binding of the antibodies onto the cells was tested by flow cytometry and the mean fluorescence intensity (MFI) was analyzed by FlowJo. 
     Human CTLA-4-Binding by FACS 
     Engineered human CTLA-4 expressing cells W316-293F.hPro1.FL were seeded at 1×10 5  cells/well in U-bottom 96-well plates (COSTAR 3799). 3.16-fold titrated Abs with 1% BSA DPBS from 200 nM to 0.002 nM were added to the cells. Plates were incubated at 4° C. for 1 hour. After wash, 100 μL 1:150 diluted PE-labeled goat anti-human antibody (Jackson 109-115-098) was added to each well and the plates were incubated at 4° C. for 1 hour. The binding of the antibodies onto the cells was tested by flow cytometry and the mean fluorescence intensity (MFI) was analyzed by FlowJo. 
     Cynomolgus CTLA-4-Binding by FACS 
     Engineered human CTLA-4 expressing cells W316-293F.cynoPro1.F1.Pool were seeded at 1×10 5  cells/well in U-bottom 96-well plates (COSTAR 3799). 4-fold titrated Abs with 1% BSA DPBS from 40 μg/ml to 0.00004 μg/ml were added to the cells. Plates were incubated at 4° C. for 1 hour. After wash, 100 μL 1:150 diluted PE-labeled goat anti-human antibody (Jackson 109-115-098) was added to each well and the plates were incubated at 4° C. for 1 hour. The binding of the antibodies onto the cells was tested by flow cytometry and the mean fluorescence intensity (MFI) was analyzed by FlowJo. 
     hPD-1 and hCTLA-4 Dual Binding by ELISA 
     In order to test whether the bispecific antibodies could bind to both hPD-1 and hCTLA-4, an ELISA assay was developed as below. A 96-well ELISA plate (Nunc MaxiSorp, ThermoFisher) was coated overnight at 4° C. with 0.5 μg/ml antigen-1 (hPD-1-ECD, W305-hPro1.ECD.mFc) in carbonate-bicarbonate buffer. After a 1 hour blocking step with 2% (w/v) bovine serum albumin (Pierce) dissolved in PBS, serial dilutions of the different PD-1×CTLA-4 bispecific antibodies in PBS containing 2% BSA PBS were incubated on the plates for 1 hour at room temperature. Following the incubation, plates were washed three times with 300 μL per well of PBS containing 0.5% (v/v) Tween 20. 0.5 μg/ml antigen-2 (hCTLA-4-ECD, W316-hPro1.ECD.hFc.Biotin) was added to plates and the mixture was incubated for 1 hour. After washing the plates three times, Streptavidin-HRP (Lifetechnologies, # SNN1004) (1:20000 diluted) was added and incubated on the plates for 1 hour at room temperature. After washing six times with 300 μL per well of PBS containing 0.5% (v/v) Tween 20, 100 μL tetramethylbenzidine (TMB) substrate was added for the detection per well. The reaction was stopped after approximately 5 minutes through the addition of 100 μL per well of 2 M HCl. The absorbance of the wells was measured at 450 nm with a multiwall plate reader (SpectraMax® M5e). 
     hPD-1 and hCTLA-4 Dual Binding by FACS 
     In order to test whether the bispecific antibodies could bind to both hPD-1 and hCTLA-4, a FACS assay was developed as below. Engineered human PD-1 and CTLA-4 expressing cells W305-CHO-S.hPro1.C6 and W316-293F.hPro1.F1 were stained with Calcein-AM (Corning-354216) at 50 nM and Far red (Invitrogen-C34572) at 20 nM, respectively, for 20 mins at 37° C. After wash with 1% (w/v) bovine serum albumin (Pierce) dissolved in PBS twice, mixed hPD-1 (5E4) and hCTLA-4 (5E4) cells were seeded at 1×10 5  cells/well in U-bottom 96-well plates (COSTAR 3799). After removal of the supernatant, 3×serially diluted antibodies with 1% BSADPBS from 7.5 nM to 0.83 nM were added to the cells. The plates were incubated at 4° C. for 1.5 hour. The cells were tested by flow cytometry and the percentage of double positive cells was analyzed by FlowJo. 
     Human PD-1-Competitive FACS 
     In order to test whether the bispecific antibodies could block hPD-L1 binding to hPD-1 protein, a competitive FACS was conducted. Briefly, engineered human PD-1 expressing cells W305-CHO-S.hPro1.C6 (in house) were seeded at 1×10 5  cells/well in U-bottom 96-well plates (COSTAR 3799), 200 nM to 0.002 nM human PD-L1 coupled with 5 ug/ml human PD-L1 protein W315-hPro1.ECD.mFc were added to the cells. Plates were incubated at 4° C. for 1 hour. After wash, the binding of W315-hPro1.ECD.mFc to cell expressive human PD-1 was detected by FITC-labeled goat anti-mouse antibody (abcam 98716 1:125). The competition binding of antibodies to the cells was tested by flow cytometry and the mean fluorescence intensity (MFI) was analyzed by FlowJo. 
     Blockage of Human/Cynomolgus CTLA-4 Binding to Human CD80 
     ELISA was used to test whether the bispecific antibodies could block hCTLA-4 binding to hCD80 protein. Briefly, flat-bottom 96-well plates (Nunc MaxiSorp, ThermoFisher) were pre-coated with 0.5 μg/ml W316-hPro1.ECD.hFc overnight at 4° C. After 2% BSA blocking, 100 μL 3.16-fold titrated Abs from 400 nM to 0.04 nM Abs coupled with 0.5 μg/ml human CD80 protein W316-hPro1L1.ECD.His were pipetted into each well and incubated for 1 hour at ambient temperature. Following the incubation, plates are washed 3 times with 300 μL per well of PBS containing 0.5% (v/v) Tween 20. 100 μL 0.5 μg/ml Biotin-labeled anti-His mAb (GenScript-A00613) was added to plate pre well and incubation 1 hour. After washing for 6 times, the binding of W315-hPro1L1.ECD.His to WBP316-hPro1.ECD.hFc was detected by Streptavidin-HRP (Lifetechnologies, # SNN1004) (1:20000 diluted). The color was developed by dispensing 100 μL of TMB substrate, and then stopped by 100 μL of 2N HCl. The absorbance was read at 450 nm using a Microplate Spectrophotometer (SpectraMax® M5e). 
     Competitive FACS was used to test whether the antibodies could block human or cynomolgus CTLA-4 binding to hCD80 on cell surface. Briefly, human CD80-expressing CHO-K1 cells were added to each well of a 96-well plate (COSTAR 3799) at 1×10 5  per well and centrifuged at 1500 rpm for 4 minutes at 4° C. before removing the supernatant. Serial dilutions of test antibodies, positive and negative controls were mixed with biotinylated human CTLA-4.ECD.hFc. Due to different density of ligands on cell surface, 0.066-0.037 μg/mL of hCTLA-4.ECD.hFc-Biotin was used for human CD80-expressing cells. Then the mixtures of antibody and CTLA-4 were added to the cells and incubated for 1 hour at 4° C. The cells were washed two times with 200 μl FACS washing buffer (DPBS containing 1% BSA). Streptavidin PE (BD Pharmingen-554061) 1 to 600 diluted in FACS buffer was added to the cells and incubated at 4° C. for 1 hour. Additional washing steps were performed two times with 200 μL FACS washing buffer followed by centrifugation at 1500 rpm for 4 minutes at 4° C. Finally, the cells were resuspended in 100 μL FACS washing buffer and fluorescence values were measured by flow cytometry and analyzed by FlowJo. 
     Affinity to CTLA-4 and PD-1 
     SPR technology was used to measure the on-rate constant (ka) and off-rate constant (kd) of the antibodies to ECD of CTLA-4 or PD-1. The affinity constant (KD) was consequently determined. 
     Biacore T200, Series S Sensor Chip CM5, Amine Coupling Kit, and 10×HBS-EP were purchased from GE Healthcare. Goat anti-human IgG Fc antibody was purchased from Jackson ImmunoResearch Lab (catalog number 109-005-098). In immobilization step, the activation buffer was prepared by mixing 400 mM EDC and 100 mM NHS immediately prior to injection. The CM5 sensor chip was activated for 420 s with the activation buffer. 30 μg/mL of goat anti-human IgG Fcγ antibody in 10 mM NaAc (pH 4.5) was then injected to Fc1-Fc4 channels for 200 s at a flow rate of 5 μL/min. The chip was deactivated by 1 M ethanolamine-HCl (GE). Then the antibodies were captured on the chip. Briefly, 4 μg/mL antibodies in running buffer (HBS-EP+) was injected individually to Fc3 channel for 30 s at a flow rate of 10 μL/min. Eight different concentrations (20, 10, 5, 2.5, 1.25, 0.625, 0.3125 and 0.15625 nM) of analyte ECD of CTLA-4 or PD-1 and blank running buffer were injected orderly to Fc1-Fc4 channels at a flow rate of 30 μL/min for an association phase of 120 s, followed by 2400 s dissociation phase. Regeneration buffer (10 mM Glycine pH 1.5) was injected at 10 μL/min for 30 s following every dissociation phase. 
     Human Serum Stability 
     The antibodies were incubated in freshly isolated human serum at 37° C. On indicated time points, an aliquot of serum treated sample was removed from the incubator and snap frozen in liquid nitrogen, and then stored at −80° C. until ready for a dual-binding ELISA test. The frozen samples were quickly thawed immediately prior to the stability test. Briefly, plates were pre-coated with 0.5 μg/mL of hCTLA4.ECD.hFc (in house) at 4° C. overnight. After 1-hour blocking, the testing antibodies were added to the plates at various concentrations. The plates were incubated at ambient temperature for 1 hour. Following the incubation, the plates were washed three times with 300 μL per well of PBS containing 0.5% (v/v) Tween 20. Then 0.1 μg/ml hPD-1-ECD. Biotin was added to plates and the mixture was incubated for 1 hour. After washing the plates three times, Streptavidin-HRP (Lifetechnologies, # SNN1004) (1:20000 diluted) was added and incubated on the plates for 1 hour at room temperature. After washing six times with 300 μL per well of PBS containing 0.5% (v/v) Tween 20, 100 μL tetramethylbenzidine (TMB) substrate is added for the detection per well. The reaction was stopped after approximately 5 minutes by addition of 100 μL per well of 2 M HCl. The absorbance of the wells was measured at 450 nm with a multiwall plate reader (SpectraMax® M5e). 
     Results 
     Expression and Purification of Bispecific Antibodies 
     The purity of the bispecific antibodies was above 90%, analyzed by both SDS-PAGE ( FIG. 56A ) and SEC-HPLC ( FIG. 56B ). 
     DSF of WuXiBody 
     DSF was used to measure T m  of WuXiBody. As shown in  FIG. 57 , W3248-U6T1.G25R-1.uIgG4.SP and WBP3248-U6T5.G25-1-uIgG4.SP have Th1 at 60.8 and 63.4° C., respectively. 
     Binding to Human and Cynomolgus PD-1 
     The bispecific antibodies could bind to human PD-1 ( FIG. 58 ) and cynomolgus PD-1 ( FIG. 59 ). The human PD-1-binding activity of W3248-U6T1.G25R-1.uIgG4.SP was slightly better than WBP3248-U6T5.G25-1-uIgG4.SP in FACS. W3248-U6T1.G25R-1.uIgG4.SP and WBP3248-U6T5.G25-1-uIgG4.SP have affinity to human PD-1 at 1.24 nM and 1.32 nM, respectively ( FIG. 62 ). 
     Binding to Human and Cynomolgus CTLA-4 
     The purified bispecific antibodies bound to human CTLA-4, as tested in FACS ( FIG. 60 ). The two bispecific antibodies also bound to cynomolgus CTLA-4 ( FIG. 61 ). W3248-U6T1.G25R-1.uIgG4.SP and WBP3248-U6T5.G25-1-uIgG4.SP have affinity to human CTLA-4 at 0.0356 nM and 0.357 nM, respectively ( FIG. 62 ). 
     Simultaneous Binding to CTLA-4 and PD-1 
     In order to test whether the bispecific antibodies can bind to both targets, ELISA and FACS were used. In the ELISA, human PD-1 was coated on the plate. After adding bispecific antibodies, biotinylated CTLA-4 was used to detect bound bispecific antibodies. As shown in  FIG. 66 , W3248-U6T1.G25R-1.uIgG4.SP and WBP3248-U6T5.G25-1-uIgG4.SP could bind to both PD-1 and CTLA-4 with EC50 at 0.1072 to 0.0710 nM, comparable with a bispecific reference antibody WBP324 BMK1 (EC50=0.0599 nM). In the FACS, both W3248-U6T1.G25R-1.uIgG4.SP and WBP3248-U6T5.G25-1-uIgG4.SP could simultaneously bind to PD-1+ and CTLA-4+ cells ( FIG. 67 ). 
     Blocking Human or Cynomolgus CTLA-4 Binding to CD80 Binding 
     A competitive FACS was used to test the bispecific antibodies&#39; blockage of CTLA-4 with its ligand CD80. W3248-U6T1.G25R-1.uIgG4.SP and WBP3248-U6T5.G25-1-uIgG4.SP blocked CTLA-4 binding to CD80 with IC 50  of 4.300 and 0.7581 nM ( FIG. 64 ). Similarly, the bispecific antibodies could also block cynomolgus CTLA-4 binding to human CD80+ cells ( FIG. 65 ). 
     Blocking PD-1 Binding to its Ligand 
     A competitive FACS was used to test the bispecific antibodies&#39; blockage of PD-1 with its ligand PD-L1. W3248-U6T1.G25R-1.uIgG4.SP and WBP3248-U6T5.G25-1-uIgG4.SP blocked PD-1 binding to PD-L1 with IC 50  of 1.670 nM and 1.917 nM ( FIG. 63 ). 
     Serum Stability 
     The two bispecific antibodies were incubated at 37° C. human serum for 14 days, and their dual binding to human CTLA-4 and PD-1 was measure in ELISA. As shown in  FIGS. 68A and 68B , W3248-U6T1.G25R-1.uIgG4.SP and WBP3248-U6T5.G25-1-uIgG4.SP dual binding to the targets did not change over time, indicating that these two bispecific antibodies were stable in 37° C. human serum for at least 14 days. 
     While the disclosure has been particularly shown and described with reference to specific embodiments, it should be understood by those having skill in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the present disclosure as disclosed herein.