Patent Publication Number: US-11642417-B2

Title: Compositions of protein complexes and methods of use thereof

Description:
CROSS-REFERENCE 
     This application is a continuation of International Application Serial No. PCT/US2021/32313, filed May 13, 2021, which claims the benefit of U.S. Provisional Patent Application No. 63/024,422, filed May 13, 2020, each of which is entirely incorporated herein by reference in its entirety. 
    
    
     SEQUENCE LISTING 
     The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jun. 21, 2021, is named 57824-701_601_SL.txt and is 870,829 bytes in size. 
     BACKGROUND 
     Many promising therapeutics that are needed locally exhibit toxicity upon systemic administration. There is a need for drugs that can be delivered systemically but can be regulated to exhibit therapeutic activity locally or in the presence of markers for disease. 
     SUMMARY 
     In various aspects, the present disclosure provides a complex comprising: a) a therapeutic domain; b) a linker; and c) a sensor domain, wherein the therapeutic domain is linked to the sensor domain by the linker, and wherein the sensor domain is capable of binding the therapeutic domain and a marker. 
     In some aspects, the sensor domain is bound to the therapeutic domain in an absence of the marker. In some aspects, the therapeutic domain is blocked from binding the sensor domain upon binding of the sensor domain to the marker. In some aspects, the activity of the therapeutic domain is reduced upon binding of the therapeutic domain to the sensor domain. In some aspects, the therapeutic domain is capable of exhibiting therapeutic activity upon binding of the sensor domain to the marker. In some aspects, the therapeutic domain is therapeutically active upon binding of the sensor domain to the marker. 
     In some aspects, the sensor domain comprises an antibody. In some aspects, the antibody is an antibody fragment or antibody derivative. In some aspects, the complex comprises an Fc domain. In some aspects, the complex comprises a domain that improves kinetic properties. In some aspects, the complex includes two heavy chains and two light chains. 
     In some aspects, the complex comprises two therapeutic domains. In some aspects, the complex comprises two sensor domains. In some aspects, the complex is a regulated therapeutic protein. In some aspects, the therapeutic domain is a cytokine, a chemokine, an antibody, an antibody fragment, a peptide agonist, a peptide antagonist, an enzyme, a soluble receptor, a growth factor, a protein toxin, a soluble ligand, a small molecule, or any combination thereof. In some aspects, the antibody or the antibody fragment comprises an IgG, a single domain antibody fragment, a nanobody, or a single chain variable fragment (scFv). 
     In some aspects, the therapeutic domain is an IL-2 receptor agonist. In some aspects, the IL-2 receptor agonist is IL-2, IL-15, or variants or fusions thereof. In some aspects, the therapeutic domain is IFNα, IFNγ IL-12, IL-4, IL-8, IL-10, IL-15, IL-18, IL-21, TGF beta, an anti-CD3 antibody, an anti-CD28 antibody or ligand, an antibody to or ligand of CD40, GITR, OX40, CD137, CD27, or Death Receptors, the extracellular domain of TGFBR2, VEGF-C, kynureninase, IL-7, TNF, MICA, MICB, CD47, an anti-CTLA4 antibody, an anti-PD-L1 antibody, or an anti-PD-1 antibody. In some aspects, the therapeutic domain binds to the sensor domain. 
     In some aspects, the linker is a polypeptide linker. In some aspects, the linker comprises from 2 to 200 amino acids in length. In some aspects, the linker is: attached to a heavy chain of the sensor domain, attached to a light chain of the sensor domain, is a fusion with an N-terminus of the sensor domain, or is a fusion with a C-terminus of the sensor domain. In some aspects, the linker is: attached to a heavy chain of the therapeutic domain, attached to a light chain of the therapeutic domain, is a fusion with an N-terminus of the therapeutic domain, or is a fusion with a C-terminus of the therapeutic domain. 
     In some aspects, the activity of the therapeutic domain is reduced when bound to the sensor domain. In some aspects, the therapeutic domain is inactive when bound to the sensor domain. In some aspects, the sensor domain blocks the activity of the therapeutic domain when bound to the therapeutic domain. In some aspects, the therapeutic domain is active when the sensor domain is bound to the marker. In some aspects, an affinity of the sensor domain for the marker is equal to or greater than an affinity of the sensor domain for the therapeutic domain. 
     In some aspects, an affinity of the sensor domain for the marker is at least 2 times, 5 times, 10 times, 100 times, 1000 times, 10000, or 100000 times greater than an affinity of the sensor domain for the therapeutic domain. 
     In some aspects, the sensor domain is an antibody or a fragment thereof. In some aspects, the sensor domain comprises one or both antigen binding domains of a bispecific antibody. In some aspects, the bispecific antibody comprises a first antigen binding domain that is capable of binding to the therapeutic domain and is capable of binding to the marker, and a second antigen binding domain that is capable of binding to the marker. In some aspects, the bispecific antibody comprises a first antigen binding domain that is capable of binding to the therapeutic domain and the marker and a second antigen binding domain that is capable of binding to a second marker. In some aspects, the bispecific antibody comprises a first antigen binding domain that is capable of binding to the therapeutic domain and the marker and a second antigen binding domain that is capable of binding to the therapeutic domain and a second marker. In some aspects, the bispecific antibody comprises a single therapeutic domain. 
     In some aspects, the therapeutic domain is IFNα, the first marker is ATP, and the second marker is CEA. In some aspects, the sensor domain binds to an IL-2 receptor agonist and to PD-1. In some aspects, the IL-2 receptor agonist is IL-2, IL-15, or variants or fusions thereof. In some aspects, the sensor domain binds to IFNα and PD-L1. 
     In some aspects, the marker is a surface protein, a cell surface marker, or soluble ATP. In some aspects, the marker is a secreted protein. In some aspects, the marker is expressed by a cancer cell. In some aspects, the marker is expressed by an immune cell. In some aspects, the marker is PD-1. In some aspects, the marker is PD-L1. In some aspects, the marker is CEACAM5. In some aspects, the marker is FAP. In some aspects, the marker is LRRC15. In some aspects, the marker is expressed by a stromal cell. In some aspects, the marker is expressed by an endothelial cell. In some aspects, the marker is a metabolite. In some aspects, the marker is adenosine, AMP, ADP, or ATP. In some aspects, the marker is kynurenine. 
     In some aspects, the sensor domain comprises a complementarity determining region selected from TABLE 13 or TABLE 18. In some aspects, the sensor domain is selected from TABLE 13. In some aspects, the complex is selected from TABLE 15. 
     In some aspects, the sensor domain comprises a complementarity determining region having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to any one of SEQ ID NO: 1-SEQ ID NO: 20 or SEQ ID NO: 142-SEQ ID NO: 173, or SEQ ID NO: 238-252. In some aspects, the sensor domain has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to any one of SEQ ID NO: 21-SEQ ID NO: 27, SEQ ID NO: 31-SEQ ID NO: 39, or SEQ ID NO: 127-SEQ ID NO: 141. In some aspects, the protein complex has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to any one of SEQ ID NO: 41, SEQ ID NO: 44, SEQ ID NO: 80-SEQ ID NO: 112, SEQ ID NO: 174-175, SEQ ID NO: 181-182, SEQ ID NO: 195-196, SEQ ID NO: 205-206, SEQ ID NO: 210-212, SEQ ID NO: 220-223, SEQ ID NO: 226-231, SEQ ID NO: 259-261, SEQ ID NO: 266-282, SEQ ID NO: 289-293, or a fragment thereof. 
     In various aspects, the present disclosure provides a method comprising administering any of the above complexes to a subject in need thereof. In various aspects, the present disclosure provides a method of treating a subject in need thereof comprising administering any of the above complexes to the subject in need thereof. In some aspects, the administering comprises intravenous, intramuscular, or subcutaneous administration. In some aspects, the subject in need thereof has cancer. In some aspects, the subject in need thereof has an autoimmune disease. In some aspects, the subject in need thereof has a viral disease. In some aspects, the therapeutic domain treats the subject in need thereof. In some aspects, the subject in need thereof is a mammal. In some aspects, the subject in need thereof is a human. 
     INCORPORATION BY REFERENCE 
     All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The novel features of the disclosure are set forth with particularity in the appended claims. The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which: 
         FIGS.  1 A and  1 B  shows a schematic of the protein complexes of the present disclosure.  FIG.  1 A  shows an exemplary dual binding protein complex in an inactive state. The protein complex has a sensor domain and a therapeutic domain. The sensor domain and therapeutic domain are linked by a linker. The sensor domain is shown bound to the therapeutic domain, rendering the therapeutic domain inactive.  FIG.  1 B  shows an exemplary dual binding protein complex in an active state. The protein complex has a sensor domain and a therapeutic domain. The sensor domain and therapeutic domain are linked by a linker. The sensor domain is shown bound to a marker (e.g., a tumor marker or other disease marker), rendering the therapeutic domain active. 
         FIG.  2    shows an example of a protein complex in an active state when bound to a tumor and examples of protein complex in an inactive state when not bound to a tumor. 
         FIG.  3    shows an exemplary gBlock sequence used for cell-free expression of scFv antibodies.  FIG.  3    discloses SEQ ID NO: 40. 
         FIG.  4    shows candidate PD-L1/IFNα DBAs bind IFNα. 
         FIG.  5    shows candidate PD-L1/IFNα DBAs bind PD-L1. 
         FIG.  6    shows inhibition of IFNα binding to IFNAR by candidate PD-L1/IFNα DBAs. 
         FIG.  7    shows IFNα can compete with PD-L1 for binding to candidate PD-L1/IFNα DBAs of SEQ ID NO: 24 and SEQ ID NO: 26. 
         FIG.  8    shows a schematic of a protein complex comprising a cytokine therapeutic domain and a DBA (at left) and said protein complex&#39;s sequence (at right).  FIG.  8    discloses SEQ ID NO: 41. 
         FIGS.  9 A-F  show schematics of other protein complexes of the present disclosure comprising one or more sensor domains and one or more therapeutic domain.  FIG.  9 A  shows a first embodiment of the protein complex disclosed herein;  FIG.  9 B  shows a second embodiment of the protein complex disclosed herein;  FIG.  9 C  shows a third embodiment of the protein complex disclosed herein;  FIG.  9 D  shows a fourth embodiment of the protein complex disclosed herein;  FIG.  9 E  shows a fifth embodiment of the protein complex disclosed herein; and  FIG.  9 F  shows a sixth embodiment of the protein complex disclosed herein. 
         FIG.  10    shows that interferon signaling by two exemplary PD-L1/IFNα DBA cytokine protein complexes (C08 IFNα and B09 IFNα, SEQ ID NO: 57-SEQ ID NO: 58 and SEQ ID NO: 59-SEQ ID NO: 60 respectively) is reduced as compared to two control IFNα-antibody protein complexes (Anti-HER2 IFNα and Anti-PD-L1 IFNα, SEQ ID NO: 63-SEQ ID NO: 64 and SEQ ID NO: 61-SEQ ID NO: 62 respectively). 
         FIG.  11    shows that IL-2 signaling by five exemplary PD-1/IL-2 DBA-cytokine protein complexes (2_A08, 2_A11, 2_B05, 2_B07, and 7_A04, SEQ ID NO: 67-SEQ ID NO: 68, SEQ ID NO: 69-SEQ ID NO: 70, SEQ ID NO: 71-SEQ ID NO: 72, SEQ ID NO: 73-SEQ ID NO: 74 and SEQ ID NO: 75-SEQ ID NO: 76 respectively) is reduced as compared to a control IL-2-Anti-HER2 protein complex (SEQ ID NO: 65-SEQ ID NO: 66). 
         FIG.  12    shows PD-L1 regulated IFNα activity of an exemplary PD-L1/IFNα DBA-cytokine protein complex. 
         FIGS.  13 A-C  show a schematic of a bispecific antibody comprising a therapeutic domain and two sensor domains such that both sensor domains must bind their target marker to allow activity of the therapeutic domain.  FIG.  13 A  shows that when neither target is present, the therapeutic domain is inactive;  FIG.  13 B  shows that when only one target is present, the therapeutic domain is inactive; and  FIG.  13 C  shows that when both targets are present, the therapeutic domain is active. 
         FIG.  14 A-C  illustrate immunoglobulin-containing protein complexes consistent with the present disclosure. 
         FIG.  15 A-D  provide IL-2 activity of IL-2-linked protein complexes comprising the structure depicted in  FIG.  14 A  in wells coated with PD-1-Fc or an IgG1 control protein. Activity was measured as growth of a 630 nm signal from HEK-Blue™ IL-2 reporter cells (an engineered human kidney cell line which generates a detectable color change in upon activation of its IL-2 receptor).  FIG.  15 A  provides the IL-2 activity of an PD-1/IL-2 DBA-IL-2 complex.  FIG.  15 B  provides the IL-2 activity of an anti-Her2 antibody-IL-2 complex.  FIG.  15 C  provides the activity of an anti-IL-2 antibody-IL-2 complex.  FIG.  15 D  provides the activity of an anti-PD-1 antibody-IL-2 complex. 
         FIG.  16 A-F  provide IL-2 activity of protein complexes comprising the structure depicted in  FIG.  14 B  in wells coated with PD-1-Fc or an IgG1 control protein. Activity was measured as growth of a 630 nm signal from HEK-Blue™ IL-2 reporter cells.  FIG.  16 A-C  provide the IL-2 activities of three different PD-1/IL-2 DBA-IL-2 complexes.  FIG.  16 D  provides the activity of an anti-PD-1 antibody-IL-2 complex.  FIG.  16 E  provides the activity of an anti-Her-2 antibody-IL-2 complex.  FIG.  16 F  provides the activity of an anti-IL-2 antibody-IL-2 complex. 
         FIG.  17 A-H  provide IL-2 activity of protein complexes comprising the structure depicted in  FIG.  14 C  in wells coated with PD-1-Fc or an IgG1 control protein. Activity was measured as growth of a 630 nm signal from HEK-Blue™ IL-2 reporter cells.  FIGS.  17 B and  17 D  provide results for two PD-1/IL-2 DBA complexes comprising anti-PD-1 domains in the Fab arms and a PD-1/IL-2 DBA scFv on the Fc arm.  FIGS.  17 A,  17 C, and  17 E -H provide results for control protein complexes. 
         FIG.  18    provides rates of serum concentration decreases in the blood of wild-type mice of a PD-1/IL-2 DBA-cytokine complex (‘2B07 IL-2 mut’) and two control complexes. 
         FIG.  19 A-D  provide CD8 +  T cell and NK cell counts in blood and spleen tissue collected from wild-type mice 5 days following treatment with a PD-1/IL-2 DBA-cytokine complex (‘2B07 IL-2 mut’) and two control complexes. 
         FIG.  20    provides tumor volume measurements as a function of the number of days post tumor cell implant in mice. Mice received various intravenous doses of a PD-1/IL-2 DBA-IL-2 complex, a PD-1/IL-2 DBA complex lacking IL-2, or an isotype control. 
         FIG.  21 A-F  provide results for IFNAR2 binding in the presence (triangles) and absence (squares) of PD-L1 for six separate DBA PDL1-IFN variants. 
         FIG.  22 A-H  provide IFNAR2 binding by five PD-L1/IFNα DBA-cytokine complexes and three control complexes. 
         FIG.  23 A-D  provide ELISA measurements for PD-L1 and IL-15 binding by four separate anti-PD-L1 and anti-IL-15 DBAs. 
         FIG.  24 A-F  provide IL-15 activity as measured by HEK-Blue™ IL-2 reporter cell colorimetric responses for four scFv DBA-IL-15 complexes and two monospecific anti-IL-15 antibody IL-15 complexes. 
         FIG.  25 A-D  provide IL-15 activity as measured by HEK-Blue™ IL-2 reporter cell colorimetric responses for two DBA-IL-15 complexes and PDL1-IFN DBA control protein complexes. 
         FIG.  26    shows ELISA binding data for five dual-binding scFvs binding to CEA. 
         FIG.  27 A-F  demonstrate IFNAR2 binding by three DBA-cytokine protein complexes, an IFNα monospecific binding scFv, and two non-IFNα binding scFvs. 
         FIG.  28 A  provides ELISA binding data for six scFvs to LRRC15. Results from  FIG.  28 A  are summarized in  FIG.  28 B . 
         FIG.  29 A-F  summarize IFNAR2 binding by four LRRC15-IFN-α DBA-IFNα complexes and two control complexes. 
     
    
    
     DETAILED DESCRIPTION 
     The present disclosure provides compositions of protein complexes and methods of use thereof. Promising therapeutics are often unable to be realized due to harmful side effects, or systemic on-target toxicity. Provided herein are protein complexes, which specifically exhibit therapeutic efficacy locally, where the relevant cells and targets are present. Moreover, protein complexes of the present disclosure are self-regulated, remaining inactive in the absence of a specific marker and activating in the presence of the specific marker. The protein complexes disclosed herein may include a sensor domain (e.g, an antibody or scFv) that is linked to a therapeutic domain (e.g., a cytokine, a therapeutic antibody domain, a receptor, a ligand) via a linker. The sensor domain may be a dual binding protein that has affinity for the therapeutic domain and a specific marker, such that the marker and the therapeutic domain compete for binding to the sensor domain. In some embodiments, the dual binding protein is a dual binding antibody. In the absence of the marker, the sensor domain binds the therapeutic domain, rendering the therapeutic domain unable to exert activity. When the sensor domain is bound to the marker, the therapeutic domain is unbound and may exert activity. In some embodiments, regulation of therapeutic activity by the complex may be reversible, that is, when the sensor domain disassociates from the marker, the sensor domain may bind the therapeutic domain, rendering the therapeutic domain once again unable to exert activity. Thus, the protein complexes of the present disclosure comprise sensor domains that regulate therapeutic domains in the presence of the marker, bind the marker, and render the therapeutic domain active. Various structures and compositions of protein complexes are disclosed herein, including pharmaceutical formulations. Also provided herein are methods for treating a subject in need thereof by administering the protein complex to the subject. 
     As used herein, a “marker” may refer to the moiety that is bound by the sensor domain of the protein complexes disclosed herein. Non-limiting examples of a “marker” include a protein, a protein modification, a carbohydrate, a metabolite, or any other molecule that can be bound by an antibody. A marker may also refer to a disease-specific marker, such as a molecular marker of a disease state (e.g., cancer). 
     As used herein, a “target” may refer to a molecule through which the therapeutic domain of the protein complexes disclosed herein may act. Non-limiting examples of a “target” include cytokine receptor, a cytokine, a ligand, an enzyme substrate, or any other molecule that, when contacted by the therapeutic domain, has a therapeutic impact on a subject (e.g., human or non-human animal) administered the protein complex. 
     As used herein, an “antibody” may refer to an antibody, an antibody derivative, or fragment(s) thereof that contains part or all of an antibody variable domain. 
     The term “recombinant nucleic acid” refers to synthetic nucleic acid having a nucleotide sequence that is not naturally occurring. A recombinant nucleic acid may be synthesized in the laboratory. A recombinant nucleic acid is prepared by using recombinant DNA technology by using enzymatic modification of DNA, such as enzymatic restriction digestion, ligation, and DNA cloning. A recombinant nucleic acid as used herein can be DNA, or RNA. A recombinant DNA may be transcribed in vitro, to generate a messenger RNA (mRNA), the recombinant mRNA may be isolated, purified and used to transfect a cell. A recombinant nucleic acid may encode a protein or a polypeptide. A recombinant nucleic acid, under suitable conditions, can be incorporated into a living cell, and can be expressed inside the living cell. As used herein, “expression” of a nucleic acid usually refers to transcription and/or translation of the nucleic acid. The product of a nucleic acid expression is usually a protein but can also be an mRNA. Detection of an mRNA encoded by a recombinant nucleic acid in a cell that has incorporated the recombinant nucleic acid, is considered positive proof that the nucleic acid is “expressed” in the cell. 
     The process of inserting or incorporating a nucleic acid into a cell can be via transformation, transfection or transduction. Transformation is the process of uptake of foreign nucleic acid by a bacterial cell. This process is adapted for propagation of plasmid DNA, protein production, and other applications. Transformation introduces recombinant plasmid DNA into competent bacterial cells that take up extracellular DNA from the environment. Some bacterial species are naturally competent under certain environmental conditions, but competence is artificially induced in a laboratory setting. Transfection is the forced introduction of small molecules such as DNA, RNA, or antibodies into eukaryotic cells. Just to make life confusing, ‘transfection’ also refers to the introduction of bacteriophage into bacterial cells. ‘Transduction’ is mostly used to describe the introduction of recombinant viral vector particles into target cells, while ‘infection’ refers to natural infections of humans or animals with wild-type viruses. 
     Protein Complexes 
     The present disclosure provides complexes that may self-regulate therapeutic activity. Protein complexes of the present disclosure may include a sensor domain and a therapeutic domain. The sensor domain and therapeutic domain may be linked by a linker. The sensor domain may regulate the activity of the therapeutic domain. Regulation of the activity of the therapeutic domain may include binding of the sensor domain to the therapeutic domain, rendering the therapeutic domain unable to exert therapeutic activity. Regulation of the activity of the therapeutic domain may further include unbinding, or release, of the therapeutic domain by the sensor domain upon binding of the sensor domain to a marker. The marker may be expressed by a cell associated with a disease. For example, the disease may be cancer, the cell may be a tumor cell, and the marker may be a tumor-specific marker that is expressed on tumor cells. Thus, the protein complexes of the present disclosure are superior drug candidates as the sensor domain-dependent activity of the therapeutic domain allows for localized activity, even upon systemic administration of the protein complex. Compared to therapeutic domains administered on their own, the protein complexes of the present disclosure exhibit regulated therapeutic activity of the therapeutic domain. As a result, compared to free therapeutic domains administered on their own, the protein complexes of the present disclosure exhibit reduced systemic on-target toxicity. 
     The protein complexes of the present disclosure can have an Fc region. The protein complexes of the present disclosure can have a domain that improves kinetic properties. For example, the protein complexes of the present disclosure may be further coupled to a half-life extender, such as an Fc region, albumin, PEG, or another zwitterionic polymer. The protein complexes of the present disclosure may have two heavy chains and two light chains. The protein complexes of the present disclosure may have two heavy chains and one light chain. The protein complexes of the present disclosure may include multiple sensor domains and multiple therapeutic domains. For example, a protein complex of the present disclosure may include two sensor domains and two therapeutic domains, all of which are linked and in which the two therapeutic domains are bound to the two sensor domains. In some embodiments, a protein complex of the present disclosure may include two sensor domains and one therapeutic domain, all of which are linked and in which the therapeutic domain may bind to both sensor domains or only one of the two sensor domains. 
     In some embodiments, the marker may be a surface protein, such as a cell surface protein. The marker may also be soluble ATP. In some embodiments, the marker may be a secreted protein. For example, the secreted protein may be a protein that is released by proliferating tumor cells. In some embodiments, the marker may be expressed by a cancer cell. The marker may be expressed by an immune cell. The marker may be expressed by a stromal cell. The marker may be expressed by an endothelial cell. Exemplary markers include PD1, PD-L1, CEACAM5, FAP, LRRC15, a metabolite, adenosine, AMP, ADP, ATP, or kynurenine. Other markers may include CRIPTO, CD19, CD20, CD22, CD30, CD33, Glycoprotein NMB, CanAg, HER2 (ErbB2/Neu), CD56 (NCAM), CD70, CD79, CD138, PSCA, PSMA (prostate specific membrane antigen), BCMA, E-selectin, EphB2, Melanotransferin, Muc16 and TMEFF2, or any other marker described in U.S. Pat. No. 10,561,739, incorporated herein by reference in its entirety. Other markers may also include BMPR1B, E16, STEAP1, 0772P, MPF, Naρi3b, Sema 5b, PSCA hlg, ETBR, MSG783, STEAP2, TrpM4, CD21, CD79b, FcRH2, NCA, MDP, IL20Rα, Brevican, EphB2R, ASLG659, PSCA, GEDA, BAFF-R, CD79a, CXCR5, HLA-DOB, P2X5, CD72, FCRHI, IRTA2, or any other marker described in WO 2005/082023, incorporated herein by reference in its entirety. 
     In some embodiments, binding of the sensor domain to the therapeutic domain versus binding of the sensor domain to a marker is regulated by the relative affinity of the sensor domain for the therapeutic domain. In some embodiments, the sensor domain may have a dissociation constant (Kd) for the marker that is lower than the dissociation constant of the sensor domain for the therapeutic domain. Thus, the sensor may have a higher affinity (lower Kd) for the marker than for the therapeutic domain. The sensor domains of the present disclosure may be engineered, for example by affinity maturation, to have a higher affinity (lower dissociation constant) for the marker than the therapeutic domain. In the absence of the marker, the sensor domain of the present disclosure may have a sufficiently high affinity for the therapeutic domain such that the therapeutic domain is bound by the sensor domain. In the presence of the marker, the affinity of the sensor domain for the marker is sufficiently high (low dissociation constant), such that the marker outcompetes the therapeutic domain for binding to the sensor domain. As a result, the equilibrium binding shifts from a state in which the sensor domain is bound to the therapeutic domain to a state in which the therapeutic domain is unbound and the sensor domain binds to the marker. 
     The sensor domain may have an affinity for the marker that is at least 2-fold higher than an affinity for the therapeutic domain. The sensor domain may have an affinity for the marker that is at least 5-fold higher than an affinity for the therapeutic domain. The sensor domain may have an affinity for the marker that is at least 10-fold higher than an affinity for the therapeutic domain. The sensor domain may have an affinity for the marker that is at least 15-fold higher than an affinity for the therapeutic domain. The sensor domain may have an affinity for the marker that is at least 20-fold higher than an affinity for the therapeutic domain. The sensor domain may have an affinity for the marker that is at least 25-fold higher than an affinity for the therapeutic domain. The sensor domain may have an affinity for the marker that is at least 30-fold higher than an affinity for the therapeutic domain. The sensor domain may have an affinity for the marker that is at least 35-fold higher than an affinity for the therapeutic domain. The sensor domain may have an affinity for the marker that is at least 40-fold higher than an affinity for the therapeutic domain. The sensor domain may have an affinity for the marker that is at least 45-fold higher than an affinity for the therapeutic domain. The sensor domain may have an affinity for the marker that is at least 50-fold higher than an affinity for the therapeutic domain. The sensor domain may have an affinity for the marker that is at least 60-fold higher than an affinity for the therapeutic domain. The sensor domain may have an affinity for the marker that is at least 70-fold higher than an affinity for the therapeutic domain. The sensor domain may have an affinity for the marker that is at least 80-fold higher than an affinity for the therapeutic domain. The sensor domain may have an affinity for the marker that is at least 90-fold higher than an affinity for the therapeutic domain. The sensor domain may have an affinity for the marker that is at least 100-fold higher than an affinity for the therapeutic domain. The sensor domain may have an affinity for the marker that is at least 150-fold higher than an affinity for the therapeutic domain. The sensor domain may have an affinity for the marker that is at least 200-fold higher than an affinity for the therapeutic domain. The sensor domain may have an affinity for the marker that is at least 250-fold higher than an affinity for the therapeutic domain. The sensor domain may have an affinity for the marker that is at least 300-fold higher than an affinity for the therapeutic domain. The sensor domain may have an affinity for the marker that is at least 350-fold higher than an affinity for the therapeutic domain. The sensor domain may have an affinity for the marker that is at least 400-fold higher than an affinity for the therapeutic domain. The sensor domain may have an affinity for the marker that is at least 450-fold higher than an affinity for the therapeutic domain. The sensor domain may have an affinity for the marker that is at least 500-fold higher than an affinity for the therapeutic domain. The sensor domain may have an affinity for the marker that is at least 1000-fold higher than an affinity for the therapeutic domain. The sensor domain may have an affinity for the marker that is at least 10000-fold higher than an affinity for the therapeutic domain. The sensor domain may have an affinity for the marker that is at least 100000-fold higher than an affinity for the therapeutic domain. The sensor domain may have an affinity for the marker that is from 2 to 10-fold higher than an affinity for the therapeutic domain. The sensor domain may have an affinity for the marker that is from 10 to 20-fold higher than an affinity for the therapeutic domain. The sensor domain may have an affinity for the marker that is from 20 to 30-fold higher than an affinity for the therapeutic domain. The sensor domain may have an affinity for the marker that is from 30 to 40-fold higher than an affinity for the therapeutic domain. The sensor domain may have an affinity for the marker that is from 40 to 50-fold higher than an affinity for the therapeutic domain. The sensor domain may have an affinity for the marker that is from 50 to 100-fold higher than an affinity for the therapeutic domain. The sensor domain may have an affinity for the marker that is from 100 to 150-fold higher than an affinity for the therapeutic domain. The sensor domain may have an affinity for the marker that is from 150 to 200-fold higher than an affinity for the therapeutic domain. The sensor domain may have an affinity for the marker that is from 200 to 250-fold higher than an affinity for the therapeutic domain. The sensor domain may have an affinity for the marker that is from 250 to 300-fold higher than an affinity for the therapeutic domain. The sensor domain may have an affinity for the marker that is from 300 to 350-fold higher than an affinity for the therapeutic domain. The sensor domain may have an affinity for the marker that is from 350 to 400-fold higher than an affinity for the therapeutic domain. The sensor domain may have an affinity for the marker that is from 400 to 450-fold higher than an affinity for the therapeutic domain. The sensor domain may have an affinity for the marker that is from 450 to 500-fold higher than an affinity for the therapeutic domain. The sensor domain may have an affinity for the marker that is from 500 to 1000-fold higher than an affinity for the therapeutic domain. The sensor domain may have an affinity for the marker that is from 10 to 80-fold higher than an affinity for the therapeutic domain. The sensor domain may have an affinity for the marker that is from 30 to 70-fold higher than an affinity for the therapeutic domain. The sensor domain may have an affinity for the marker that is from 40 to 60-fold higher than an affinity for the therapeutic domain. The sensor domain may have an affinity for the marker that is from 20 to 50-fold higher than an affinity for the therapeutic domain. The sensor domain may have an affinity for the marker that is from 10 to 1000-fold higher than an affinity for the therapeutic domain. The sensor domain may have an affinity for the marker that is from 70 to 500-fold higher than an affinity for the therapeutic domain. The sensor domain may have an affinity for the marker that is from 100 to 500-fold higher than an affinity for the therapeutic domain. The sensor domain may have an affinity for the marker that is from 500 to 750-fold higher than an affinity for the therapeutic domain. The sensor domain may have an affinity for the marker that is from 250 to 750-fold higher than an affinity for the therapeutic domain. The sensor domain may have an affinity for the marker that is from 1000 to 100000-fold higher than an affinity for the therapeutic domain. The sensor domain may have an affinity for the marker that is from 2 to 100000-fold higher than an affinity for the therapeutic domain. 
     A protein complex of the present disclosure, or a fragment thereof, may comprise one or more complementary determining regions (CDRs) having have at least 80% sequence identity to any one of the CDRs disclosed herein. For example, a protein complex of the present disclosure, or a fragment thereof, may comprise one or more CDRs having at least 80% sequence identity to any one of SEQ ID NO: 1-SEQ ID NO: 20, SEQ ID NO: 142-173, or SEQ ID NO: 238-252. A protein complex of the present disclosure, or a fragment thereof, may comprise one or more CDRs having at least 85% sequence identity to any one of SEQ ID NO: 1-SEQ ID NO: 20, SEQ ID NO: 142-173, or SEQ ID NO: 238-252. A protein complex of the present disclosure, or a fragment thereof, may comprise one or more CDRs having at least 90% sequence identity to any one of SEQ ID NO: 1-SEQ ID NO: 20, SEQ ID NO: 142-173, or SEQ ID NO: 238-252. A protein complex of the present disclosure, or a fragment thereof, may comprise one or more CDRs having at least 92% sequence identity to any one of SEQ ID NO: 1-SEQ ID NO: 20, SEQ ID NO: 142-173, or SEQ ID NO: 238-252. A protein complex of the present disclosure, or a fragment thereof, may comprise one or more CDRs having at least 95% sequence identity to any one of SEQ ID NO: 1-SEQ ID NO: 20, SEQ ID NO: 142-173, or SEQ ID NO: 238-252. A protein complex of the present disclosure, or a fragment thereof, may comprise one or more CDRs having at least 97% sequence identity to any one of SEQ ID NO: 1-SEQ ID NO: 20, SEQ ID NO: 142-173, or SEQ ID NO: 238-252. A protein complex of the present disclosure, or a fragment thereof, may comprise one or more CDRs having at least 99% sequence identity to any one of SEQ ID NO: 1-SEQ ID NO: 20, SEQ ID NO: 142-173, or SEQ ID NO: 238-252. A protein complex of the present disclosure, or a fragment thereof, may comprise one or more CDRs having any one of SEQ ID NO: 1-SEQ ID NO: 20, SEQ ID NO: 142-173, or SEQ ID NO: 238-252. 
     A protein complex, or a fragment thereof, can have at least 80% sequence identity to any one of SEQ ID NO: 41, SEQ ID NO: 44, SEQ ID NO: 80-SEQ ID NO: 112, SEQ ID NO: 174-175, SEQ ID NO: 181-182, SEQ ID NO: 195-196, SEQ ID NO: 205-206, SEQ ID NO: 210-212, SEQ ID NO: 220-223, SEQ ID NO: 226-231, SEQ ID NO: 259-261, SEQ ID NO: 266-282, or SEQ ID NO: 289-293, or a fragment thereof. A protein complex can have at least 85% sequence identity to any one of SEQ ID NO: 41, SEQ ID NO: 44, SEQ ID NO: 80-SEQ ID NO: 112, SEQ ID NO: 174-175, SEQ ID NO: 181-182, SEQ ID NO: 195-196, SEQ ID NO: 205-206, SEQ ID NO: 210-212, SEQ ID NO: 220-223, SEQ ID NO: 226-231, SEQ ID NO: 259-261, SEQ ID NO: 266-282, or SEQ ID NO: 289-293, or a fragment thereof. A protein complex can have at least 90% sequence identity to any one of SEQ ID NO: 41, SEQ ID NO: 44, SEQ ID NO: 80-SEQ ID NO: 112, SEQ ID NO: 174-175, SEQ ID NO: 181-182, SEQ ID NO: 195-196, SEQ ID NO: 205-206, SEQ ID NO: 210-212, SEQ ID NO: 220-223, SEQ ID NO: 226-231, SEQ ID NO: 259-261, SEQ ID NO: 266-282, or SEQ ID NO: 289-293, or a fragment thereof. A protein complex can have at least 92% sequence identity to any one of SEQ ID NO: 41, SEQ ID NO: 44, SEQ ID NO: 80-SEQ ID NO: 112, SEQ ID NO: 174-175, SEQ ID NO: 181-182, SEQ ID NO: 195-196, SEQ ID NO: 205-206, SEQ ID NO: 210-212, SEQ ID NO: 220-223, SEQ ID NO: 226-231, SEQ ID NO: 259-261, SEQ ID NO: 266-282, or SEQ ID NO: 289-293, or a fragment thereof. A protein complex can have at least 95% sequence identity to any one of SEQ ID NO: 41, SEQ ID NO: 44, SEQ ID NO: 80-SEQ ID NO: 112, SEQ ID NO: 174-175, SEQ ID NO: 181-182, SEQ ID NO: 195-196, SEQ ID NO: 205-206, SEQ ID NO: 210-212, SEQ ID NO: 220-223, SEQ ID NO: 226-231, SEQ ID NO: 259-261, SEQ ID NO: 266-282, or SEQ ID NO: 289-293, or a fragment thereof. A protein complex can have at least 97% sequence identity to any one of SEQ ID NO: 41, SEQ ID NO: 44, SEQ ID NO: 80-SEQ ID NO: 112, SEQ ID NO: 174-175, SEQ ID NO: 181-182, SEQ ID NO: 195-196, SEQ ID NO: 205-206, SEQ ID NO: 210-212, SEQ ID NO: 220-223, SEQ ID NO: 226-231, SEQ ID NO: 259-261, SEQ ID NO: 266-282, or SEQ ID NO: 289-293, or a fragment thereof. A protein complex can have at least 99% sequence identity to any one of SEQ ID NO: 41, SEQ ID NO: 44, SEQ ID NO: 80-SEQ ID NO: 112, SEQ ID NO: 174-175, SEQ ID NO: 181-182, SEQ ID NO: 195-196, SEQ ID NO: 205-206, SEQ ID NO: 210-212, SEQ ID NO: 220-223, SEQ ID NO: 226-231, SEQ ID NO: 259-261, SEQ ID NO: 266-282, or SEQ ID NO: 289-293, or a fragment thereof. A protein complex is any one of SEQ ID NO: 41, SEQ ID NO: 44, SEQ ID NO: 80-SEQ ID NO: 112, SEQ ID NO: 174-175, SEQ ID NO: 181-182, SEQ ID NO: 195-196, SEQ ID NO: 205-206, SEQ ID NO: 210-212, SEQ ID NO: 220-223, SEQ ID NO: 226-231, SEQ ID NO: 259-261, SEQ ID NO: 266-282, or SEQ ID NO: 289-293, or a fragment thereof. 
     A protein complex of the present disclosure may have at least 95% sequence identity to any one of SEQ ID NO: 41, SEQ ID NO: 44, SEQ ID NO: 80-SEQ ID NO: 112, SEQ ID NO: 174-175, SEQ ID NO: 181-182, SEQ ID NO: 195-196, SEQ ID NO: 205-206, SEQ ID NO: 210-212, SEQ ID NO: 220-223, SEQ ID NO: 226-231, SEQ ID NO: 259-261, SEQ ID NO: 266-282, or SEQ ID NO: 289-293, or a fragment thereof and have one or more CDRs with at least 80% sequence identity to any one SEQ ID NO: 1-SEQ ID NO: 20, SEQ ID NO: 142-173, or SEQ ID NO: 238-252. The protein complexes of the present disclosure can have CDRs selected from SEQ ID NO: 1-SEQ ID NO: 20, SEQ ID NO: 142-173, or SEQ ID NO: 238-252 arranged in any combination or order. 
     A fragment of any of the above may retain the functional binding domains of the sensor or any functional therapeutic domains of the therapeutic. For example, a dual binding antibody protein complex can include the entire antibody or a fragment having regions of the antibody that are capable of binding to a marker and the therapeutic domain. In the latter case, the fragment may be an scFv that can bind to a marker and the therapeutic domain. Exemplary sequence of protein complexes of the present disclosure is shown below in TABLE 1. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Exemplary Protein Complexes 
               
            
           
           
               
               
               
            
               
                 SEQ ID NO 
                 Sequence 
                 Description 
               
               
                   
               
               
                 SEQ ID NO: 41 
                 MSTSTCDLPQTHSLGSRRTLMLLAQMRRISL 
                 Protein complex 
               
               
                   
                 FSCLKDRHDFGFPQEEFGNQFQKAETIPVLHE 
                 comprising a 
               
               
                   
                 MIQQIFNLFSTKDSSAAWDETLLDKFYTELY 
                 DBA/cytokine 
               
               
                   
                 QQLNDLEACVIQGVGVTETPLMKEDSILAVR 
                 complex having a 
               
               
                   
                 KYFQRITLYLKEKKYSPCAWEVVRAEIMRSF 
                 PD-L1/IFNα scFv 
               
               
                   
                 SLSTNLQESLRSKEGGGGSGGGGSGGGGSGG 
                 sensor domain and 
               
               
                   
                 GGSQVQLVQSGAEVKKPGASVKVSCKASGY 
                 an IFNα therapeutic 
               
               
                   
                 TFSNYYVHWVRQAPGQGLEWMGWMDPNS 
                 domain 
               
               
                   
                 GGTGYAHQFQGRVTMTRDTSTSTVYMELSS 
                 scFv_IFN- 
               
               
                   
                 LRSEDTAVYYCAKEVFSGWYDYWGQGTLV 
                 Heavy_GS20_PDL1- 
               
               
                   
                 TVSSASGGGGSGGGGSGGGGSHASDIQMTQS 
                 IFN_1A05_H_I39V_ 
               
               
                   
                 PSSLSASVGDRVTITCRASQSISSYLNWYQQK 
                 S58P_Q69H_K70Q 
               
               
                   
                 PGKAPKLLIYAASSLQSGVPSRFSGSGSGTDF 
                   
               
               
                   
                 TLTISSLQPEDFATYYCQQSYSTPYTFGQGTK 
                   
               
               
                   
                 VEIKGKPIPNPLLGLDST 
                   
               
               
                   
               
               
                 SEQ ID NO: 42 
                 QVQLVQSGAEVKKPGASVKVSCKASGYTFT 
                 Protein complex 
               
               
                   
                 KNYMHWVRQAPGQGLEWLGWVSPDSGYTG 
                 comprising a 
               
               
                   
                 YAQKFQGRVTMTRDTSTSTVYMELSSLRSED 
                 DBA/cytokine 
               
               
                   
                 TAVYYCTTDLLSLELDDAFDIWGQGTMVTV 
                 complex having a 
               
               
                   
                 SSASGGGGSGGGGSGGGGSHASDIQMTQSPS 
                 PD-L1 antibody 
               
               
                   
                 SLSASVGDRVTITCRASQSISSWLAWYQQKP 
                 sensor domain and 
               
               
                   
                 GKAPKLLIYAASTLQSGVPSRFSGSGSGTDFT 
                 an IFNα therapeutic 
               
               
                   
                 LTISSLQPEDFATYYCQQSYSTPLTFGGGTKL 
                 domain, where the 
               
               
                   
                 EIKPRGPTIKPCPPCKCPAPNAAGGPSVFIFPP 
                 DBA is an scFv 
               
               
                   
                 KIKDVLMISLSPIVTCVVVDVSEDDPDVQISW 
                 PDL1- 
               
               
                   
                 FVNNVEVHTAQTQTHREDYNSTLRVVSALPI 
                 IFN_uIFN_2D10scFv_ 
               
               
                   
                 QHQDWMSGKEFKCKVNNKDLGAPIERTISKP 
                 KiH_PDL1- 
               
               
                   
                 KGSVRAPQVYVLPPCEEEMTKKQVTLSCAV 
                 IFN_1A05_H_N36G_ 
               
               
                   
                 TDFMPEDIYVEWTNNGKTELNYKNTEPVLDS 
                 Pep1 
               
               
                   
                 DGSYFMVSKLRVEKKNWVERNSYSCSVVHE 
                   
               
               
                   
                 GLHNHHTTKSFSRTPGK 
                   
               
               
                   
               
               
                 SEQ ID NO: 43 
                 CDLPQTHSLGSRRTLMLLAQMRRISLFSCLK 
                 First heavy chain of 
               
               
                   
                 DRHDFGFPQEEFGNQFQKAETIPVLHEMIQQI 
                 a protein complex 
               
               
                   
                 FNLFSTKDSSAAWDETLLDKFCTELYQQLND 
                 comprising a 
               
               
                   
                 LEACVMQEERVGETPLMNADSILAVKKYFR 
                 DBA/cytokine 
               
               
                   
                 RITLYLTEKKYSPCAWEVVRAEIVRSLSLSTN 
                 complex having a 
               
               
                   
                 LQERLRRKEGGGGSGGGGSGGGGSGGGGSQ 
                 PD-L1 binding 
               
               
                   
                 VQLVQSGAEVKKPGASVKVSCKASGYTFSG 
                 domain and an IFNα 
               
               
                   
                 YYIHWVRQAPGQGLEWMGWMDSNSGGTGY 
                 therapeutic domain 
               
               
                   
                 AQKFQGRVTMTRDTSTSTVYMELSSLRSEDT 
                 PDL1- 
               
               
                   
                 AVYYCAKEVFSGWYDYWGQGTLVTVSSAK 
                 IFN_uIFN_2D10scFv_ 
               
               
                   
                 TTAPSVYPLAPVCGDTTGSSVTLGCLVKGYF 
                 KiH_PDL1- 
               
               
                   
                 PEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLS 
                 IFN_1A05_H_N36G_ 
               
               
                   
                 SSVTVTSSTWPSQSITCNVAHPASSTKVDKKI 
                 Pep2 
               
               
                   
                 EPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKI 
                   
               
               
                   
                 KDVLMISLSPIVTCVVVDVSEDDPDVQISWF 
                   
               
               
                   
                 VNNVEVHTAQTQTHREDYNSTLRVVSALPIQ 
                   
               
               
                   
                 HQDWMSGKEFKCKVNNKDLGAPIERTISKPK 
                   
               
               
                   
                 GSVRAPQVCVLPPPEEEMTKKQVTLWCMVT 
                   
               
               
                   
                 DFMPEDIYVEWTNNGKTELNYKNTEPVLDS 
                   
               
               
                   
                 DGSYFMYSKLRVEKKNWVERNSYSCSVVHE 
                   
               
               
                   
                 GLHNHHTTKSFSRTPGK 
                   
               
               
                   
               
               
                 SEQ ID NO: 44 
                 DIQMTQSPSSLSASVGDRVTITCRASQSISSYL 
                 Light chain of a 
               
               
                   
                 NWYQQKPGKAPKLLIYAASSLQSGVPSRFSG 
                 protein complex 
               
               
                   
                 SGSGTDFTLTISSLQPEDFATYYCQQSYSTPY 
                 comprising a 
               
               
                   
                 TFGQGTKVEIKRADAAPTVSIFPPSSEQLTSG 
                 DBA/cytokine 
               
               
                   
                 GASVVCFLNNFYPKDINVKWKIDGSERQNG 
                 complex having a 
               
               
                   
                 VLNSWTDQDSKDSTYSMSSTLTLTKDEYERH 
                 PD-L1 scFv sensor 
               
               
                   
                 NSYTCEATHKTSTSPIVKSFNRNEC 
                 domain and an IFNα 
               
               
                   
                   
                 therapeutic domain 
               
               
                   
                   
                 PDL1- 
               
               
                   
                   
                 IFN_uIFN_2D10scFv_ 
               
               
                   
                   
                 KiH_PDL1- 
               
               
                   
                   
                 IFN_1A05_H_N36G_ 
               
               
                   
                   
                 Pep3 
               
               
                   
               
               
                 SEQ ID NO: 
                 QVQLVESGGGVVQPGRSLRLDCKASGITFSN 
                 PD1- 
               
               
                 303 
                 SGMHWVRQAPGKGLEWVAVIWYDGSKRYY 
                 IL2_3x_Cterm_Nivo_ 
               
               
                   
                 ADSVKGRFTISRDNSKNTLFLQMNSLRAEDT 
                 2B07_H_H37Y_L_ 
               
               
                   
                 AVYYCATNDDYWGQGTLVTVSSAKTTAPSV 
                 A107Y_S109R; 
               
               
                   
                 YPLAPVCGDTTGSSVTLGCLVKGYFPEPVTL 
                 AF4505_pep2 
               
               
                   
                 TWNSGSLSSGVHTFPAVLQSDLYTLSSSVTV 
                   
               
               
                   
                 TSSTWPSQSITCNVAHPASSTKVDKKIEPRGP 
                   
               
               
                   
                 TIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLM 
                   
               
               
                   
                 ISLSPIVTCVVVDVSEDDPDVQISWFVNNVEV 
                   
               
               
                   
                 HTAQTQTHREDYNSTLRVVSALPIQHQDWM 
                   
               
               
                   
                 SGKEFKCKVNNKDLGAPIERTISKPKGSVRAP 
                   
               
               
                   
                 QVYVLPPPEKEMTKKQVSLTCLVKDFMPEDI 
                   
               
               
                   
                 YVEWTNNGKTELNYKNTEPVLKSDGSYFMY 
                   
               
               
                   
                 SKLTVEKKNWVERNSYSCSVVHEGLHNHHT 
                   
               
               
                   
                 TKSFSRTPGGGGGSGGGGSGGGGSGGGGSQ 
                   
               
               
                   
                 VQLVQSGAEVKKPGASVKVSCKASGDTFTR 
                   
               
               
                   
                 YYVHWVRQAPGQGLEWMGIINPSGGYASYA 
                   
               
               
                   
                 QKFQGRVTMTRDTSTSTVYMELSSLRSEDTA 
                   
               
               
                   
                 VYYCAAGLFIWGQGTLVTVSSASGGGGSGG 
                   
               
               
                   
                 GGSGGGGSHASDIQMTQSPSSLSASVGDRVTI 
                   
               
               
                   
                 TCRASQSIGRWLAWYQQKPGKAPKLLIYSAS 
                   
               
               
                   
                 NLETGVPSRFSGSGSGTDFTLTISSLQPEDFAT 
                   
               
               
                   
                 YYCQQYNRFPVTFGPGTKVDIK 
                   
               
               
                   
               
               
                 SEQ ID NO: 
                 QVQLVESGGGVVQPGRSLRLDCKASGITFSN 
                 PD1- 
               
               
                 304 
                 SGMHWVRQAPGKGLEWVAVIWYDGSKRYY 
                 IL2_3x_Cterm_Nivo_ 
               
               
                   
                 ADSVKGRFTISRDNSKNTLFLQMNSLRAEDT 
                 704var 
               
               
                   
                 AVYYCATNDDYWGQGTLVTVSSAKTTAPSV 
                 AF4504_pep2 
               
               
                   
                 YPLAPVCGDTTGSSVTLGCLVKGYFPEPVTL 
                   
               
               
                   
                 TWNSGSLSSGVHTFPAVLQSDLYTLSSSVTV 
                   
               
               
                   
                 TSSTWPSQSITCNVAHPASSTKVDKKIEPRGP 
                   
               
               
                   
                 TIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLM 
                   
               
               
                   
                 ISLSPIVTCVVVDVSEDDPDVQISWFVNNVEV 
                   
               
               
                   
                 HTAQTQTHREDYNSTLRVVSALPIQHQDWM 
                   
               
               
                   
                 SGKEFKCKVNNKDLGAPIERTISKPKGSVRAP 
                   
               
               
                   
                 QVYVLPPPEKEMTKKQVSLTCLVKDFMPEDI 
                   
               
               
                   
                 YVEWTNNGKTELNYKNTEPVLKSDGSYFMY 
                   
               
               
                   
                 SKLTVEKKNWVERNSYSCSVVHEGLHNHHT 
                   
               
               
                   
                 TKSFSRTPGGGGGSGGGGSGGGGSGGGGSQ 
                   
               
               
                   
                 VQLVQSGAEVKKPGASVKVSCKASGYTFTR 
                   
               
               
                   
                 YYMHWVRQAPGQGLEWMGIINPRAGYTSY 
                   
               
               
                   
                 ALKFQGRVTMTRDTSTSTVYMELSSLRSEDT 
                   
               
               
                   
                 AVYYCTSGWDVWGQGTLVTVSSASGGGGS 
                   
               
               
                   
                 GGGGSGGGGSHASDIQMTQSPSSLSASVGDR 
                   
               
               
                   
                 VTITCRASQSISTWLAWYQQKPGKAPKLLIY 
                   
               
               
                   
                 AASSLDSGVPSRFSGSGSGTDFTLTISSLQPED 
                   
               
               
                   
                 FATYYCQQSYSFPVTFGQGTKVEIK 
                   
               
               
                   
               
               
                 SEQ ID NO: 
                 APTSSSTKKTQLQLEHLLLDLQMILNGINNYK 
                 PD1- 
               
               
                 181 
                 NPKLTDMLTFEFYMPKKATELKHLQCLEREL 
                 IL2_3x_Asym_PD1- 
               
               
                   
                 KPLEEVLNLAQSKNFHLRPRDLISNINVIVLEL 
                 IL2_2B07_H_H37Y_L_ 
               
               
                   
                 KGSETTFMCEYADETATIVEFLNRWITFCQSII 
                 W38Y_A107Y 
               
               
                   
                 STLTGGGGSGGGGSGGGGSGGGGSQVQLVQ 
                   
               
               
                   
                 SGAEVKKPGASVKVSCKASGDTFTRYYVHW 
                   
               
               
                   
                 VRQAPGQGLEWMGIINPSGGYASYAQKFQG 
                   
               
               
                   
                 RVTMTRDTSTSTVYMELSSLRSEDTAVYYCA 
                   
               
               
                   
                 AGLFIWGQGTLVTVSSAKTTAPSVYPLAPVC 
                   
               
               
                   
                 GDTTGSSVTLGCLVKGYFPEPVTLTWNSGSL 
                   
               
               
                   
                 SSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQ 
                   
               
               
                   
                 SITCNVAHPASSTKVDKKIEPRGPTIKPCPPCK 
                   
               
               
                   
                 CPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTC 
                   
               
               
                   
                 VVVDVSEDDPDVQISWFVNNVEVHTAQTQT 
                   
               
               
                   
                 HREDYNSTLRVVSALPIQHQDWMSGKEFKC 
                   
               
               
                   
                 KVNNKDLGAPIERTISKPKGSVRAPQVYVLPP 
                   
               
               
                   
                 PEEEMTKKQVTLTCMVTDFMPEDIYVEWTN 
                   
               
               
                   
                 NGKTELNYKNTEPVLDSDGSYFMYSDLRVE 
                   
               
               
                   
                 KKNWVERNSYSCSVVHEGLHNHHTTESFSR 
                   
               
               
                   
                 TPGK 
                   
               
               
                   
               
               
                 SEQ ID NO: 
                 QVQLVQSGAEVKKPGASVKVSCKASGDTFTRYYVHWV 
                 PD1- 
               
               
                 182 
                 RQAPGQGLEWMGIINPSGGYASYAQKFQGRVTMTRD 
                 IL2_3x_Asym_PD1- 
               
               
                   
                 TSTSTVYMELSSLRSEDTAVYYCAAGLFIWGQGTLVTVS 
                 IL2_2B07_H_H37Y_L_ 
               
               
                   
                 SAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVT 
                 W38Y_A107Y 
               
               
                   
                 LTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPS 
                   
               
               
                   
                 QSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPN 
                   
               
               
                   
                 AAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDV 
                   
               
               
                   
                 QISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQH 
                   
               
               
                   
                 QDWMSGKEFKCKVNNKDLGAPIERTISKPKGSVRAPQ 
                   
               
               
                   
                 VYVLPPPEKEMTKKQVSLTCLVKDFMPEDIYVEWTNN 
                   
               
               
                   
                 GKTELNYKNTEPVLKSDGSYFMYSKLTVEKKNWVERNS 
                   
               
               
                   
                 YSCSVVHEGLHNHHTTKSFSRTPGGGGSGGGSHHHHH 
                   
               
               
                   
                 H 
                   
               
               
                   
               
               
                 SEQ ID NO: 
                 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDM 
                 PD1- 
               
               
                 183 
                 LTFEFYMPKKATELKHLQCLERELKPLEEVLNLAQSKNF 
                 IL2_3x_Asym_PD1- 
               
               
                   
                 HLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLN 
                 IL2_7A04_H_M115W_L_ 
               
               
                   
                 RWITFCQSIISTLTGGGGSGGGGSGGGGSGGGGSQVQ 
                 Q68D 
               
               
                   
                 LVQSGAEVKKPGASVKVSCKASGYTFTDYYMHWVRQ 
                   
               
               
                   
                 APGQGLEWMGIINPRAGYTSYALKFQGRVTMTRDTST 
                   
               
               
                   
                 STVYMELSSLRSEDTAVYYCTSGWDVWGQGTLVTVSS 
                   
               
               
                   
                 AKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTL 
                   
               
               
                   
                 TWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPS 
                   
               
               
                   
                 QSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPN 
                   
               
               
                   
                 AAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDV 
                   
               
               
                   
                 QISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQH 
                   
               
               
                   
                 QDWMSGKEFKCKVNNKDLGAPIERTISKPKGSVRAPQ 
                   
               
               
                   
                 VYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNN 
                   
               
               
                   
                 GKTELNYKNTEPVLDSDGSYFMYSDLRVEKKNWVERN 
                   
               
               
                   
                 SYSCSVVHEGLHNHHTTESFSRTPGK 
                   
               
               
                   
               
               
                 SEQ ID NO: 
                 QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMHW 
                 PD1- 
               
               
                 184 
                 VRQAPGQGLEWMGIINPRAGYTSYALKFQGRVTMTR 
                 IL2_3x_Asym_PD1- 
               
               
                   
                 DTSTSTVYMELSSLRSEDTAVYYCTSGWDVWGQGTLV 
                 IL2_7A04_H_M115W_L_ 
               
               
                   
                 TVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPE 
                 Q68D 
               
               
                   
                 PVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSST 
                   
               
               
                   
                 WPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCP 
                   
               
               
                   
                 APNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSED 
                   
               
               
                   
                 DPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSAL 
                   
               
               
                   
                 PIQHQDWMSGKEFKCKVNNKDLGAPIERTISKPKGSVR 
                   
               
               
                   
                 APQVYVLPPPEKEMTKKQVSLTCLVKDFMPEDIYVEWT 
                   
               
               
                   
                 NNGKTELNYKNTEPVLKSDGSYFMYSKLTVEKKNWVE 
                   
               
               
                   
                 RNSYSCSVVHEGLHNHHTTKSFSRTPGGGGSGGGSHH 
                   
               
               
                   
                 HHHH 
               
               
                   
               
            
           
         
       
     
     A. Sensor Domains 
     Protein complexes of the present disclosure include sensor domains. A sensor domain may be any protein that is capable of sensing the presence of a first moiety and regulating a second moiety, where the first moiety is a marker (e.g., a tumor cell marker) and the second moiety is a therapeutic domain (e.g., a cytokine therapeutic domain). For example, the present disclosure provides a sensor domain that may be an antibody or antibody fragment capable of binding a first moiety and binding and blocking the activity of a second moiety, wherein the first moiety is a marker (e.g., a tumor marker) and the second moiety is a therapeutic domain (e.g., a cytokine therapeutic domain). In the absence of the first moiety, the sensor domain binds the second moiety. If the first moiety is introduced into the system, the sensor domain binds the first moiety and unbinds the second moiety. Thus, the binding and unbinding of the second moiety is reversible. The sensor domain inactivates or blocks the activity of the therapeutic domain by binding the therapeutic domain and preventing it from binding to its target (e.g., a receptor, a ligand, or a substrate). The sensor domain regulates the therapeutic domain by releasing it to act on its target upon binding of a marker. 
     In some embodiments, the sensor domain is a dual binding protein such as a dual binding antibody. A dual binding protein may be capable of binding the marker and the therapeutic domain. A dual binding protein of the present disclosure may be selected or engineered to bind the marker and the therapeutic domain. The dual binding protein may have a higher affinity for the marker as compared to the therapeutic domain. The dual binding protein may be affinity matured to have a higher affinity for the marker as compared to the therapeutic domain. 
     In some embodiments, the sensor domain is an antibody. The sensor domain may also be a fragment of an antibody. A fragment of an antibody consistent with the sensor domains disclosed herein retains its ability to exhibit dual binding to both a marker and a therapeutic domain. One or both domains of a bispecific antibody may be sensor domains of the protein complexes of the present disclosure. In the instance that bispecific antibodies are used, the bispecific antibody may include a first antigen binding domain that may bind a therapeutic domain and a marker and may also include a second antigen binding domain capable of binding the marker. In some embodiments, the bispecific antibody may have a first antigen binding domain that binds a therapeutic domain and a first marker, and a second antigen binding domain that binds a second marker. In some embodiments, the bispecific antibody may have a first antigen binding domain that binds a therapeutic domain and a first marker, and a second antigen binding domain that binds a therapeutic domain and a second marker. In some embodiments the first and second antigen binding domains may bind to the same therapeutic domain ( FIG.  13   ). 
     In some embodiments the two sensor domains may bind to a single IFNα domain attached by a linker to two antibody domains; a first antibody domain that may bind to CEA (a first marker) and to the IFNα domain, and a second antibody domain that may bind to ATP (a second marker), and to the IFNα domain such that the IFNα is able to bind its receptor only in the presence of CEA and ATP ( FIG.  13   ). 
     In some embodiments, the sensor domain is an anti-PD1 or anti-PDL1 antibody or fragment thereof (e.g., an scFv that binds PD1 or PD-L1). In some embodiments, the sensor domain binds to a marker comprising a surface protein, such as a cell surface protein, soluble ATP, a secreted protein, PD1, PD-L1, CEACAM5, FAP, LRRC15, a metabolite, adenosine, AMP, ADP, ATP, or kynurenine, or CRIPTO, CD19, CD20, CD22, CD30, CD33, Glycoprotein NMB, CanAg, HER2 (ErbB2/Neu), CD56 (NCAM), CD70, CD79, CD138, PSCA, PSMA (prostate specific membrane antigen), BCMA, E-selectin, EphB2, Melanotransferin, Muc16 and TMEFF2, BMPR1B, E16, STEAP1, 0772P, MPF, Naρi3b, Sema 5b, PSCA hlg, ETBR, MSG783, STEAP2, TrpM4, CD21, CD79b, FcRH2, NCA, MDP, IL20Rα, Brevican, EphB2R, ASLG659, PSCA, GEDA, BAFF-R, CD79a, CXCR5, HLA-DOB, P2X5, CD72, FCRHI, IRTA2, a sialic acid, or any other marker described in U.S. Pat. No. 10,561,739, incorporated herein by reference in its entirety or WO 2005/082023, incorporated herein by reference in its entirety. 
     In some embodiments, the sensor domain comprises a condition-dependent target affinity. Many cell types, including a range of cancer cell types, generate specific extracellular and tissue-specific microenvironments distinct from those of healthy cells. Recently, significant attention has been placed on the link between extracellular sodium depletion and certain brain cancers. As a further example, some cancers generate low pH microenvironments which can affect changes in the membranome protonation and conformational patterns. Accordingly, a sensor domain may comprise enhanced affinity for a target marker in the presence of a particular condition. A sensor domain may be responsive to pH, temperature, salinity, osmotic pressure, or any combination thereof. For example, a sensor domain may comprise an order of magnitude greater affinity for a target molecule or an order of magnitude lower affinity for a therapeutic domain in the presence of a particular condition. The particular condition may affect the sensor (e.g., a charge or conformation of the sensor), the target (e.g., a charge or solubility of the target), or both. 
     B. Therapeutic Domains 
     Protein complexes of the present disclosure include therapeutic domains. A therapeutic domain of the present disclosure is linked to a sensor domain via a linker to form a protein complex. The therapeutic domain may exert therapeutic activity by binding to a target. For example, the therapeutic domain may be a cytokine and its target may be a receptor target. Upon binding of the cytokine to its receptor target, the cytokine may modulate cellular proliferation, activation, differentiation, and/or may exert anti-tumor or anti-viral activity. Therapeutic domains consistent with the protein complexes of the present disclosure include a cytokine, a chemokine, an antibody, an antibody fragment, a peptide agonist, a peptide antagonist, an enzyme, a soluble receptor, a growth factor, a protein toxin, a soluble ligand, a small molecule, or combinations thereof. In some embodiments, an antibody or antibody fragment comprises an IgG, an IgA, an IgD, an IgE, an IgM, an Fab, an F(ab)′2, a single domain antibody fragment (e.g., a nanobody), a diabody, an scFab, an scFv, an (scFv) 2 , or any fragment (e.g., an Fe domain or CH domain) or combination thereof. 
     In some embodiments, the protein complexes of the present disclosure comprise a therapeutic domain comprising an IL-2 receptor agonist, IL-12 receptor agonist, or IFNα, or variants or fusions of these cytokines. In some embodiments, the therapeutic domain may be IFNα, IFNγ IL-12 IL-4, IL-8, IL-10, IL-15, IL-18, IL-21, TGF beta, an anti-CD3 antibody, an anti-CD28 antibody or ligand, an antibody to or ligand of CD40, GITR, OX40, CD137, CD27, or Death Receptors, the extracellular domain of TGFBR2, VEGF-C, kynureninase, IL-7, TNF, MICA, MICB, CD47, an anti-CTLA4 antibody, an anti-PD-L1 antibody, or an anti-PD-1 antibody. The therapeutic domain may also be a fragment of any of the above mentioned moieties. A fragment retains functional regions of the moiety needed for binding to its target (e.g., IL-2 receptor) and any functional regions needed for activity. 
     C. Linkers 
     A protein complex disclosed herein may comprise a linker. The linker may connect two domains, such as a sensor domain and a therapeutic domain. The linker may connect two portions of a sensor domain, for example a light chain variable domain and a heavy chain variable domain. Various linkers are consistent with the protein complexes of the present disclosure. In some embodiments, the linker may be an amino acid linker or a chemical linker. 
     The linker may be a stable linker. For example, a linker may maintain a connection between a therapeutic domain and a sensor domain even upon binding of the sensor domain to a marker and, thereby, unbinding of the therapeutic domain from the sensor domain. For example, although the sensor domain may unbind the therapeutic domain, the therapeutic domain may remain linked to the sensor domain via the linker. Examples of linkers that are consistent with this activity may include non-cleavable linkers. 
     The linker may also be a flexible linker. A flexible linker is a linker that is long enough to allow for the therapeutic domain to bind to its target, once it is unbound from the sensor domain. Flexibility of the linker may affect therapeutic efficacy. For example, upon binding of the sensor domain to a marker and unbinding of the therapeutic domain, the therapeutic domain needs to be able to encounter and bind its therapeutic target (e.g., a receptor on the same cell surface as the marker or a receptor on an adjacent cell surface to the marker). If the linker is not flexible enough to allow for the therapeutic domain to binds its therapeutic target, therapeutic efficacy may be reduced or not exerted. When the linker is flexible, therapeutic domains may be able to bind their therapeutic target and exert high therapeutic efficacy. Flexibility of a linker may arise from the length of the linker. For example, short linkers may sterically hinder the therapeutic domain from binding its target. Longer linkers may allow for the protein complex to be more flexible and allow for therapeutic domains to bind their target. In some embodiments, a linker that is too long may impact the ability of the sensor domain to bind the therapeutic domain and inhibit activity in the absence of the marker. In some embodiments, a linker that is too long may impact the stability of a protein therapeutic domain or the half-life of the protein therapeutic domain in vivo. 
     In some embodiments, the linker may be attached to a heavy chain of the sensor domain or a light chain of the sensor domain. A linker may be fused to the N-terminus or C-terminus of the sensor domain. In some embodiments, the linker may be attached to a heavy chain or light chain of the therapeutic domain or is fused with the N-terminus or C-terminus of the therapeutic domain. For example, a linker may be attached to an N-terminus or C-terminus of an scFV or an ScFab. 
     Amino Acid Linkers. An amino acid linker may comprise any amino acid residues. In some embodiments, favored amino acid residues are amino acid residues that are entropically flexible. Favored amino acid residues in an amino acid linker of the present disclosure may include glycine and serine. Other preferred amino acid residues may include alanine, proline, threonine, and glutamic acid. In preferred embodiments, the amino acid linker may comprise from 3 to 60 amino acid residues in length. In some embodiments, the amino acid linker may comprise 20 amino acid residues. In some embodiments, the amino acid linker may comprise 40 amino acid residues. In some embodiments, the amino acid linker may comprise 60 amino acid residues. In some embodiments, the amino acid linker may comprise 80 amino acid residues. An amino acid linker may comprise at least 5 amino acid residues. An amino acid linker may comprise at least 10 amino acid residues. An amino acid linker may comprise at least 15 amino acid residues. An amino acid linker may comprise at least 20 amino acid residues. An amino acid linker may comprise at least 25 amino acid residues. An amino acid linker may comprise at least 30 amino acid residues. An amino acid linker may comprise at least 35 amino acid residues. An amino acid linker may comprise at least 40 amino acid residues. An amino acid linker may comprise at least 45 amino acid residues. An amino acid linker may comprise at least 50 amino acid residues. An amino acid linker may comprise at least 55 amino acid residues. An amino acid linker may comprise at least 60 amino acid residues. An amino acid linker may comprise at least 65 amino acid residues. An amino acid linker may comprise at least 70 amino acid residues. An amino acid linker may comprise at least 75 amino acid residues. An amino acid linker may comprise at least 80 amino acid residues. An amino acid linker may comprise at least 85 amino acid residues. An amino acid linker may comprise at least 90 amino acid residues. An amino acid linker may comprise at least 95 amino acid residues. An amino acid linker may comprise at least 100 amino acid residues. An amino acid linker may comprise at least 110 amino acid residues. An amino acid linker may comprise at least 120 amino acid residues. An amino acid linker may comprise at least 130 amino acid residues. An amino acid linker may comprise at least 140 amino acid residues. An amino acid linker may comprise at least 150 amino acid residues. An amino acid linker may comprise at least 160 amino acid residues. An amino acid linker may comprise at least 170 amino acid residues. An amino acid linker may comprise at least 180 amino acid residues. An amino acid linker may comprise at least 190 amino acid residues. An amino acid linker may comprise at least 200 amino acid residues. An amino acid linker may comprise at least 300 amino acid residues. An amino acid linker may comprise at least 400 amino acid residues. An amino acid linker may comprise at least 500 amino acid residues. An amino acid linker may comprise from 5 to 10 amino acid residues. An amino acid linker may comprise from 10 to 15 amino acid residues. An amino acid linker may comprise from 15 to 20 amino acid residues. An amino acid linker may comprise from 20 to 25 amino acid residues. An amino acid linker may comprise from 25 to 30 amino acid residues. An amino acid linker may comprise from 30 to 35 amino acid residues. An amino acid linker may comprise from 35 to 40 amino acid residues. An amino acid linker may comprise from 40 to 45 amino acid residues. An amino acid linker may comprise from 45 to 50 amino acid residues. An amino acid linker may comprise from 50 to 55 amino acid residues. An amino acid linker may comprise from 55 to 60 amino acid residues. An amino acid linker may comprise from 60 to 65 amino acid residues. An amino acid linker may comprise from 65 to 70 amino acid residues. An amino acid linker may comprise from 70 to 75 amino acid residues. An amino acid linker may comprise from 75 to 80 amino acid residues. An amino acid linker may comprise from 80 to 85 amino acid residues. An amino acid linker may comprise from 85 to 90 amino acid residues. An amino acid linker may comprise from 90 to 95 amino acid residues. An amino acid linker may comprise from 95 to 100 amino acid residues. An amino acid linker may comprise from 5 to 80 amino acid residues. An amino acid linker may comprise from 20 to 40 amino acid residues. An amino acid linker may comprise from 20 to 80 amino acid residues. An amino acid linker may comprise from 30 to 60 amino acid residues. An amino acid linker may comprise from 40 to 50 amino acid residues. An amino acid linker may comprise from 10 to 30 amino acid residues. An amino acid linker may comprise from 10 to 20 amino acid residues. An amino acid linker may comprise from 5 to 25 amino acid residues. An amino acid linker may comprise from 25 to 75 amino acid residues. An amino acid linker may comprise from 100 to 500 amino acid residues. An amino acid linker may comprise from 100 to 300 amino acid residues. An amino acid linker may comprise from 5 to 500 amino acid residues. An amino acid linker may comprise no more than 100 amino acid residues. An amino acid linker may comprise no more than 90 amino acid residues. An amino acid linker may comprise no more than 80 amino acid residues. An amino acid linker may comprise no more than 70 amino acid residues. An amino acid linker may comprise no more than 60 amino acid residues. An amino acid linker may comprise no more than 50 amino acid residues. An amino acid linker may comprise no more than 40 amino acid residues. An amino acid linker may comprise no more than 30 amino acid residues. An amino acid linker may comprise no more than 20 amino acid residues. An amino acid linker may comprise no more than 10 amino acid residues. An amino acid linker may comprise no more than 95 amino acid residues. An amino acid linker may comprise no more than 90 amino acid residues. An amino acid linker may comprise no more than 85 amino acid residues. An amino acid linker may comprise no more than 80 amino acid residues. An amino acid linker may comprise no more than 75 amino acid residues. An amino acid linker may comprise no more than 70 amino acid residues. An amino acid linker may comprise no more than 65 amino acid residues. An amino acid linker may comprise no more than 60 amino acid residues. An amino acid linker may comprise no more than 55 amino acid residues. An amino acid linker may comprise no more than 50 amino acid residues. An amino acid linker may comprise no more than 45 amino acid residues. An amino acid linker may comprise no more than 40 amino acid residues. An amino acid linker may comprise no more than 35 amino acid residues. An amino acid linker may comprise no more than 30 amino acid residues. An amino acid linker may comprise no more than 25 amino acid residues. An amino acid linker may comprise no more than 20 amino acid residues. An amino acid linker may comprise no more than 15 amino acid residues. An amino acid linker may comprise no more than 10 amino acid residues. An amino acid linker may comprise no more than 200 amino acid residues. An amino acid linker may comprise no more than 300 amino acid residues. An amino acid linker may comprise no more than 400 amino acid residues. An amino acid linker may comprise no more than 500 amino acid residues. 
     Non-Cleavable Linkers. 
     A non-cleavable linker of the present disclosure may include a chemical linker that is stable. Examples of non-cleavable linkers consistent for use in protein complexes of the present disclosure to link the sensor domain and the therapeutic domain may include a thioether linker, an alkyl linker, a polymeric linker. A linker may be an SMCC linker or a PEG linker. In preferred embodiments, the linker may be a PEG linker. 
     A non-cleavable linker may also include a non-proteolytically cleavable peptide. A non-proteolytically cleavable peptide may be inert to proteases present in a given sample or organism. For example, a peptide may be inert to all human protease cleavage sequences, and thereby may comprise a high degree of stability within humans and human samples. Such a peptide may also comprise a secondary structure which renders a protease cleavage site inert or inaccessible to a protease. A non-cleavable linker of the present disclosure may comprise a half-life for cleavage of at least 1 hour, at least 2 hours, at least 4 hours, at least 8 hours, at least 12 hours, at least 16 hours, at least 1 day, at least 2 days, at least 3 days, at least 1 week, at least 2 weeks, or at least 1 month in the presence of human proteases at 25° C. in pH 7 buffer. 
     D. Protein Complex Structures 
     The present disclosure provides a wide variety of protein complexes spanning a range of structures. A protein complex of the present disclosure may comprise a therapeutic domain and a sensor domain expressed as a single unit. A therapeutic domain may be expressed as an N-terminal extension of a sensor domain, as a C-terminal extension of a sensor domain, or disposed within a sensor domain. For example, a protein complex may comprise a peptide which comprises, from N-terminus to C-terminus, a therapeutic domain, a peptide linker, an scFv domain, and optionally a tag, such as a purification tag (e.g., a V5 or myc tag) or a localization signal. Alternatively, a therapeutic domain and a sensor domain may be coupled (e.g., chemically coupled) subsequent to expression. 
     A protein complex may comprise a plurality of protein subunits. The plurality of protein subunits (e.g., a therapeutic domain and a sensor domain, two sensor domains, or two subunits of a sensor domain) may be chemically or physically coupled following expression. The plurality of protein subunits may comprise a plurality of sensor and/or therapeutic domains. A sensor and/or a therapeutic domain may be comprised of a single protein subunit, of multiple protein subunits, or by portions thereof. For example, a sensor domain may comprise an antibody Fab region comprising portions of an immunoglobulin light chain and an immunoglobulin heavy chain. 
     A plurality of protein subunits may comprise physical handles which facilitate their selective coupling. The physical handles may enable spontaneous, irreversible, and/or non-mediated (e.g., not requiring a chaperone protein or a catalytic complex) coupling between the protein subunits, thereby enabling complex and asymmetric protein complexes. For example, two distinct protein complex subunits expressed in a single Chinese hamster ovary (CHO) cell, may comprise physical handles which spontaneously and irreversibly couple prior to cellular export. Such physical handles may comprise a ‘knob-into-hole’ (KIH) construct or a charge-swap construct, in which two protein subunits comprise physical structures with mutual binding affinities and specificities. Such physical handles may comprise a covalently binding pair, such as a plurality of thiols configured to form disulfide bonds. Physical handles may enable facile production of protein complexes comprising identical or distinct domains. 
     A protein complex may comprise two or more identical domains. An example of such a protein complex is provided in  FIG.  14 A , which illustrates an antibody (multi-sensor domain) coupled to two IL-2 therapeutic domains. In this example, the protein complex comprises two protein immunoglobulin light chain subunits and two immunoglobulin heavy chain subunits complexed to form a competent antibody. The two immunoglobulin heavy chain subunits comprise N-terminal linkers coupled to IL-2 therapeutic domains. Each immunoglobulin heavy chain is coupled to an immunoglobulin light chain, such that the protein complex comprises two Fab regions, each separately coupled to a therapeutic domain by a linker. 
     While the above example provides a symmetric protein complex with two identical sensor domains and two identical therapeutic domains, a protein complex may also comprise a plurality of distinct sensor and/or therapeutic domains. Such a protein complex may comprise an immunoglobulin unit with a first arm comprised of a heavy chain-light chain pair, and a second arm comprised of an antibody fragment such as an scFv, an scFab, a VH, or a fragment thereof. In such cases, the heavy chain, the antibody fragment, or the light chain may comprise an N-terminal extension with a linker and a therapeutic domain, as illustrated in  FIGS.  9 A , C, and F, respectively. Alternatively, the heavy chain, the antibody fragment, or the light chain may comprise a C-terminal extension with a linker and a therapeutic domain. A protein complex may also comprise a symmetric immunoglobulin unit with a single therapeutic domain. For example, as shown in  FIG.  9 B , an immunoglobulin unit may comprise an N-terminal linker and therapeutic unit on a single heavy chain. Alternatively, an immunoglobulin unit may comprise an N-terminal linker and therapeutic unit on a single light chain. An immunoglobulin unit may also comprise a pair of antibody fragments coupled to a single Fc region. An immunoglobulin unit may comprise a nanobody. An immunoglobulin unit may comprise a diabody. 
     In some cases, a plurality of distinct sensor domains are associated with a plurality of distinct therapeutic domains. Such a plurality of sensor domains may comprise common targets. For example, a protein complex may comprise a first sensor domain associated with an IL-2 therapeutic domain and comprising affinities for IL-2 and PD-1, and a second sensor domain associated with an IFNα therapeutic domain and comprising an affinity for IFNα and PD-1. Alternatively, a plurality of sensor domains may comprise separate targets. For example, a protein complex may comprise a first sensor domain associated with an IL-2 therapeutic domain and comprising affinities for IL-2 and PD-1, and a second sensor domain associated with an IFNα therapeutic domain and comprising an affinity for IFNα and CEACAM5. 
     A protein complex may comprise a therapeutic domain targeted by one or more than one sensor domain. A protein complex comprising such a plurality of sensor domains may comprise a multi-target dependence for activity. This concept is illustrated in  FIGS.  13 A- 13 C , which provides a protein complex comprising a therapeutic domain, a first sensor domain targeting the therapeutic domain and a first target (‘Marker 1’), and a second sensor domain targeting the therapeutic domain and a second target (‘Marker 2’). In this example, the presence of Marker 1 leads to therapeutic domain binding to DBA 2, while the presence of Marker 2 leads to therapeutic domain binding to DBA 1. However, the presence of Marker 1 and Marker 2 liberates the therapeutic domain, enhancing its activity. Accordingly, the activity of the protein complex is requisite upon the presence of both of its markers. An example of such a system may be a protein complex comprising a first sensor domain which targets IL-2 and PD-1, and a second sensor domain which targets IL-2 and CEACAM5, such that CEACAM5 and PD-1 are requisite for IL-2 activity by the protein complex. A protein complex may comprise a dependence for at least 2, at least 3, at least 4, or at least 5 target markers. A protein complex may comprise a dependence for at most 5, at most 4, at most 3, at most 2, or for a single target marker. 
     Multi-marker activity dependence may enhance the selectivity of a protein complex. Some cells, including many forms of cancerous cells, comprise minor variations in their surfaceomes relative to healthy cells, rendering monospecific targeting unfeasible for distinguishing diseased cells. Accordingly, selectively targeting a particular diseased cell or tissue may require targeting a plurality of markers. A protein complex of the present disclosure may target at least 2, at least 3, at least 4, or at least 5 markers. A protein complex of the present disclosure may target at most 5, at most 4, at most 3, or at most 2 markers. In some cases, at least one of the markers targeted by a protein complex is commonly shared between a target cell or tissue and a healthy cell or tissue. In some cases, all of the markers targeted by a protein complex are commonly shared between a target cell or tissue and a healthy cell or tissue. 
     A sensor domain of the present disclosure may target at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 markers and no therapeutic domain. A sensor domain of the present disclosure may target at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, or at most 10 markers and no therapeutic domain. A sensor domain of the present disclosure may target a single therapeutic domain and at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 markers. A sensor domain of the present disclosure may target a single therapeutic domain and at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, or at most 10 markers. A sensor domain of the present disclosure may target at least two therapeutic domains and at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 markers. A sensor domain of the present disclosure may target at least two therapeutic domains and at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, or at most 10 markers. 
     Two sensor domains may comprise identical affinities for a therapeutic domain, or may comprise different affinities for the therapeutic domain. Two sensor domains may comprise affinities for a therapeutic domain differing by at least 1 order of magnitude, at least 2 orders of magnitude, at least 3 orders of magnitude, or at least 4 orders of magnitude for a therapeutic domain. Two sensor domains may comprise affinities for a therapeutic domain which differ by at most 4 orders of magnitude, at most 3 orders of magnitude, at most 2 orders of magnitude, or at most 1 order of magnitude. A combination of different therapeutic domain affinities by a plurality of sensor domains may enhance the affinity of a protein complex for a target marker. For example, a protein complex may comprise a first sensor domain which weakly targets a first cell surface marker and weakly targets a therapeutic domain, and a second sensor domain which strongly targets a second cell surface marker and strongly targets the therapeutic domain, such that the protein complex exhibits weak activity in the presence of the cell surface second marker and strong activity in the presence of the first and the second cell surface markers. 
     Two sensor domains of a protein complex may also target separate therapeutic domains. For example, a protein complex may comprise a first sensor domain which targets IL-2 and PD-1, and a second sensor domain which targets IFNα and CEACAM5. A protein complex may comprise a sensor domain which does not target a therapeutic domain. Such a sensor domain may aid in target localization, or may enhance the activity of a separate sensor domain for a therapeutic domain. An example of a protein complex comprising a sensor domain which does not target a therapeutic domain is provided in  FIG.  14 C . This system comprises a monospecific anti-PD-1 antibody, wherein a first heavy chain comprises a C-terminal linker coupled to a therapeutic domain, and a second heavy chain comprises a C-terminal linker coupled to a sensor domain with dual specificity for the therapeutic domain and for a target marker. 
     A protein complex may comprise a single target, 2 targets, 3 targets, 4 targets, or more than 4 targets. A protein complex may comprise at least 2 targets, at least 3 targets, or at least 4 targets. A protein complex may comprise at most 4 targets, at most 3 targets, or at most 2 targets. A protein complex may comprise a single sensor domain, 2 sensor domains, 3 sensor domains, 4 sensor domains, or more than 4 sensor domains. A protein complex may comprise at least 2 sensor domains, at least 3 sensor domains, or at least 4 sensor domains. For example, a protein complex may comprise an IgM antibody comprising Fab region sensor domains, or an IgA antibody comprising 4 Fab region sensor domains. 
     A protein complex may comprise a range of sensor-to-therapeutic domain ratios. A protein complex may comprise equal numbers of sensor domains and therapeutic domains, examples of which are provided by  FIG.  14 A , which illustrates a protein complex with 2 sensor domains and 2 therapeutic domains, and  FIG.  8   , which illustrates a protein complex with a single sensor domain and a single therapeutic domain. A protein complex may comprise a greater number of sensor domains than therapeutic domains, such as the protein complexes of  FIGS.  9 A,  9 B,  9 C,  9 F, and  14 B , which each comprise two sensor domains and one therapeutic domain. In such cases, a therapeutic domain may be capable of interacting with multiple sensor domains, or may be constrained from interacting with more than one sensor domain. The number of therapeutic domains with which a sensor domain may interact may depend on its linker. A linker may be sufficiently short so as to prevent a therapeutic domain from interacting with a sensor domain, or may be sufficiently long so as to allow a therapeutic domain to interact with multiple sensor domains. 
     In specific cases, a protein complex may comprise an antibody with Fc-coupled therapeutic and sensor domains. As illustrated in  FIG.  14 C , a protein complex may comprise an antibody with a first heavy chain C-terminal extension comprising a linker and a therapeutic domain, and a second heavy chain C-terminal extension comprising a linker and a sensor domain. An antibody of this design may comprise common targets across its Fab and C-terminal extension sensor domain. For example, the antibody Fab regions and C-terminal extension sensor domain may each target PD-1. Conversely, an antibody of this design may comprise separate targets across its Fab regions and C-terminal extension sensor domain. 
     In some embodiments, an amino acid in the protein complex described herein may comprise a conservative substitution. A conservative substitution may comprise a substitution of one amino acid with a different amino acid with similar biochemical properties (e.g. charge, hydrophobicity, and size). Examples of conservative substitutions, as well as substitutions that may be, but are not necessarily, preferred, are provided in TABLE 33. 
     
       
         
           
               
             
               
                 TABLE 33 
               
             
            
               
                   
               
               
                 Exemplary Conservative Substitutions 
               
            
           
           
               
               
               
            
               
                 Original 
                   
                 Preferred 
               
               
                 Residue 
                 Exemplary Substitutions 
                 Substitutions 
               
               
                   
               
               
                 Ala (A) 
                 Val; Leu; Ile 
                 Val 
               
               
                 Arg (R) 
                 Lys; Gln; Asn 
                 Lys 
               
               
                 Asn (N) 
                 Gln; His; Lys; Arg 
                 Gln 
               
               
                 Asp (D) 
                 Glu 
                 Glu 
               
               
                 Cys (C) 
                 Ser 
                 Ser 
               
               
                 Gln (Q) 
                 Asn 
                 Asn 
               
               
                 Glu (E) 
                 Asp 
                 Asp 
               
               
                 Gly (G) 
                 Pro; Ala 
                 Ala 
               
               
                 His (H) 
                 Asn; Gln; Lys; Arg 
                 Arg 
               
               
                 Ile (I) 
                 Leu; Val; Met; Ala; Phe; Norleucine 
                 Leu 
               
               
                 Leu (L) 
                 Norleucine; Ile; Val; Met; Ala; Phe 
                 Ile 
               
               
                 Lys (K) 
                 Arg; Gln; Asn 
                 Arg 
               
               
                 Met (M) 
                 Leu; Phe; Ile 
                 Leu 
               
               
                 Phe (F) 
                 Leu; Val; Ile; Ala; Tyr 
                 Leu 
               
               
                 Pro (P) 
                 Ala 
                 Ala 
               
               
                 Ser (S) 
                 Thr 
                 Thr 
               
               
                 Thr (T) 
                 Ser 
                 Ser 
               
               
                 Trp (W) 
                 Tyr; Phe 
                 Tyr 
               
               
                 Tyr (Y) 
                 Trp; Phe; Thr; Ser 
                 Phe 
               
               
                 Val (V) 
                 Ile; Leu; Met; Phe; Ala; Norleucine 
                 Leu 
               
               
                   
               
            
           
         
       
     
     In some embodiments, the present disclosure describes a recombinant nucleic acid that encodes the protein complex disclosed herein. In some embodiments, the recombinant nucleic acid comprises a plasmid or a vector that encodes the entire protein complex. In some embodiments, the recombinant nucleic acid comprises plasmids or vectors that encode the therapeutic domain, the sensor domain, and the linker respectively. In some embodiments, the recombinant nucleic acid comprises plasmids or vectors that encode any two of the therapeutic domain, the sensor domain, and the linker together. 
     Pharmaceutical Formulations 
     A protein complex or a recombinant nucleic acid encoding the protein complex of the present disclosure may be formulated as a pharmaceutical composition. A pharmaceutical composition may comprise a pharmaceutically acceptable carrier or excipient. As used herein “pharmaceutically acceptable” or “pharmacologically acceptable” includes molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a subject, as appropriate. “Pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients are often also incorporated into the compositions. 
     Applications 
     A protein complex of the present disclosure may be used for various therapeutic applications. A protein complex of the present disclosure may be used as a therapeutic to administer to a subject in need thereof. The subject may be a human or non-human mammal. The subject may have a disease. The disease may be cancer. The cancer may be acute lymphoblastic leukemia (ALL); acute myeloid leukemia (AML); cancer in adolescents; adrenocortical carcinoma; aids-related cancers; kaposi sarcoma (soft tissue sarcoma); aids-related lymphoma (lymphoma); primary cns lymphoma (lymphoma); anal cancer; appendix cancer—see gastrointestinal carcinoid tumors; astrocytomas, childhood (brain cancer); atypical teratoid/rhabdoid tumor, childhood, central nervous system (brain cancer); basal cell carcinoma of the skin—see skin cancer; bile duct cancer; bladder cancer; bone cancer (includes ewing sarcoma and osteosarcoma and malignant fibrous histiocytoma); brain tumors; breast cancer; bronchial tumors (lung cancer); burkitt lymphoma—see non-hodgkin lymphoma; carcinoid tumor (gastrointestinal); carcinoma of unknown primary; cardiac (heart) tumors, childhood; central nervous system; atypical teratoid/rhabdoid tumor, childhood (brain cancer); medulloblastoma and other cns embryonal tumors, childhood (brain cancer); germ cell tumor, childhood (brain cancer); primary cns lymphoma; cervical cancer; childhood cancers; cancers of childhood, unusual; cholangiocarcinoma—see bile duct cancer; chordoma, childhood (bone cancer); chronic lymphocytic leukemia (CLL); chronic myelogenous leukemia (CMIL); chronic myeloproliferative neoplasms; colorectal cancer; craniopharyngioma, childhood (brain cancer); cutaneous t-cell lymphoma—see lymphoma (mycosis fungoides and sezary syndrome); ductal carcinoma in situ (DCIS)—see breast cancer; embryonal tumors, medulloblastoma and other central nervous system, childhood (brain cancer); endometrial cancer (uterine cancer); ependymoma, childhood (brain cancer); esophageal cancer; esthesioneuroblastoma (head and neck cancer); ewing sarcoma (bone cancer); extracranial germ cell tumor, childhood; extragonadal germ cell tumor; eye cancer; intraocular melanoma; retinoblastoma; fallopian tube cancer; fibrous histiocytoma of bone, malignant, and osteosarcoma; gallbladder cancer; gastric (stomach) cancer; gastrointestinal carcinoid tumor; gastrointestinal stromal tumors (GIST) (soft tissue sarcoma); germ cell tumors; childhood central nervous system germ cell tumors (brain cancer); childhood extracranial germ cell tumors; extragonadal germ cell tumors; ovarian germ cell tumors; testicular cancer; gestational trophoblastic disease; hairy cell leukemia; head and neck cancer; heart tumors, childhood; hepatocellular (liver) cancer; histiocytosis, langerhans cell; hodgkin lymphoma; hypopharyngeal cancer (head and neck cancer); intraocular melanoma; islet cell tumors, pancreatic neuroendocrine tumors; kaposi sarcoma (soft tissue sarcoma); kidney (renal cell) cancer; langerhans cell histiocytosis; laryngeal cancer (head and neck cancer); leukemia; lip and oral cavity cancer (head and neck cancer); liver cancer; lung cancer (non-small cell, small cell, pleuropulmonary blastoma, and tracheobronchial tumor); lymphoma; male breast cancer; malignant fibrous histiocytoma of bone and osteosarcoma; melanoma; melanoma, intraocular (eye); merkel cell carcinoma (skin cancer); mesothelioma, malignant; metastatic cancer; metastatic squamous neck cancer with occult primary (head and neck cancer); midline tract carcinoma with nut gene changes; mouth cancer (head and neck cancer); multiple endocrine neoplasia syndromes; multiple myeloma/plasma cell neoplasms; mycosis fungoides (lymphoma); myelodysplastic syndromes, myelodysplastic/myeloproliferative neoplasms; myelogenous leukemia, chronic (CML); myeloid leukemia, acute (AML); myeloproliferative neoplasms, chronic; nasal cavity and paranasal sinus cancer (head and neck cancer); nasopharyngeal cancer (head and neck cancer); neuroblastoma; non-hodgkin lymphoma; non-small cell lung cancer; oral cancer, lip and oral cavity cancer and oropharyngeal cancer (head and neck cancer); osteosarcoma and malignant fibrous histiocytoma of bone; ovarian cancer; pancreatic cancer; pancreatic neuroendocrine tumors (islet cell tumors); papillomatosis (childhood laryngeal); paraganglioma; paranasal sinus and nasal cavity cancer (head and neck cancer); parathyroid cancer; penile cancer; pharyngeal cancer (head and neck cancer); pheochromocytoma; pituitary tumor; plasma cell neoplasm/multiple myeloma; pleuropulmonary blastoma (lung cancer); pregnancy and breast cancer; primary central nervous system (CNS) lymphoma; primary peritoneal cancer; prostate cancer; rectal cancer; recurrent cancer; renal cell (kidney) cancer; retinoblastoma; rhabdomyosarcoma, childhood (soft tissue sarcoma); salivary gland cancer (head and neck cancer); sarcoma; childhood rhabdomyosarcoma (soft tissue sarcoma); childhood vascular tumors (soft tissue sarcoma); ewing sarcoma (bone cancer); kaposi sarcoma (soft tissue sarcoma); osteosarcoma (bone cancer); soft tissue sarcoma; uterine sarcoma; Sezary syndrome (lymphoma); skin cancer; small cell lung cancer; small intestine cancer; soft tissue sarcoma; squamous cell carcinoma of the skin—see skin cancer; squamous neck cancer with occult primary, metastatic (head and neck cancer); stomach (gastric) cancer; t-cell lymphoma, cutaneous—see lymphoma (mycosis fungoides and Sezary syndrome); testicular cancer; throat cancer (head and neck cancer); nasopharyngeal cancer; oropharyngeal cancer; hypopharyngeal cancer; thymoma and thymic carcinoma; thyroid cancer; tracheobronchial tumors (lung cancer); transitional cell cancer of the renal pelvis and ureter (kidney (renal cell) cancer); unknown primary carcinoma; unusual cancers of childhood; ureter and renal pelvis, transitional cell cancer (kidney (renal cell) cancer; urethral cancer; uterine cancer, endometrial; uterine sarcoma; vaginal cancer; vascular tumors (soft tissue sarcoma); vulvar cancer; Wilms tumor and other childhood kidney tumors; or cancer in young adults or any cancer mentioned at https://www.cancer.gov/types. 
     In addition to the treatment of cancer, the protein complexes of the present disclosure have potential applications in a variety of other settings where targeted, conditional activity may be advantageous. In autoimmune and inflammatory disease, therapeutics that act through global immune suppression have the disadvantage of leaving patients more susceptible to a variety of opportunistic infections. Additionally, the short half-life and lack of accumulation in disease tissues may limit the efficacy of immune-dampening recombinant cytokines. The protein complexes of the present disclosure may address these shortcomings by allowing targeted delivery of immune modulators including IL-4, IL-10, TGF-β, and TNFR2 selectively to affected anatomical locations while remaining silent in the periphery. Additional applications may include cell type-specific therapeutic targeting, such Treg cell-directed IL-2. Targeted, conditional activation of opioid agonists in specific organs or in the presence of markers of inflammation may reduce the addictive risk of pain control. 
     A protein complex may be administered as a pharmaceutical composition. A pharmaceutical composition of the disclosure can be a combination of any protein complex described herein with other chemical components, such as carriers, stabilizers, diluents, dispersing agents, suspending agents, thickening agents, and/or excipients. The pharmaceutical composition facilitates administration of a protein complex described herein to an organism. Pharmaceutical compositions can be administered in therapeutically-effective amounts as pharmaceutical compositions by various forms and routes including, for example, intravenous, subcutaneous, intramuscular, rectal, aerosol, parenteral, ophthalmic, pulmonary, transdermal, vaginal, optic, nasal, oral, inhalation, dermal, intra-articular, intrathecal, intranasal, and topical administration. A pharmaceutical composition can be administered in a local or systemic manner, for example, via injection of the protein complex described herein directly into an organ, optionally in a depot. 
     Parenteral injections can be formulated for bolus injection or continuous infusion. The pharmaceutical compositions can be in a form suitable for parenteral injection as a sterile suspension, solution or emulsion in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Pharmaceutical formulations for parenteral administration include aqueous solutions of a protein complex described herein in water-soluble form. Suspensions of protein complexes described herein can be prepared as oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions can contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. The suspension can also contain suitable stabilizers or agents which increase the solubility and/or reduces the aggregation of such protein complexes described herein to allow for the preparation of highly concentrated solutions. Alternatively, the protein complexes described herein can be lyophilized or in powder form for re-constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use. In some embodiments, a purified protein complex is administered intravenously. A protein complex of the present disclosure may comprise a sufficiently long serum half life (e.g., as demonstrated in EXAMPLE 17) to enable dosing regimens comprising daily, alternating day, twice weekly, weekly, biweekly, or monthly dosing frequencies. A protein complex of the present disclosure may comprise a serum half-life of at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 72 hours, at least 96 hours, at least 120 hours, at least 168 hours, at least 250 hours, at least 320 hours, or at least 400 hours. The serum half-life may be a human serum half-life, a murine serum half-life, a porcine serum-half life, a bovine serum half-life, a canine serum half-life, a feline serum half-life, or a leporine serum half-life. 
     A protein complex of the disclosure can be applied directly to an organ, or an organ tissue or cells, during a surgical procedure, or via transdermal, subcutaneous, intramuscular, intratumoral, intrathecal, topical, or local delivery. In some embodiments, a protein complex of the present disclosure may be injected directly into the synovium (e.g., for administration of a protein complex comprising IL-10 for rheumatoid arthritis). In some embodiments, a protein complex may be applied directly to a cancerous tissue (e.g., a tumor). The protein complexes described herein can be administered topically and can be formulated into a variety of topically administrable compositions, such as solutions, suspensions, lotions, gels, pastes, medicated sticks, balms, creams, and ointments. Such pharmaceutical compositions can contain solubilizers, stabilizers, tonicity enhancing agents, buffers and preservatives. 
     In practicing the methods of treatment or use provided herein, therapeutically-effective amounts of the protein complex described herein are administered in pharmaceutical compositions to a subject suffering from a condition. In some instances the pharmaceutical composition will affect the physiology of the animal, such as the immune system, inflammatory response, or other physiologic affect. In some embodiments, the subject is a mammal such as a human. A therapeutically-effective amount can vary widely depending on the severity of the disease, the age and relative health of the subject, the potency of the compounds used, and other factors. 
     Pharmaceutical compositions can be formulated using one or more physiologically-acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active compounds into preparations that can be used pharmaceutically. Formulation can be modified depending upon the route of administration chosen. Pharmaceutical compositions comprising a protein complex described herein can be manufactured, for example, by expressing the protein complex in a recombinant system, purifying the protein complex, lyophilizing the protein complex, mixing, or dissolving. The pharmaceutical compositions can include at least one pharmaceutically acceptable carrier, diluent, or excipient and compounds described herein as free-base or pharmaceutically-acceptable salt form. 
     Methods for the preparation of protein complexes described herein include formulating the protein complex described herein with one or more inert, pharmaceutically-acceptable excipients or carriers to form a solid, semi-solid, or liquid composition. Solid compositions include, for example, powders, tablets, dispersible granules, capsules, cachets, and suppositories. These compositions can also contain minor amounts of nontoxic, auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, and other pharmaceutically-acceptable additives. 
     Certain methods described herein comprise administering to the subject an intravenous pharmaceutical composition comprising a protein complex of the present disclosure, for example, as described herein. Intravenous pharmaceutical compositions of protein complexes include any formulation suitable for administration to a subject via any intravenous method, including a bolus, an infusion which occurs over time or any other intravenous method known in the art. In some aspects, the rate of infusion is such that the dose is administered over a period of less than five minutes, more than five minutes but less than 15 minutes or greater than 15 minutes. In other aspects, the rate of infusion is such that the dose is administered over a period of less than 5 minutes. In other aspects, the rate of infusion is such that the dose is administered over a period of greater than 5 minutes and less than 15 minutes. In some other aspects, the rate of infusion is such that the dose is administered over a period of greater than 15 minutes. 
     “Product” or “dosage form” as used herein refers to any solid, semi-solid, lyophilized, aqueous, liquid or frozen formulation or preparation used for administration. Upon administration, the rate of release of an active moiety from a product is often greatly influenced by the excipients and/or product characteristics which make up the product itself. For example, an enteric coat on a tablet is designed to separate that tablet&#39;s contents from the stomach contents to prevent, for example, degradation of the stomach which often induces gastrointestinal discomfort or injury. According to the currently accepted conventional understanding, systemic exposure of the active moiety will be relatively insensitive to the small formulation changes. 
     Non-limiting examples of pharmaceutically-acceptable excipients can be found, for example, in Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington&#39;s Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. 1975; Liberman, H. A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams &amp; Wilkins 1999), each of which is incorporated by reference in its entirety. 
     A protein complex of the present disclosure may be administered to a patient in an effective amount. The term “effective amount,” as used herein, can refer to a sufficient amount of an agent or a compound being administered which will relieve to some extent one or more of the symptoms of the disease or condition being treated. The result can be reduction and/or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. Compositions containing such agents or compounds can be administered for prophylactic, enhancing, and/or therapeutic treatments. An appropriate “effective” amount in any individual case can be determined using techniques, such as a dose escalation study. 
     The methods, compositions, and kits of this disclosure can comprise a method to prevent, treat, arrest, reverse, or ameliorate the symptoms of a condition. The treatment can comprise treating a subject (e.g., an individual, a domestic animal, a wild animal or a lab animal afflicted with a disease or condition) with a protein complex of the disclosure. Protein complexes of the present disclosure may be administered to treat a disease in a subject. The subject can be a human. A subject can be a human; a non-human primate such as a chimpanzee, or other ape or monkey species; a farm animal such as a cattle, horse, sheep, goat, swine; a domestic animal such as a rabbit, dog, and cat; a laboratory animal including a rodent, such as a rat, mouse and guinea pig, or the like. A subject can be of any age. A subject can be, for example, an elderly adult, adult, adolescent, pre-adolescent, child, toddler, infant, or fetus in utero. 
     Treatment can be provided to the subject before clinical onset of disease. Treatment can be provided to the subject after clinical onset of disease. Treatment can be provided to the subject after 1 day, 1 week, 6 months, 12 months, or 2 years or more after clinical onset of the disease. Treatment may be provided to the subject for more than 1 day, 1 week, 1 month, 6 months, 12 months, 2 years or more after clinical onset of disease. Treatment may be provided to the subject for less than 1 day, 1 week, 1 month, 6 months, 12 months, or 2 years after clinical onset of the disease. Treatment can also include treating a human in a clinical trial. A treatment can comprise administering to a subject a pharmaceutical composition, such as one or more of the pharmaceutical compositions described throughout the disclosure. A treatment can comprise a once daily dosing. A treatment can comprise delivering a protein complex of the disclosure to a subject, either intravenously, subcutaneously, intramuscularly, by inhalation, dermally, intra-articular injection, orally, intrathecally, transdermally, intranasally, via a peritoneal route, or directly onto or into a diseased tissue, e.g., via topical, intra-articular injection route or injection route of application. 
     In some embodiments, the present disclosure provides a method for treating a cancer, the method comprising administering to a subject in need thereof an effective amount of a protein complex of the present disclosure. 
     In some embodiments, the present disclosure provides a method for treating a cancer, the method comprising administering to a patient in need thereof an effective amount of a pharmaceutical composition comprising a protein complex of the present disclosure and a pharmaceutically acceptable carrier. 
     Kits 
     A protein complex of the present disclosure may be provided in various kits. In some embodiments, pharmaceutical compositions comprising a protein complex of the present disclosure may be supplied as a kit. A kit may comprise a container that comprises a protein complex. Therapeutic protein complexes can be provided in the form of an injectable solution for single or multiple doses, or as a sterile powder that will be reconstituted before injection. Alternatively, such a kit can include a dry-powder disperser, liquid aerosol generator, or nebulizer for administration of a therapeutic protein complexes. Such a kit may further comprise written information on indications and usage of the pharmaceutical composition. 
     Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Any reference to “or” herein is intended to encompass “and/or” unless otherwise stated. 
     Whenever the term “at least,” “greater than,” or “greater than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “at least,” “greater than” or “greater than or equal to” applies to each of the numerical values in that series of numerical values. For example, greater than or equal to 1, 2, or 3 is equivalent to greater than or equal to 1, greater than or equal to 2, or greater than or equal to 3. 
     Whenever the term “no more than,” “less than,” “less than or equal to,” or “at most” precedes the first numerical value in a series of two or more numerical values, the term “no more than,” “less than” or “less than or equal to,” or “at most” applies to each of the numerical values in that series of numerical values. For example, less than or equal to 3, 2, or 1 is equivalent to less than or equal to 3, less than or equal to 2, or less than or equal to 1. 
     Where values are described as ranges, it will be understood that such disclosure includes the disclosure of all possible sub-ranges within such ranges, as well as specific numerical values that fall within such ranges irrespective of whether a specific numerical value or specific sub-range is expressly stated. 
     EXAMPLES 
     The following examples are illustrative and non-limiting to the scope of the devices, methods, systems, and kits described herein. 
     Example 1 
     Selection of IFNα and PD-L1 Specific Dual Binding Antibodies (DBAs) 
     This example describes isolation of sensor domains of the present disclosure, specifically, selection of IFNα and PD-L1 specific dual binding antibodies (DBAs). Anti-PD-L1 and anti-IFNα DBAs were isolated from a Tumbler antibody phage display library (Distributed Bio, Inc.). The antibody phage display library was constructed to incorporate the heavy chain CDR1, heavy chain CDR2, and light chain diversity of the Superhuman 2.0 antibody library combined with 10 heavy chain (“HC”) CDR3 sequences (SEQ ID NO: 1-SEQ ID NO: 10) from the PD-L1 binding antibodies described, as shown below in TABLE 2. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 HC-CDR3 of PD-L1 binders 
               
            
           
           
               
               
               
            
               
                 SEQ ID NO 
                 Sequence 
                 Description 
               
               
                   
               
               
                 SEQ ID NO: 1 
                 CARDRIAVAGFDYW 
                 HC-CDR3 of PD-L1 binder 
               
               
                   
               
               
                 SEQ ID NO: 2 
                 CAKEVFSGWYDYW 
                 HC-CDR3 of PD-L1 binder 
               
               
                   
               
               
                 SEQ ID NO: 3 
                 CTTDLLSLELDDAFDIW 
                 HC-CDR3 of PD-L1 binder 
               
               
                   
               
               
                 SEQ ID NO: 4 
                 CARSLFPTIFGVEVAFDIW 
                 HC-CDR3 of PD-L1 binder 
               
               
                   
               
               
                 SEQ ID NO: 5 
                 CARDSYYYDSFDYW 
                 HC-CDR3 of PD-L1 binder 
               
               
                   
               
               
                 SEQ ID NO: 6 
                 CARHGEWGSGWPFDYW 
                 HC-CDR3 of PD-L1 binder 
               
               
                   
               
               
                 SEQ ID NO: 7 
                 CARDLLPAIFSGEVNDAFDIW 
                 HC-CDR3 of PD-L1 binder 
               
               
                   
               
               
                 SEQ ID NO: 8 
                 CARETIAVAGFDPW 
                 HC-CDR3 of PD-L1 binder 
               
               
                   
               
               
                 SEQ ID NO: 9 
                 CARDVLPTIFGVVSDAFDIW 
                 HC-CDR3 of PD-L1 binder 
               
               
                   
               
               
                 SEQ ID NO: 10 
                 CARGDYGDYFDYW 
                 HC-CDR3 of PD-L1 binder 
               
               
                   
               
            
           
         
       
     
     The library was subjected to four rounds of selection alternating between PD-L1 (to develop the PD-L1 binding, where PD-L1 serves as a marker) and IFNα (to develop IFNα binding, where IFNα is the therapeutic domain regulated by the sensor domain). Each round the phage library was incubated with the antigen (PD-L1 or IFNα), captured on magnetic beads, washed on a Kingfisher magnetic particle processor, eluted from the magnetic beads, and amplified by passaging in  E. coli . In Round 1, the phage library was incubated with 50 nM of a human PD-L1-Fc fusion (R&amp;D Systems, Prod. Num. 156-B7) and captured on protein G magnetic beads. In Round 2, the phage library was incubated with 100 nM of biotinylated human IFNα (Genscript, Prod. Num. Z03003, biotinylated using standard protocols) and captured on streptavidin magnetic beads. In Round 3, the phage library was incubated with 50 nM of a cynomolgus PD-L1-Fc fusion and captured on protein G magnetic beads. In Round 4, the phage library was incubated with 50 nM of biotinylated human IFNα and captured on streptavidin magnetic beads. The final selection was plated as single colonies and 380 colonies were picked for Sanger sequencing. Forty-one unique clones were chosen for expression. The scFv sequence for each clone was codon-optimized for  E. coli  expression and the corresponding DNA sequences synthesized as gBlocks (Integrated DNA Technologies, Inc.) with a T7 promoter, a translation initiation site, a Myc tag, the scFv sequence, a V5 tag sequence, and a T7 terminator. If the framework sequence of the antibody variable regions differed from the germline sequence, a second version of the clone was synthesized with the germline sequence. An exemplary sequence of a gBlock expression fragment is shown in 
                    FIG. 3       (SEQ ID NO: 40,               GCGAATTAATACGACTCACTATAGGGCTTAAGTATAAGGAGAATAATAT               ATGTCTACTTCAACAGAACAAAAGTTAATTAGTGAAGAAGATTTACAGG               TCCAGTTGGTTCAGTCAGGCGCAGAAGTCAAAAAGCCGGGAGCGAGTGT               CAAAGTATCTTGTAAAGCGAGCGGTGGTACTTTTAGTAGTTATGCGATT               TCCTGGGTTCGCCAAGCCCCGGGACAGGGTCTGGAATGGATGGGTATTA               TTGACCCTTCCGTGACTTACACCCGCTACGCTCAGAAATTCCAGGGACG               TGTTACCATGACCCGCGATACCAGCACCAGTACCGTTTACATGGAACTT               TCCTCCCTGAGATCGGAAGACACGGCCGTGTATTATTGCGCTCGCTCAC               TCTTTCCGACCATCTTCGGCGTTGAAGTCGCCTTCGACATCTGGGGCCA               GGGCACGCTGGTTACGGTAAGTTCCGCAAGTGGCGGTGGTGGTAGTGGT               GGAGGTGGATCAGGAGGAGGTGGTTCTCACGCATCAGACATTCAAATGA               CACAGAGTCCATCATCCCTTTCTGCCTCCGTGGGTGACCGGGTGACGAT               AACCTGCCAAGCTAGCCAAGACATTAGCAACTATCTGAACTGGTACCAG               CAAAAGCCTGGGAAAGCTCCGAAACTATTGATTTACGGTGCGTCGACTC               TCCAGAGTGGGGTACCTAGTCGTTTTTCCGGTTCAGGGTCGGGTACAGA               TTTTACCCTTACTATTTCCTCTCTGCAGCCAGAAGACTTTGCTACTTAT               TACTGCCAACAGACTTATTCGACTCCGATTACGTTTGGCCAGGGAACCA               AAGTCGAAATCAAAGGCAAGCCGATCCCGAACCCTCTGCTGGGATTAGA               CAGCACGTAACTAGCATAACCCCTCTCTAAACGGAGGGGTTT).            
Proteins from each of the gBlock fragments were expressed using a cell-free transcription/translation system (Cosmo Bio USA, Inc., PUREfrex2.1, Product #GFK-PF213 with DS Supplement, Prod. #GFK-PFof5).
 
     The cell-free expression samples containing V5-tagged scFvs were serially diluted in a 384-well plate. Alexa Fluor 647-labeled anti-V5 antibody was added to each well along with Eu-labeled IFNα 2a or PD-L. Plates were incubated at room temperature for 2 hours and the HTRF signal was read on an Envision (Perkin Elmer) equipped with an HTRF laser module. To examine the ability of DBA binding domains to block Interferon alpha Receptor 2 (dFNAR2) binding to IFNα, V5-tagged DBA scFvs were synthesized using the PUREfrex 2.1 in vitro translation system and serially diluted in a 384 well plate. Eu-labeled IFNα was added to each well along with IFNAR2-Fc and an APC-labeled anti-Fc antibody. Plates were incubated at room temperature for 2 hours, and the HTRF signal was read on an Envision (Perkin Elmer) as a measure of IFNα:IFNAR2 binding. Fluorescence signal values from the binding and inhibition curves is summarized in TABLE 4 and binding curves for IFNα and PD-L1 are shown in  FIG.  4    and  FIG.  5   , respectively. IFNAR2 blocking curves are shown in  FIG.  6   . DBAs having sequences of SEQ TD NO: 21-SEQ TD NO: 27 were able to bind both IFNα ( FIG.  4   ) and PD-L1 ( FIG.  5   ). SEQ TD NO: 24 blocked binding of IFNα to IFNAR2, and SEQ TD NO: 26 reduced binding of IFNα to IFNAR2 ( FIG.  6   ). 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 DBAs and Controls 
               
            
           
           
               
               
               
            
               
                 SEQ ID NO 
                 Sequence 
                 Description 
               
               
                   
               
               
                 SEQ ID NO: 21 
                 QVQLVQSGAEVKKPGASVKVSCKASGYTF 
                 DBA capable of 
               
               
                   
                 SNYYIHWVRQAPGQGLEWMGWMDSNSG 
                 binding a PD-L1 
               
               
                   
                 GTGYAQKFQGRVTMTRDTSTSTVYMELSS 
                 marker and IFNα 
               
               
                   
                 LRSEDTAVYYCAKEVFSGWYDYWGQGTL 
                 therapeutic domain 
               
               
                   
                 VTVSSASGGGGSGGGGSGGGGSHASDIQM 
                   
               
               
                   
                 TQSPSSLSASVGDRVTITCRASQSISSYLNW 
                   
               
               
                   
                 YQQKPGKAPKLLIYAASSLQSGVPSRFSGS 
                   
               
               
                   
                 GSGTDFTLTISSLQPEDFATYYCQQSYSTPY 
                   
               
               
                   
                 TFGQGTKVEIK 
                   
               
               
                   
               
               
                 SEQ ID NO: 22 
                 QVQLVQSGAEVKKPGASVKVSCKASGGTF 
                 DBA capable of 
               
               
                   
                 SSYAISWVRQAPGQGLEWVGWMDPKSGN 
                 binding a PD-L1 
               
               
                   
                 TGYAQKFQGRVTMTRDTSTSTVYMELSSL 
                 marker and IFNα 
               
               
                   
                 RSEDTAVYYCARSLFPTIFGVEVAFDIWGQ 
                 therapeutic domain 
               
               
                   
                 GTLVTVSSASGGGGSGGGGSGGGGSHASD 
                   
               
               
                   
                 IQMTQSPSSLSASVGDRVTITCQASQDINNY 
                   
               
               
                   
                 LNWYQQKPGKAPKLLIYKASSLEPGVPSRF 
                   
               
               
                   
                 SGSGSGTDFTLTISSLQPEDFATYYCQKSND 
                   
               
               
                   
                 VPFTFGQGTKVEIK 
                   
               
               
                   
               
               
                 SEQ ID NO: 23 
                 QVQLVQSGAEVKKPGASVKVSCKASGGTF 
                 DBA capable of 
               
               
                   
                 SSYYMHWVRQAPGQGLEWMGWMNPNSG 
                 binding a PD-L1 
               
               
                   
                 NTGYAQKFQGRVTMTRDTSTSTVYMELSS 
                 marker and IFNα 
               
               
                   
                 LRSEDTAVYYCARSLFPTIFGVEVAFDIWG 
                 therapeutic domain 
               
               
                   
                 QGTLVTVSSASGGGGSGGGGSGGGGSHAS 
                   
               
               
                   
                 DIQMTQSPSSLSASVGDRVTITCQASQDITN 
                   
               
               
                   
                 YLNWYQQKPGKAPKLLIYAASSLESGVPSR 
                   
               
               
                   
                 FSGSGSGTDFTLTISSLQPEDFATYYCQQSY 
                   
               
               
                   
                 SIPITFGQGTRLEIK 
                   
               
               
                   
               
               
                 SEQ ID NO: 24 
                 QVQLVQSGAEVKKPGASVKVSCKASGGTF 
                 DBA capable of 
               
               
                   
                 TGYYMHWVRQAPGQGLEWMGWVNPNSG 
                 binding a PD-L1 
               
               
                   
                 NTGYAQKFQGRVTMTRDTSTSTVYMELSS 
                 marker and IFNα 
               
               
                   
                 LRSEDTAVYYCARSLFPTIFGVEVAFDIWG 
                 therapeutic domain 
               
               
                   
                 QGTLVTVSSASGGGGSGGGGSGGGGSHAS 
                   
               
               
                   
                 DIQMTQSPSSLSASVGDRVTITCQASQDISN 
                   
               
               
                   
                 YLNWYQQKPGKAPKLLIYAASSLQSGVPS 
                   
               
               
                   
                 RFSGSGSGTDFTLTISSLQPEDFATYYCQQS 
                   
               
               
                   
                 YSPPPTFGQGTKLEIK 
                   
               
               
                   
               
               
                 SEQ ID NO: 25 
                 QVQLVQSGAEVKKPGASVKVSCKASGNTF 
                 DBA capable of 
               
               
                   
                 TDYYMHWVRQAPGQGLEWMGWMNPNS 
                 binding a PD-L1 
               
               
                   
                 GNTGYAQKFQGRVTMTRDTSTSTVYMELS 
                 marker and IFNα 
               
               
                   
                 SLRSEDTAVYYCARSLFPTIFGVEVAFDIW 
                 therapeutic domain 
               
               
                   
                 GQGTLVTVSSASGGGGSGGGGSGGGGSHA 
                   
               
               
                   
                 SDIQMTQSPSSLSASVGDRVTITCQASQDIS 
                   
               
               
                   
                 NYLNWYQQKPGKAPKLLIYAASSLQSGVP 
                   
               
               
                   
                 SRFSGSGSGTDFTLTISSLQPEDFATYYCQQ 
                   
               
               
                   
                 SYSTPPTFGQGTRLEIK 
                   
               
               
                   
               
               
                 SEQ ID NO: 26 
                 QVQLVQSGAEVKKPGASVKVSCKASGGTF 
                 DBA capable of 
               
               
                   
                 SSYAISWVRQAPGQGLEWMGIIDPSVTYTR 
                 binding a PD-L1 
               
               
                   
                 YAQKFQGRVTMTRDTSTSTVYMELSSLRS 
                 marker and IFNα 
               
               
                   
                 EDTAVYYCARSLFPTIFGVEVAFDIWGQGT 
                 therapeutic domain 
               
               
                   
                 LVTVSSASGGGGSGGGGSGGGGSHASDIQ 
                   
               
               
                   
                 MTQSPSSLSASVGDRVTITCQASQDISNYL 
                   
               
               
                   
                 NWYQQKPGKAPKLLIYGASTLQSGVPSRFS 
                   
               
               
                   
                 GSGSGTDFTLTISSLQPEDFATYYCQQTYST 
                   
               
               
                   
                 PITFGQGTKVEIK 
                   
               
               
                   
               
               
                 SEQ ID NO: 27 
                 QVQLVQSGAEVKKPGASVKVSCKASGGTF 
                 DBA capable of 
               
               
                   
                 SSYAISWVRQAPGQGLEWMGWMDANNG 
                 binding a PD-L1 
               
               
                   
                 NTGYAQKFQGRVTMTRDTSTSTVYMELSS 
                 marker and IFNα 
               
               
                   
                 LRSEDTAVYYCARSLFPTIFGVEVAFDIWG 
                 therapeutic domain 
               
               
                   
                 QGTLVTVSSASGGGGSGGGGSGGGGSHAS 
                   
               
               
                   
                 DIQMTQSPSSLSASVGDRVTITCRASQSVSS 
                   
               
               
                   
                 YLNWYQQKPGKAPKLLIYKASSLESGVPSR 
                   
               
               
                   
                 FSGSGSGTDFTLTISSLQPEDFATYYCQQSS 
                   
               
               
                   
                 STPLSFGGGTKVEIK 
                   
               
               
                   
               
               
                 SEQ ID NO: 28 
                 EVQLVESGGGLVQPGGSLRLSCAASGFNIK 
                 Anti-HER2 control 
               
               
                   
                 DTYIHWVRQAPGKGLEWVARIYPTNGYTR 
                   
               
               
                   
                 YADSVKGRFTISADTSKNTAYLQMNSLRA 
                   
               
               
                   
                 EDTAVYYCSRWGGDGFYAMDYWGQGTL 
                   
               
               
                   
                 VTVSSASGGGGSGGGGSGGGGSHASDIQM 
                   
               
               
                   
                 TQSPSSLSASVGDRVTITCRASQDVNTAVA 
                   
               
               
                   
                 WYQQKPGKAPKLLIYSASFLYSGVPSRFSG 
                   
               
               
                   
                 SRSGTDFTLTISSLQPEDFATYYCQQHYTTP 
                   
               
               
                   
                 PTFGQGTKVEIK 
                   
               
               
                   
               
               
                 SEQ ID NO: 29 
                 EVQLVESGGGLVQPGGSLRLSCAASGFTFS 
                 Anti-PD-L1 control 
               
               
                   
                 DSWIHWVRQAPGKGLEWVAWISPYGGST 
                   
               
               
                   
                 YYADSVKGRFTISADTSKNTAYLQMNSLR 
                   
               
               
                   
                 AEDTAVYYCARRHWPGGFDYWGQGTLVT 
                   
               
               
                   
                 VSSAASGGGGSGGGGSGGGGSHASDIQMT 
                   
               
               
                   
                 QSPSSLSASVGDRVTITCRASQDVSTAVAW 
                   
               
               
                   
                 YQQKPGKAPKLLIYSASFLYSGVPSRFSGS 
                   
               
               
                   
                 GSGTDFTLTISSLQPEDFATYYCQQYLYHP 
                   
               
               
                   
                 ATFGQGTKVEIK 
                   
               
               
                   
               
               
                 SEQ ID NO: 30 
                 EVQLVESGGGLVQPGGSLRLSCATSGYTFT 
                 Anti-IFNα control 
               
               
                   
                 EYIIHWVRQAPGKGLEWVASINPDYDITNY 
                   
               
               
                   
                 NQRFKGRFTISLDKSKRTAYLQMNSLRAED 
                   
               
               
                   
                 TAVYYCASWISDFFDYWGQGTLVTVSSAS 
                   
               
               
                   
                 GGGGSGGGGSGGGGSHASDIQMTQSPSSL 
                   
               
               
                   
                 SASVGDRVTITCRASQSVSTSSYSYMHWY 
                   
               
               
                   
                 QQKPGKAPKVLISYASNLESGVPSRFSGSG 
                   
               
               
                   
                 SGTDFTLTISSLQPEDFATYYCQHSWGIPRT 
                   
               
               
                   
                 FGQGTKVEIK 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Fluorescence Signal Values from Binding and Inhibition Curves 
               
            
           
           
               
               
               
               
            
               
                   
                 PDL1 
                 IFNα 
                 IFNAR2- 
               
               
                 Name 
                 Binding 
                 Binding 
                 IFN binding 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 PDL1-IFN-R01-A03 
                 2,715 
                 20,447 
                 42,528 
               
               
                 PDL1-IFN-R01-A04 
                 2,377 
                 10,890 
                 45,590 
               
               
                 PDL1-IFN-R01-A05 (SEQ ID NO: 21) 
                 3,113 
                 7,115 
                 50,505 
               
               
                 PDL1-IFN-R01-A06 (SEQ ID NO: 22) 
                 2,781 
                 8,563 
                 44,019 
               
               
                 PDL1-IFN-R01-A07 
                 3,010 
                 4,496 
                 52,343 
               
               
                 PDL1-IFN-R01-A08 
                 2,274 
                 19,905 
                 40,314 
               
               
                 PDL1-IFN-R01-A09 
                 1,628 
                 6,585 
                 50,070 
               
               
                 PDL1-IFN-R01-A09V2 
                 1,894 
                 5,264 
                 49,709 
               
               
                 PDL1-IFN-R01-A10 
                 1,969 
                 6,105 
                 48,781 
               
               
                 PDL1-IFN-R01-A11 (SEQ ID NO: 23) 
                 2,965 
                 14,613 
                 41,796 
               
               
                 PDL1-IFN-R01-A12 
                 2,133 
                 9,478 
                 53,273 
               
               
                 PDL1-IFN-R01-B01 
                 3,172 
                 7,460 
                 44,230 
               
               
                 PDL1-IFN-R01-B02 
                 2,695 
                 7,190 
                 42,135 
               
               
                 PDL1-IFN-R01-B03 
                 2,383 
                 4,072 
                 44,076 
               
               
                 PDL1-IFN-R01-B04 
                 2,518 
                 12,736 
                 42,948 
               
               
                 PDL1-IFN-R01-B04V2 
                 2,703 
                 12,952 
                 43,748 
               
               
                 PDL1-IFN-R01-B05 
                 2,074 
                 3,480 
                 50,563 
               
               
                 PDL1-IFN-R01-B06 
                 3,084 
                 17,212 
                 43,958 
               
               
                 PDL1-IFN-R01-B07 
                 2,897 
                 6,271 
                 40,647 
               
               
                 PDL1-IFN-R01-B07V2 
                 2,907 
                 6,111 
                 42,880 
               
               
                 PDL1-IFN-R01-B08 
                 2,924 
                 4,042 
                 46,089 
               
               
                 PDL1-IFN-R01-B09 (SEQ ID NO: 24) 
                 1,378 
                 35,717 
                 31,232 
               
               
                 PDL1-IFN-R01-B10 
                 899 
                 4,118 
                 46,508 
               
               
                 PDL1-IFN-R01-B11 
                 2,525 
                 14,580 
                 43,204 
               
               
                 PDL1-IFN-R01-B12 (SEQ ID NO: 25) 
                 2,977 
                 9,230 
                 44,505 
               
               
                 PDL1-IFN-R01-C01 
                 2,780 
                 6,975 
                 47,032 
               
               
                 PDL1-IFN-R01-C02 
                 2,923 
                 4,123 
                 49,398 
               
               
                 PDL1-IFN-R01-C03 
                 2,671 
                 6,522 
                 46,323 
               
               
                 PDLI-IFN-R01-C04 
                 2,917 
                 7,761 
                 48,699 
               
               
                 PDL1-IFN-R01-C05 
                 2,802 
                 3,427 
                 47,470 
               
               
                 PDL1-IFN-R01-C06 
                 399 
                 4,380 
                 49,878 
               
               
                 PDL1-IFN-R01-C06V2 
                 391 
                 4,130 
                 47,685 
               
               
                 PDL1-IFN-R01-C07 
                 2,139 
                 24,494 
                 43,262 
               
               
                 PDL1-IFN-R01-C08 (SEQ ID NO: 26) 
                 2,746 
                 45,175 
                 14,739 
               
               
                 PDL1-IFN-R01-C08V2 
                 2,812 
                 55,319 
                 16,753 
               
               
                 PDL1-IFN-R01-C09 
                 2,388 
                 4,346 
                 48,696 
               
               
                 PDL1-IFN-R01-C10 
                 2,489 
                 12,727 
                 43,275 
               
               
                 PDL1-IFN-R01-C10V2 
                 2,665 
                 11,972 
                 43,576 
               
               
                 PDL1-IFN-R01-C11 (SEQ ID NO: 27) 
                 3,043 
                 7,730 
                 40,503 
               
               
                 PDL1-IFN-R01-C12 
                 1,006 
                 13,135 
                 44,871 
               
               
                 PDL1-IFN-R01-D01 
                 582 
                 7,018 
                 39,109 
               
               
                 PDL1-IFN-R01-D03 
                 2,904 
                 6,084 
                 42,638 
               
               
                 PDL1-IFN-R01-D05 
                 2,325 
                 6,402 
                 49,246 
               
               
                 PDL1-IFN-R01-D06 
                 2,907 
                 8,326 
                 45,581 
               
               
                 PDL1-IFN-R01-D07 
                 414 
                 4,974 
                 38,164 
               
               
                 anti-Her2 control (SEQ ID NO: 28) 
                 425 
                 4,275 
                 52,922 
               
               
                 Anti-PD-Ll control (SEQ ID NO: 29) 
                 2,445 
                 1,803 
                 39,121 
               
               
                 Anti-IFN control (SEQ ID NO: 30) 
                 416 
                 19,098 
                 51,190 
               
               
                 No DNA control 
                 422 
                 4,135 
                 45,042 
               
               
                   
               
            
           
         
       
     
     Example 2 
     Isolation of a Set of Dual-Binding Antibodies (DBAs) that Bind Human PD-1 and Human IL-2 
     This example describes the isolation of sensor domains of the present disclosure, specifically, a set of DBAs that bind human PD-1 and human IL-2. Anti-PD-1 and anti-IL-2 DBAs were isolated from a Tumbler antibody phage display library (Distributed Bio, Inc.). The antibody phage display library was constructed to incorporate the heavy chain CDR1, heavy chain CDR2, and light chain diversity of the Superhuman 2.0 antibody library combined with 10 heavy chain CDR3 sequences from PD-1 binding antibodies (SEQ ID NO: 11-SEQ ID NO: 20). 
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 HC-CDR3 of PD-1 binders 
               
            
           
           
               
               
               
            
               
                 SEQ ID NO 
                 Sequence 
                 Description 
               
               
                   
               
               
                 SEQ ID NO: 11 
                 CAAGLFIW 
                 HC-CDR3 of PD-1 binder 
               
               
                   
               
               
                 SEQ ID NO: 12 
                 CAGGWLDW 
                 HC-CDR3 of PD-1 binder 
               
               
                   
               
               
                 SEQ ID NO: 13 
                 CARDHLGGSYQPW 
                 HC-CDR3 of PD-1 binder 
               
               
                   
               
               
                 SEQ ID NO: 14 
                 CARDLVGVSPGINYVPRYYYY 
                 HC-CDR3 of PD-1 binder 
               
               
                   
                 YYGMDVW 
                   
               
               
                   
               
               
                 SEQ ID NO: 15 
                 CARDTGLGYYYGSGDFDYW 
                 HC-CDR3 of PD-1 binder 
               
               
                   
               
               
                 SEQ ID NO: 16 
                 CARSGYSYGYYFDYW 
                 HC-CDR3 of PD-1 binder 
               
               
                   
               
               
                 SEQ ID NO: 17 
                 CARTGGYPAIDSW 
                 HC-CDR3 of PD-1 binder 
               
               
                   
               
               
                 SEQ ID NO: 18 
                 CASGWDVW 
                 HC-CDR3 of PD-1 binder 
               
               
                   
               
               
                 SEQ ID NO: 19 
                 CASSPLQWVDVW 
                 HC-CDR3 of PD-1 binder 
               
               
                   
               
               
                 SEQ ID NO: 20 
                 CTSGMDVW 
                 HC-CDR3 of PD-1 binder 
               
               
                   
               
            
           
         
       
     
     This library was subjected to four rounds of selection with standard protocols. In brief, the phage library was incubated with the antigen, then captured on magnetic beads and washed on a Kingfisher magnetic particle processor, eluted form the magnetic beads and amplified by passaging in  E. coli . Round 1 was incubated with 50 nM human PD-1-His fusion (R&amp;D Systems, Prod. Num. 8986-PD) and captured with TRIS NTA Biotin (Sigma-Aldrich Prod. Num. 75543) and streptavidin magnetic beads. Round 2 was incubated with 100 nM biotinylated IL-2 (Creative Biomart, Prod. Num. IL2-501H, biotinylated using standard protocols) and captured on streptavidin magnetic beads. Round 3 was incubated with 50 nM cynomolgus PD-1-Fc fusion (R&amp;D Systems, Prod. Num. 8578-PD) and captured on protein G magnetic beads. Round 4 was incubated with 50 nM biotinylated human IL-2 and captured on streptavidin magnetic beads. The final selection was plated as single colonies and 380 colonies picked for Sanger sequencing. One hundred and fifty-one unique clones were chosen for expression. The scFv sequence for each clone was codon-optimized for  E. coli  expression and the corresponding DNA sequences sent to Integrated DNA Technologies, Inc. (IDT) for synthesis as gBlocks with a T7 promoter, a translation initiation site and a T7 terminator (see an exemplary gBlock sequence in  FIG.  3   ). Protein from each gBlock encoding an scFv was expressed using the PURExpress In vitro Protein Synthesis Kit (New England Biolabs, Inc., Prod. Num. E6800). The PURExpress scFv proteins were used directly in HTRF binding assays and cell-based functional assays. Each scFv was tested for binding to PD-1 and to human IL-2. Eighty-one of the antibodies showed dual-binding activity for both PD-1 and IL-2 and a summary of fluorescence signal values of binding curves is shown in TABLE 7. To examine the ability of DBA binding domains to block IL-2 receptor binding, V5-tagged DBA scFvs were serially diluted in a 384 well plate. Europium-labeled Streptavidin, biotin-labeled IL-2 (Acro Biosystems, Prod. Num. IL2-H82E4), IL-2 Receptor beta (Fc-IL2RB) (Acro Biosystems, Prod. Num. ILB-H5253), and APC-labeled anti-Fc antibody. Plates were incubated at room temperature for 2 hours, and the HTRF signal was read on an Envision (Perkin Elmer) as a measure of IL-2:IL2RB binding. Four scFvs (SEQ TD NO: 31-SEQ TD NO: 34) bound PD-1, bound IL-2 and blocked binding of IL-2 to IL-2RB (TABLE 7). 
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 DBAs and Controls 
               
            
           
           
               
               
               
            
               
                 SEQ ID NO 
                 Sequence 
                 Description 
               
               
                   
               
               
                 SEQ ID NO: 31 
                 QVQLVQSGAEVKKPGVSVKVSCKASGYTF 
                 DBA capable of 
               
               
                   
                 PRSYIHWVRQAPGQGLEWMGWINPHSGDT 
                 binding a PD-1 marker 
               
               
                   
                 YYAQNFQGRVTMTRDTSTSTVYMELSSLR 
                 and IL-2 therapeutic 
               
               
                   
                 SEDTAVYYCARDTGLGYYYGSGDFDYWG 
                   
               
               
                   
                 QGTLVTVSSASGGGGSGGGGSGGGGSHAS 
                   
               
               
                   
                 DIQMTQSPSSLSASVGDRVTITCRASQSISR 
                   
               
               
                   
                 YLNWYQQKPGKAPKLLIYTASSLQSGVPSR 
                   
               
               
                   
                 FSGSGSGTDFTLTISSLQPEDFATYYCQQAN 
                   
               
               
                   
                 RFPLTFGPGTKVDIK 
                   
               
               
                   
               
               
                 SEQ ID NO: 32 
                 QVQLVQSGAEVKKPGASVKVSCKASGYTF 
                 DBA capable of 
               
               
                   
                 PRYHIHWVRQAPGQGLEWMGMINPSGGTT 
                 binding a PD-1 marker 
               
               
                   
                 TYAQKFQGRVTMTRDTSTSTVYMELSSLR 
                 and IL-2 therapeutic 
               
               
                   
                 SEDTAVYYCARDTGLGYYYGSGDFDYWG 
                   
               
               
                   
                 QGTLVTVSSASGGGGSGGGGSGGGGSHAS 
                   
               
               
                   
                 DIQMTQSPSSLSASVGDRVTITCRASQSISS 
                   
               
               
                   
                 WLAWYQQKPGKAPKLLIYAASSLQSGVPS 
                   
               
               
                   
                 RFSGSGSGTDFTLTISSLQPEDFATYYCQQS 
                   
               
               
                   
                 HSFPLTFGGGTKVEIK 
                   
               
               
                   
               
               
                 SEQ ID NO: 33 
                 QVQLVQSGAEVKKPGASVKVSCKASGYTF 
                 DBA capable of 
               
               
                   
                 TRYYIHWVRQAPGQGLEWMGWINAYNGD 
                 binding a PD-1 marker 
               
               
                   
                 TNYAQKLQGRVTMTRDTSTSTVYMELSSL 
                 and IL-2 therapeutic 
               
               
                   
                 RSEDTAVYYCARDSYYYDSFDYWGQGTL 
                   
               
               
                   
                 VTVSSASGGGGSGGGGSGGGGSHASDIQM 
                   
               
               
                   
                 TQSPSSLSASVGDRVTITCRASQTITDWLA 
                   
               
               
                   
                 WYQQKPGKAPKLLIYGASNLQGGVPSRFS 
                   
               
               
                   
                 GSGSGTDFTLTISSLQPEDFATYYCQQYYSS 
                   
               
               
                   
                 WTFGQGTKVEIK 
                   
               
               
                   
               
               
                 SEQ ID NO: 34 
                 QVQLVQSGAEVKKPGASVKVSCKASGYTF 
                 DBA capable of 
               
               
                   
                 TSYYMHWVRQAPGQGLEWMGIINPSDGST 
                 binding a PD-1 marker 
               
               
                   
                 TYAQSFQGRVTMTRDTSTSTVYMELSSLRS 
                 and IL-2 therapeutic 
               
               
                   
                 EDTAVYYCASGWDVWGQGTLVTVSSASG 
                   
               
               
                   
                 GGGSGGGGSGGGGSHASDIVMTQSPDSLA 
                   
               
               
                   
                 VSLGERATINCKSSQSVFSSANNKNYLAW 
                   
               
               
                   
                 YQQKPGQPPKLLIYWASTRESGVPDRF SGS 
                   
               
               
                   
                 GSGTDFTLTISSLQAEDVAVYYCQQYFGTP 
                   
               
               
                   
                 VTFGGGTKVEIK 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
               
             
               
                 TABLE 7 
               
               
                   
               
               
                 Name 
                 PD1 Binding 
                 IL2 Binding 
                 IL2RB Blocking 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
            
               
                 No DNA 
                 5 
                 6 
                 1,490 
               
               
                 PD1-IL2-R01-H08 
                 576 
                 −9 
                 1,829 
               
               
                 PD1-IL2-R01-H09 
                 1,015 
                 131 
                 1,772 
               
               
                 PD1-IL2-R02-A03 
                 1,508 
                 635 
                 1,714 
               
               
                 PD1-IL2-R02-A04 
                 909 
                 978 
                 1,618 
               
               
                 PD1-IL2-R02-A05 
                 1,557 
                 23 
                 1,735 
               
               
                 PD1-IL2-R02-A06 
                 357 
                 515 
                 1,772 
               
               
                 PD1-IL2-R02-A08 
                 995 
                 520 
                 1,612 
               
               
                 PD1-IL2-R02-A09 
                 1,421 
                 1,470 
                 1,495 
               
               
                 PD1-IL2-R02-A10 
                 500 
                 838 
                 1,847 
               
               
                 PD1-IL2-R02-A11 
                 1,625 
                 1,559 
                 1,783 
               
               
                 PD1-IL2-R02-A12 
                 1,725 
                 130 
                 1,586 
               
               
                 PD1-IL2-R02-B01 
                 746 
                 1,077 
                 1,516 
               
               
                 PD1-IL2-R02-B02 
                 1,740 
                 1,107 
                 1,849 
               
               
                 PD1-IL2-R02-B04 
                 11 
                 2,346 
                 1,536 
               
               
                 PD1-IL2-R02-B05 
                 1,665 
                 2,489 
                 1,613 
               
               
                 PD1-IL2-R02-B06 
                 1,527 
                 32 
                 1,605 
               
               
                 PD1-IL2-R02-B07 
                 1,685 
                 628 
                 1,814 
               
               
                 PD1-IL2-R02-B08 
                 1,446 
                 92 
                 1,680 
               
               
                 PD1-IL2-R02-B10 
                 211 
                 343 
                 1,607 
               
               
                 PD1-IL2-R02-B11 
                 1,426 
                 915 
                 1,509 
               
               
                 PD1-IL2-R02-B12 
                 1,264 
                 316 
                 1,762 
               
               
                 PD1-IL2-R02-C01 
                 1,463 
                 296 
                 1,743 
               
               
                 PD1-IL2-R02-C02 
                 1,299 
                 298 
                 1,069 
               
               
                 (SEQ ID NO: 31) 
                   
                   
                   
               
               
                 PD1-IL2-R02-C03 
                 1,383 
                 293 
                 1,211 
               
               
                 (SEQ ID NO: 32) 
                   
                   
                   
               
               
                 PD1-IL2-R02-C04 
                 1,622 
                 575 
                 1,857 
               
               
                 PD1-IL2-R02-C06 
                 1,376 
                 34 
                 1,684 
               
               
                 PD1-IL2-R02-C07 
                 34 
                 87 
                 1,607 
               
               
                 PD1-IL2-R02-C08 
                 1,468 
                 619 
                 1,671 
               
               
                 PD1-IL2-R02-C10 
                 174 
                 256 
                 1,757 
               
               
                 PD1-IL2-R02-C12 
                 1,367 
                 340 
                 1,723 
               
               
                 PD1-IL2-R02-D01 
                 1,421 
                 68 
                 1,614 
               
               
                 PD1-IL2-R02-D02 
                 1,473 
                 539 
                 1,726 
               
               
                 PD1-IL2-R06-A10 
                 1,269 
                 9 
                 1,796 
               
               
                 PD1-IL2-R06-A11 
                 1,376 
                 34 
                 1,762 
               
               
                 PD1-IL2-R06-A12 
                 1,305 
                 7 
                 1,681 
               
               
                 PD1-IL2-R06-B01 
                 10 
                 2,109 
                 1,307 
               
               
                 PD1-IL2-R06-B02 
                 1,666 
                 15 
                 1,799 
               
               
                 PD1-IL2-R06-B03 
                 923 
                 4 
                 1,661 
               
               
                 PD1-IL2-R06-B04 
                 1,782 
                 28 
                 1,666 
               
               
                 PD1-IL2-R06-B06 
                 1,223 
                 17 
                 1,648 
               
               
                 PD1-IL2-R06-B08 
                 1,777 
                 1,160 
                 1,738 
               
               
                 PD1-IL2-R06-B10 
                 13 
                 31 
                 1,847 
               
               
                 PD1-IL2-R06-B11 
                 1,534 
                 24 
                 1,699 
               
               
                 PD1-IL2-R06-B12 
                 822 
                 1,125 
                 1,604 
               
               
                 PD1-IL2-R06-C02 
                 1,667 
                 26 
                 1,671 
               
               
                 PD1-IL2-R06-C04 
                 1,491 
                 7 
                 1,759 
               
               
                 PD1-IL2-R06-C08 
                 1,448 
                 8 
                 1,693 
               
               
                 PD1-IL2-R06-C09 
                 1,158 
                 1,525 
                 1,602 
               
               
                 PD1-IL2-R06-C11 
                 1,879 
                 −2 
                 1,785 
               
               
                 PD1-IL2-R06-C12 
                 1,669 
                 1,998 
                 1,033 
               
               
                 PD1-IL2-R06-D02 
                 280 
                 432 
                 1,677 
               
               
                 PD1-IL2-R06-D03 
                 9 
                 93 
                 1,606 
               
               
                 PD1-IL2-R06-D05 
                 505 
                 −3 
                 1,786 
               
               
                 PD1-IL2-R06-D07 
                 1,577 
                 24 
                 1,820 
               
               
                 PD1-IL2-R06-D10 
                 1,751 
                 49 
                 1,719 
               
               
                 PD1-IL2-R06-D11 
                 405 
                 593 
                 1,576 
               
               
                 PD1-IL2-R06-D12 
                 1,024 
                 1,423 
                 1,649 
               
               
                 PD1-IL2-R06-E01 
                 1,628 
                 3 
                 1,724 
               
               
                 PD1-IL2-R06-E02 
                 1,554 
                 16 
                 1,598 
               
               
                 PD1-IL2-R06-E04 
                 50 
                 247 
                 1,108 
               
               
                 (SEQ ID NO: 33) 
                   
                   
                   
               
               
                 PD1-IL2-R06-E05 
                 1,364 
                 14 
                 1,734 
               
               
                 PD1-IL2-R06-E06 
                 1,627 
                 15 
                 1,735 
               
               
                 PD1-IL2-R06-E07 
                 1,801 
                 12 
                 1,698 
               
               
                 PD1-IL2-R06-E09 
                 1,467 
                 11 
                 1,511 
               
               
                 PD1-IL2-R06-E11 
                 1,805 
                 294 
                 1,767 
               
               
                 PD1-IL2-R06-E12 
                 4 
                 −7 
                 1,735 
               
               
                 PD1-IL2-R06-F01 
                 196 
                 280 
                 1,629 
               
               
                 PD1-IL2-R06-F03 
                 1,377 
                 28 
                 1,642 
               
               
                 PD1-IL2-R06-F04 
                 26 
                 779 
                 1,726 
               
               
                 PD1-IL2-R06-F05 
                 1,493 
                 18 
                 1,625 
               
               
                 PD1-IL2-R06-F06 
                 1,577 
                 46 
                 1,595 
               
               
                 PD1-IL2-R06-F07 
                 1,544 
                 335 
                 1,682 
               
               
                 PD1-IL2-R06-F08 
                 1,570 
                 9 
                 1,780 
               
               
                 PD1-IL2-R06-F09 
                 30 
                 41 
                 1,776 
               
               
                 PD1-IL2-R06-F10 
                 1,745 
                 24 
                 1,607 
               
               
                 PD1-IL2-R06-F11 
                 1,586 
                 12 
                 1,574 
               
               
                 PD1-IL2-R06-F12 
                 623 
                 8 
                 1,645 
               
               
                 PD1-IL2-R06-G01 
                 130 
                 184 
                 1,640 
               
               
                 PD1-IL2-R06-G02 
                 1,754 
                 20 
                 1,623 
               
               
                 PD1-IL2-R06-G04 
                 1,348 
                 13 
                 1,596 
               
               
                 PD1-IL2-R06-G05 
                 1,382 
                 10 
                 1,846 
               
               
                 PD1-IL2-R06-G06 
                 1,383 
                 4 
                 1,744 
               
               
                 PD1-IL2-R06-G08 
                 1,708 
                 124 
                 1,533 
               
               
                 PD1-IL2-R06-G09 
                 557 
                 756 
                 1,527 
               
               
                 PD1-IL2-R06-G10 
                 1,595 
                 35 
                 1,703 
               
               
                 PD1-IL2-R06-G11 
                 1,469 
                 17 
                 1,709 
               
               
                 PD1-IL2-R06-G12 
                 1,281 
                 1,479 
                 1,713 
               
               
                 PD1-IL2-R06-H01 
                 381 
                 4 
                 1,647 
               
               
                 PD1-IL2-R06-H02 
                 1,501 
                 20 
                 1,748 
               
               
                 PD1-IL2-R06-H03 
                 1,132 
                 1,449 
                 1,617 
               
               
                 PD1-IL2-R06-H04 
                 355 
                 1 
                 1,677 
               
               
                 PD1-IL2-R06-H05 
                 1,409 
                 21 
                 1,561 
               
               
                 PD1-IL2-R06-H06 
                 1,491 
                 23 
                 1,650 
               
               
                 PD1-IL2-R06-H07 
                 12 
                 13 
                 1,701 
               
               
                 PD1-IL2-R06-H08 
                 847 
                 1,118 
                 1,746 
               
               
                 PD1-IL2-R06-H09 
                 1,732 
                 22 
                 1,662 
               
               
                 PD1-IL2-R06-H10 
                 830 
                 1,151 
                 1,569 
               
               
                 PD1-IL2-R07-A03 
                 1,786 
                 28 
                 1,511 
               
               
                 PD1-IL2-R07-A04 
                 730 
                 973 
                 1,613 
               
               
                 PD1-IL2-R07-A05 
                 477 
                 663 
                 1,327 
               
               
                 PD1-IL2-R07-A08 
                 1,628 
                 841 
                 1,618 
               
               
                 PD1-IL2-R07-A09 
                 1,235 
                 2,040 
                 910 
               
               
                 (SEQ ID NO: 34) 
                   
                   
                   
               
               
                 PD1-IL2-R07-A10 
                 1,716 
                 63 
                 1,518 
               
               
                 PD1-IL2-R07-B01 
                 1,397 
                 32 
                 1,565 
               
               
                 PD1-IL2-R07-B02 
                 192 
                 321 
                 1,634 
               
               
                 PD1-IL2-R07-B03 
                 65 
                 202 
                 1,604 
               
               
                 PD1-IL2-R07-B04 
                 1,862 
                 410 
                 1,527 
               
               
                 PD1-IL2-R07-B05 
                 965 
                 351 
                 1,389 
               
               
                 PD1-IL2-R07-B06 
                 1,882 
                 44 
                 1,497 
               
               
                 PD1-IL2-R07-B07 
                 6 
                 2,549 
                 1,517 
               
               
                 PD1-IL2-R07-B08 
                 906 
                 1,047 
                 1,475 
               
               
                 PD1-IL2-R07-B09 
                 1,788 
                 27 
                 1,384 
               
               
                 PD1-IL2-R07-B10 
                 18 
                 19 
                 1,635 
               
               
                 PD1-IL2-R07-B11 
                 1,765 
                 9 
                 1,641 
               
               
                 PD1-IL2-R07-C01 
                 230 
                 367 
                 1,536 
               
               
                 PD1-IL2-R07-C02 
                 236 
                 304 
                 1,500 
               
               
                 PD1-IL2-R07-C03 
                 20 
                 1,347 
                 1,536 
               
               
                 PD1-IL2-R07-C07 
                 15 
                 275 
                 1,665 
               
               
                 PD1-IL2-R07-C10 
                 1,064 
                 317 
                 1,550 
               
               
                 PD1-IL2-R07-C11 
                 1,523 
                 642 
                 1,460 
               
               
                 PD1-IL2-R07-C12 
                 1,377 
                 49 
                 1,707 
               
               
                 PD1-IL2-R07-D01 
                 1,541 
                 79 
                 1,657 
               
               
                 PD1-IL2-R07-D03 
                 1,483 
                 33 
                 1,481 
               
               
                 PD1-IL2-R07-D04 
                 923 
                 1,104 
                 1,517 
               
               
                 PD1-IL2-R07-D06 
                 1,664 
                 416 
                 1,734 
               
               
                 PD1-IL2-R07-D07 
                 6 
                 835 
                 1,512 
               
               
                 PD1-IL2-R07-D10 
                 1,580 
                 193 
                 1,572 
               
               
                 PD1-IL2-R07-D11 
                 1,401 
                 798 
                 1,614 
               
               
                 PD1-IL2-R07-E02 
                 1,473 
                 992 
                 1,830 
               
               
                 PD1-IL2-R07-E03 
                 1,459 
                 422 
                 1,683 
               
               
                 PD1-IL2-R07-E05 
                 512 
                 913 
                 1,513 
               
               
                 PD1-IL2-R07-E06 
                 1,483 
                 1,178 
                 1,526 
               
               
                 PD1-IL2-R07-E07 
                 1,181 
                 1,060 
                 1,524 
               
               
                 PD1-IL2-R07-E08 
                 1,604 
                 472 
                 1,717 
               
               
                 PD1-IL2-R07-E09 
                 1,733 
                 23 
                 1,569 
               
               
                 PD1-IL2-R07-E10 
                 1,472 
                 251 
                 1,545 
               
               
                 PD1-IL2-R07-E11 
                 1,146 
                 56 
                 1,777 
               
               
                 PD1-IL2-R07-E12 
                 1,698 
                 106 
                 1,764 
               
               
                 PD1-IL2-R07-F01 
                 3 
                 17 
                 1,529 
               
               
                 PD1-IL2-R07-F02 
                 348 
                 752 
                 1,537 
               
               
                 PD1-IL2-R07-F03 
                 1,788 
                 520 
                 1,750 
               
               
                 PD1-IL2-R07-F04 
                 1,416 
                 145 
                 1,767 
               
               
                 PD1-IL2-R07-F06 
                 1,422 
                 438 
                 1,579 
               
               
                 PD1-IL2-R07-F09 
                 1,589 
                 17 
                 1,456 
               
               
                 PD1-IL2-R07-F10 
                 24 
                 19 
                 1,778 
               
               
                 PD1-IL2-R07-F12 
                 505 
                 196 
                 1,553 
               
               
                 PD1-IL2-R07-G01 
                 4 
                 214 
                 1,560 
               
               
                 PD1-IL2-R07-G02 
                 1,610 
                 61 
                 1,735 
               
               
                 PD1-IL2-R07-G04 
                 82 
                 147 
                 1,600 
               
               
                 PD1-IL2-R07-G05 
                 981 
                 216 
                 1,475 
               
               
                 PD1-IL2-R07-G06 
                 860 
                 512 
                 1,655 
               
               
                 PD1-R04-C10 
                 1,552 
                 4 
                 1,550 
               
               
                 PD1-R07-A05 
                 653 
                 19 
                 1,730 
               
               
                 PD1-R07-A10 
                 484 
                 25 
                 2,290 
               
               
                 PD1-R07-C09 
                 1,911 
                 20 
                 2,080 
               
               
                 PD1-R07-D03 
                 1,733 
                 22 
                 2,208 
               
               
                 PD1-R07-D05 
                 1,760 
                 16 
                 1,578 
               
               
                 PD1-R07-D06 
                 1,997 
                 22 
                 1,749 
               
               
                 PD1-R07-E05 
                 633 
                 24 
                 2,246 
               
               
                 PD1-R07-G12 
                 907 
                 11 
                 1,577 
               
               
                 PD1-R15-B02 
                 1,671 
                 28 
                 1,797 
               
               
                 PDL1-DB03-H02 
                 18 
                 11 
                 1,725 
               
               
                 Anti-Her2 (SEQ ID 
                 4 
                 20 
                 1,636 
               
               
                 NO: 28) 
               
               
                   
               
            
           
         
       
     
     Example 3 
     Competitive Binding for Targets of Dual Binding Antibodies (DBAs) 
     This example describes competitive binding between the marker and the therapeutic domain of dual binding antibodies (DBAs). To test the ability of PD-L1 (marker) to compete with IFNα (therapeutic) for binding to the DBA binding domains, V5-tagged DBA or control scFvs were synthesized using the PUREfrex 2.1 in vitro translation system and added to a 384 well plate at a single dilution. Eu-labeled PD-L1 and Alexa Fluor 647-labeled anti-V5 antibody were added to all wells and incubated for 30 minutes at room temperature. Titrated concentrations of IFNα were added to all wells and the plate was incubated for 1 hour at room temperature. The HTRF signal was read on Envision (Perkin Elmer). As shown in  FIG.  7   , IFNα competed with PD-L1 for binding to DBA clones B09 (SEQ ID NO: 24) and C08 (SEQ ID NO: 26), whereas the binding of an anti-PD-L1 mono-specific antibody was unaffected by the concentration of IFNα. 
     Example 4 
     Screening Dual Binding Antibodies (DBAs) for Improved Binding Affinity 
     This example illustrates screening dual binding antibodies (DBAs) for improved binding affinity. The sequences for each DBA were aligned with the parental, single specificity antibody from which it was derived and with other DBAs derived from the same parental, single specificity antibody. Using this sequence information, variants with amino acid substitutions in and adjacent to the CDR regions were designed to test for altered binding to either antigen. Additionally, consideration was given to sequence variants that may improve stability. Representative variants are shown in TABLE 8 and TABLE 9. CDR sequences provided in TABLE 8 and TABLE 9 correspond to HCDR1-HCDR2-HCDR3, with substitutions underlined and in bold. Sequences of dual binding PD-L1 and IFNα variants are provided in TABLE 10. 
     
       
         
           
               
             
               
                 TABLE 8 
               
             
            
               
                   
               
               
                 Heavy chain CDR regions of anti-PDL1 02_A08, DBA PDL1-IFN_1A05, and 
               
               
                 variants 
               
            
           
           
               
               
               
            
               
                 SEQ ID NO 
                 Description 
                 CDR Sequence 
               
               
                   
               
               
                 SEQ ID NO: 305 
                 Parental monospecific antibody 
                 CKASGYTFSGYYMHW- 
               
               
                   
                 PDL1_02_A08 
                 WMGWMDPNSGYTGYAHQFQGRV- 
               
               
                   
                   
                 CAKEVFSGWYDYWGQ 
               
               
                   
               
               
                 SEQ ID NO: 306 
                 Dual-binding antibody (DBA) 
                 CKASGYTFSNYYIHW- 
               
               
                   
                 PDL1-IFN R01 A05 
                 WMGWMDSNSGGTGYAQKFQGRV- 
               
               
                   
                   
                 CAKEVFSGWYDYWGQ 
               
               
                   
               
               
                 SEQ ID NO: 307 
                 DBA variant H_N36G 
                 CKASGYTFS   G   YYIHW- 
               
               
                   
                   
                 WMGWMDSNSGGTGYAQKFQGRV- 
               
               
                   
                   
                 CAKEVFSGWYDYWGQ 
               
               
                   
               
               
                 SEQ ID NO: 308 
                 DBA variant 
                 CKASGYTFSNYY   V   HW- 
               
               
                   
                 H_I39V_S58P_Q69H_K70Q 
                 WMGWMD   P   NSGGTGYA   HQ   FOGRV- 
               
               
                   
                   
                 CAKEVFSGWYDYWGQ 
               
               
                   
               
               
                 SEQ ID NO: 309 
                 DBA variant H_G64Y_Q69H 
                 CKASGYTFSNYYIHW- 
               
               
                   
                   
                 WMGWMDSNSG   Y   TGYA   H   KFQGRV- 
               
               
                   
                   
                 CAKEVFSGWYDYWGQ 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 9 
               
             
            
               
                   
               
               
                 Light chain CDR regions of anti-PDL1 02_A08, DBA PDL1-IFN_1A05, and 
               
               
                 variants 
               
            
           
           
               
               
               
            
               
                 SEQ ID NO 
                 Construct 
                 CDR Sequence 
               
               
                   
               
               
                 SEQ ID NO: 310 
                 Parental monospecific antibody 
                 CRASQTISSYLNWY- 
               
               
                   
                 PDL1 02 A08 
                 IYAASTLESGVPSR- 
               
               
                   
                   
                 YYCQQGYSTPITFGPGTKVDIK 
               
               
                   
               
               
                 SEQ ID NO: 311 
                 Dual-binding antibody (DBA) 
                 CRASQSISSYLNWY- 
               
               
                   
                 PDL1-IFN_R01_A05 
                 IYAASSLQSGVPSR- 
               
               
                   
                   
                 YYCQQSYSTPYTFGQGTKVEIK 
               
               
                   
               
               
                 SEQ ID NO: 312 
                 L_Q68E 
                 CRASQSISSYLNWY- 
               
               
                   
                   
                 IYAASSLESGVPSR- 
               
               
                   
                   
                 YYCQQSYSTPYTFGQGTKVEIK 
               
               
                   
               
               
                 SEQ ID NO: 313 
                 L_Q68E_E125D 
                 CRASQSISSYLNWY- 
               
               
                   
                   
                 IYAASSL   E   SGVPSR- 
               
               
                   
                   
                 YYCQQSYSTPYTFGQGTKV   D   IK 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 10 
               
             
            
               
                   
               
               
                 Sequences of dual binding PD-L1 and IFNα variants 
               
            
           
           
               
               
               
            
               
                 SEQ ID NO 
                 Variant 
                 Sequence 
               
               
                   
               
               
                 SEQ ID NO: 35 
                 H_N36G 
                 QVQLVQSGAEVKKPGASVKVSCKASGYTFSGYYIHW 
               
               
                   
                   
                 VRQAPGQGLEWMGWMDSNSGGTGYAQKFQGRVTMT 
               
               
                   
                   
                 RDTSTSTVYMELSSLRSEDTAVYYCAKEVFSGWYDY 
               
               
                   
                   
                 WGQGTLVTVSSASGGGGSGGGGSGGGGSHASDIQMT 
               
               
                   
                   
                 QSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKA 
               
               
                   
                   
                 PKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDF 
               
               
                   
                   
                 ATYYCQQSYSTPYTFGQGTKVEIK 
               
               
                   
               
               
                 SEQ ID NO: 36 
                 H_I39V_ 
                 QVQLVQSGAEVKKPGASVKVSCKASGYTFSNYYVHW 
               
               
                   
                 S58P_ 
                 VRQAPGQGLEWMGWMDPNSGGTGYAHQFQGRVTMT 
               
               
                   
                 Q69H_K70Q 
                 RDTSTSTVYMELSSLRSEDTAVYYCAKEVFSGWYDY 
               
               
                   
                   
                 WGQGTLVTVSSASGGGGSGGGGSGGGGSHASDIQMT 
               
               
                   
                   
                 QSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKA 
               
               
                   
                   
                 PKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDF 
               
               
                   
                   
                 ATYYCQQSYSTPYTFGQGTKVEIK 
               
               
                   
               
               
                 SEQ ID NO: 37 
                 H_G64Y_ 
                 QVQLVQSGAEVKKPGASVKVSCKASGYTFSNYYIHW 
               
               
                   
                 Q69H 
                 VRQAPGQGLEWMGWMDSNSGYTGYAHKFQGRVTMT 
               
               
                   
                   
                 RDTSTSTVYMELSSLRSEDTAVYYCAKEVFSGWYDY 
               
               
                   
                   
                 WGQGTLVTVSSASGGGGSGGGGSGGGGSHASDIQMT 
               
               
                   
                   
                 QSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKA 
               
               
                   
                   
                 PKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDF 
               
               
                   
                   
                 ATYYCQQSYSTPYTFGQGTKVEIK 
               
               
                   
               
               
                 SEQ ID NO: 38 
                 LQ68E 
                 QVQLVQSGAEVKKPGASVKVSCKASGYTFSNYYIHW 
               
               
                   
                   
                 VRQAPGQGLEWMGWMDSNSGGTGYAQKFQGRVTMT 
               
               
                   
                   
                 RDTSTSTVYMELSSLRSEDTAVYYCAKEVFSGWYDY 
               
               
                   
                   
                 WGQGTLVTVSSASGGGGSGGGGSGGGGSHASDIQMT 
               
               
                   
                   
                 QSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKA 
               
               
                   
                   
                 PKLLIYAASSLESGVPSRFSGSGSGTDFTLTISSLQPEDF 
               
               
                   
                   
                 ATYYCQQSYSTPYTFGQGTKVEIK 
               
               
                   
               
               
                 SEQ ID NO: 39 
                 LQ68E_ 
                 QVQLVQSGAEVKKPGASVKVSCKASGYTFSNYYIHW 
               
               
                   
                 E125D 
                 VRQAPGQGLEWMGWMDSNSGGTGYAQKFQGRVTMT 
               
               
                   
                   
                 RDTSTSTVYMELSSLRSEDTAVYYCAKEVFSGWYDY 
               
               
                   
                   
                 WGQGTLVTVSSASGGGGSGGGGSGGGGSHASDIQMT 
               
               
                   
                   
                 QSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKA 
               
               
                   
                   
                 PKLLIYAASSLESGVPSRFSGSGSGTDFTLTISSLQPEDF 
               
               
                   
                   
                 ATYYCQQSYSTPYTFGQGTKVDIK 
               
               
                   
               
            
           
         
       
     
     The scFv sequence for each variant was codon-optimized for  E. coli  expression and the corresponding DNA sequences synthesized as gBlocks (Integrated DNA Technologies, Inc.) with a T7 promoter, a translation initiation site, a Myc tag, the scFv sequence, a V5 tag sequence and a T7 terminator. Proteins encoded by the gBlock fragments were expressed using a cell-free transcription/translation system (Cosmo Bio USA, Inc., PUREfrex2.1, Product #GFK-PF213 with DS Supplement, Prod. #GFK-PF005). The cell-free expression samples were assayed for PD-L1 and IFNα binding as described in EXAMPLE 1. 
     Variants with different binding affinities may also be generated by display methods, such as phage display and mRNA display. Libraries for use in these methods may be created from the parental antibody by varying CDRs with random amino acid changes or by varying positions in the CDRs identified as suitable for change. 
     Example 5 
     Binding Affinity of Dual Binding Antibodies (DBAs) 
     This example describes binding affinity of protein complexes, specifically, dual binding antibodies (DBAs). Variants were synthesized as V5-tagged scFvs using the PUREfrex 2.1 in vitro translation system and diluted 1:30 in 1× kinetics buffer (KB, forteBIO) for testing. Biotinylated anti-V5 antibody (clone SV5-Pk1 BioRad, biotinylated using EZ-Link® Sulfo-NHS-LC-Biotin, THERMO) was diluted to 80 nM in 1× KB, then loaded onto streptavidin biosensor tips (SA, forteBIO) on an OctetRED96e instrument. These tips were then loaded with scFv for 240 seconds. The loaded tips were transferred to 1× KB for 60 seconds to establish baseline, then dipped into 160 nM ACRO human PDL1-his for 150 seconds to measure association, then into 1× KB for 180 seconds to measure dissociation. Immediately following this dissociation, baseline signal in 1× KB was measured, the tips were dipped into 5,000 nM IFNα2b (GenScript) for 150 seconds to measure association, then into 1× KB for 240 seconds to measure dissociation. Data were fit to a 1:1 kinetics model. Response for binding of IFNα2b was qualitative negative or positive. Sequences of the DBA variants used in TABLE 11 are provided in TABLE 8 and TABLE 9. 
     
       
         
           
               
             
               
                 TABLE 11 
               
             
            
               
                   
               
               
                 Kinetics of binding of DBA variants 
               
            
           
           
               
               
               
            
               
                   
                 KD (PD-L1, nM) 
                 IFNα binding (@5 uM) 
               
               
                   
               
            
           
           
               
               
               
            
               
                 H_G64Y_Q69H 
                 13 
                 +/− 
               
               
                 H_I39V_S58P_Q69H_K70Q 
                 8 
                 − 
               
               
                 H_N36G 
                 37 
                 + 
               
               
                 L_Q68E 
                 21 
                 + 
               
               
                 L_Q68E_E125D 
                 21 
                 + 
               
               
                 PDL1-IFN_R01_A05 
                 120 
                 Not tested 
               
               
                   
               
            
           
         
       
     
     The affinity of the DBA in this or other antibody formats may be measured in a similar manner to generate quantitative or semi-quantitative measurements using standard methods of measurement of protein interaction, including biolayer interferometry (e.g., Octet, Molecular Devices LLC.) surface plasmon resonance (e.g., BiaCore, GE Healthcare Life Sciences), kinetic exclusion (KinExA, Sapidyne Instruments, Inc.), or other biophysical methods. In addition, apparent affinity of a target for the DBA in the covalently linked construct may be measured with these methods by competition for a nonlinked binding agent. 
     Example 6 
     Dual Binding Antibody (DBA)-Cytokine Protein Complexes 
     This example describes dual binding antibody (DBA)-cytokine protein complexes of the present disclosure. Various DBA-cytokine protein complexes of the present disclosure were designed to include a cytokine, a linker, and one or more dual binding antibody domains. An exemplary protein complex is shown in  FIG.  8    (at left) and its amino acid sequence is shown in  FIG.  8    (at right). Pictorial representations of other exemplary constructs are shown in  FIG.  9    (SEQ ID NO: 42-SEQ ID NO: 54 and SEQ ID NO: 77-SEQ ID NO: 79). 
     A series of DBA-cytokine protein complexes may be designed with two marker binding domains and one therapeutic domain. The DBAs used in this series, provided in TABLE 13 with sequences provided in TABLE 14, exhibit a range of affinities for the marker and the therapeutic domain. Exemplary DBA complexes are provided in TABLE 12, TABLE 15, and TABLE 16. 
     
       
         
           
               
             
               
                 TABLE 12 
               
             
            
               
                   
               
               
                 Exemplary DBA Cytokine Protein Complexes 
               
            
           
           
               
               
               
            
               
                 SEQ ID NO 
                 Sequences 
                 Description 
               
               
                   
               
               
                 SEQ ID NO: 42 
                 QVQLVQSGAEVKKPGASVKVSCKASGYTFT 
                 PD-L1/IFNα 
               
               
                   
                 KNYMHWVRQAPGQGLEWLGWVSPDSGYTG 
                 protein complexes 
               
               
                   
                 YAQKFQGRVTMTRDTSTSTVYMELSSLRSED 
                 having a DBA 
               
               
                   
                 TAVYYCTTDLLSLELDDAFDIWGQGTMVTVS 
                 sensor domain 
               
               
                   
                 SASGGGGSGGGGSGGGGSHASDIQMTQSPSS 
                 capable of binding 
               
               
                   
                 LSASVGDRVTITCRASQSISSWLAWYQQKPG 
                 a PD-L1 marker 
               
               
                   
                 KAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLT 
                 and IFNα 
               
               
                   
                 ISSLQPEDFATYYCQQSYSTPLTFGGGTKLEIK 
                 therapeutic domain 
               
               
                   
                 PRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIK 
                 and having an IFNα 
               
               
                   
                 DVLMISLSPIVTCVVVDVSEDDPDVQISWFVN 
                 therapeutic activity 
               
               
                   
                 NVEVHTAQTQTHREDYNSTLRVVSALPIQHQ 
                   
               
               
                   
                 DWMSGKEFKCKVNNKDLGAPIERTISKPKGS 
                   
               
               
                   
                 VRAPQVYVLPPCEEEMTKKQVTLSCAVTDFM 
                   
               
               
                   
                 PEDIYVEWTNNGKTELNYKNTEPVLDSDGSY 
                   
               
               
                   
                 FMVSKLRVEKKNWVERNSYSCSVVHEGLHN 
                   
               
               
                   
                 HHTTKSFSRTPGK 
                   
               
               
                   
               
               
                 SEQ ID NO: 43 
                 CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKD 
                 PD-L1/IFNα 
               
               
                   
                 RHDFGFPQEEFGNQFQKAETIPVLHEMIQQIF 
                 protein complexes 
               
               
                   
                 NLFSTKDSSAAWDETLLDKFCTELYQQLNDL 
                 having a DBA 
               
               
                   
                 EACVMQEERVGETPLMNADSILAVKKYFRRI 
                 sensor domain 
               
               
                   
                 TLYLTEKKYSPCAWEVVRAEIVRSLSLSTNLQ 
                 capable of binding 
               
               
                   
                 ERLRRKEGGGGSGGGGSGGGGSGGGGSQVQ 
                 a PD-L1 marker 
               
               
                   
                 LVQSGAEVKKPGASVKVSCKASGYTFSGYYI 
                 and IFNα 
               
               
                   
                 HWVRQAPGQGLEWMGWMDSNSGGTGYAQ 
                 therapeutic domain 
               
               
                   
                 KFQGRVTMTRDTSTSTVYMELSSLRSEDTAV 
                 and having an IFNα 
               
               
                   
                 YYCAKEVFSGWYDYWGQGTLVTVSSAKTTA 
                 therapeutic activity 
               
               
                   
                 PSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPV 
                   
               
               
                   
                 TLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVT 
                   
               
               
                   
                 VTSSTWPSQSITCNVAHPASSTKVDKKIEPRG 
                   
               
               
                   
                 PTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVL 
                   
               
               
                   
                 MISLSPIVTCVVVDVSEDDPDVQISWFVNNVE 
                   
               
               
                   
                 VHTAQTQTHREDYNSTLRVVSALPIQHQDW 
                   
               
               
                   
                 MSGKEFKCKVNNKDLGAPIERTISKPKGSVR 
                   
               
               
                   
                 APQVCVLPPPEEEMTKKQVTLWCMVTDFMP 
                   
               
               
                   
                 EDIYVEWTNNGKTELNYKNTEPVLDSDGSYF 
                   
               
               
                   
                 MYSKLRVEKKNWVERNSYSCSVVHEGLHNH 
                   
               
               
                   
                 HTTKSFSRTPGK 
                   
               
               
                   
               
               
                 SEQ ID NO: 44 
                 DIQMTQSPSSLSASVGDRVTITCRASQSISSYL 
                 PD-L1/IFNα 
               
               
                   
                 NWYQQKPGKAPKLLIYAASSLQSGVPSRFSG 
                 protein complexes 
               
               
                   
                 SGSGTDFTLTISSLQPEDFATYYCQQSYSTPYT 
                 having a DBA 
               
               
                   
                 FGQGTKVEIKRADAAPTVSIFPPSSEQLTSGGA 
                 sensor domain 
               
               
                   
                 SVVCFLNNFYPKDINVKWKIDGSERQNGVLN 
                 capable of binding 
               
               
                   
                 SWTDQDSKDSTYSMSSTLTLTKDEYERHNSY 
                 a PD-L1 marker 
               
               
                   
                 TCEATHKTSTSPIVKSFNRNEC 
                 and IFNα 
               
               
                   
                   
                 therapeutic domain 
               
               
                   
                   
                 and having an IFNα 
               
               
                   
                   
                 therapeutic activity 
               
               
                   
               
               
                 SEQ ID NO: 45 
                 QVQLVQSGAEVKKPGASVKVSCKASGGTFSS 
                 PD-L1/IFNα 
               
               
                   
                 YAISWVRQAPGQGLEWMGIIDPSVTYTRYAQ 
                 protein complexes 
               
               
                   
                 KFQGRVTMTRDTSTSTVYMELSSLRSEDTAV 
                 having a DBA 
               
               
                   
                 YYCARSLFPTIFGVEVAFDIWGQGTLVTVSSA 
                 sensor domain 
               
               
                   
                 KTTAPSVYPLAPVCGDTTGSSVTLGCLVKGY 
                 capable of binding 
               
               
                   
                 FPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTL 
                 a PD-L1 marker 
               
               
                   
                 SSSVTVTSSTWPSQSITCNVAHPASSTKVDKKI 
                 and IFNα 
               
               
                   
                 EPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIK 
                 therapeutic domain 
               
               
                   
                 DVLMISLSPIVTCVVVDVSEDDPDVQISWFVN 
                 and having an IFNα 
               
               
                   
                 NVEVHTAQTQTHREDYNSTLRVVSALPIQHQ 
                 therapeutic activity 
               
               
                   
                 DWMSGKEFKCKVNNKDLGAPIERTISKPKGS 
                   
               
               
                   
                 VRAPQVYVLPPCEEEMTKKQVTLSCAVTDFM 
                   
               
               
                   
                 PEDIYVEWTNNGKTELNYKNTEPVLDSDGSY 
                   
               
               
                   
                 FMVSKLRVEKKNWVERNSYSCSVVHEGLHN 
                   
               
               
                   
                 HHTTKSFSRTPGK 
                   
               
               
                   
               
               
                 SEQ ID NO: 46 
                 CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKD 
                 PD-L1/IFNα 
               
               
                   
                 RHDFGFPQEEFGNQFQKAETIPVLHEMIQQIF 
                 protein complexes 
               
               
                   
                 NLFSTKDSSAAWDETLLDKFCTELYQQLNDL 
                 having a DBA 
               
               
                   
                 EACVMQEERVGETPLMNADSILAVKKYFRRI 
                 sensor domain 
               
               
                   
                 TLYLTEKKYSPCAWEVVRAEIVRSLSLSTNLQ 
                 capable of binding 
               
               
                   
                 ERLRRKEGGGGSGGGGSGGGGSGGGGSQVQ 
                 a PD-L1 marker 
               
               
                   
                 LVQSGAEVKKPGASVKVSCKASGGTFSSYAIS 
                 and IFNα 
               
               
                   
                 WVRQAPGQGLEWMGIIDPSVTYTRYAQKFQ 
                 therapeutic domain 
               
               
                   
                 GRVTMTRDTSTSTVYMELSSLRSEDTAVYYC 
                 and having an IFNα 
               
               
                   
                 ARSLFPTIFGVEVAFDIWGQGTLVTVSSAKTT 
                 therapeutic activity 
               
               
                   
                 APSVYPLAPVCGDTTGSSVTLGCLVKGYFPEP 
                   
               
               
                   
                 VTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSV 
                   
               
               
                   
                 TVTSSTWPSQSITCNVAHPASSTKVDKKIEPR 
                   
               
               
                   
                 GPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDV 
                   
               
               
                   
                 LMISLSPIVTCVVVDVSEDDPDVQISWFVNNV 
                   
               
               
                   
                 EVHTAQTQTHREDYNSTLRVVSALPIQHQDW 
                   
               
               
                   
                 MSGKEFKCKVNNKDLGAPIERTISKPKGSVR 
                   
               
               
                   
                 APQVCVLPPPEEEMTKKQVTLWCMVTDFMP 
                   
               
               
                   
                 EDIYVEWTNNGKTELNYKNTEPVLDSDGSYF 
                   
               
               
                   
                 MYSKLRVEKKNWVERNSYSCSVVHEGLHNH 
                   
               
               
                   
                 HTTKSFSRTPGK 
                   
               
               
                   
               
               
                 SEQ ID NO: 47 
                 DIQMTQSPSSLSASVGDRVTITCQASQSISNYL 
                 PD-L1/IFNα 
               
               
                   
                 AWYQQKPGKAPKLLIYKASSLESGVPSRFSGS 
                 protein complexes 
               
               
                   
                 GSGTDFTLTISSLQPEDFATYYCQQTYSTPITF 
                 having a DBA 
               
               
                   
                 GQGTKVEIKRADAAPTVSIFPPSSEQLTSGGAS 
                 sensor domain 
               
               
                   
                 VVCFLNNFYPKDINVKWKIDGSERQNGVLNS 
                 capable of binding 
               
               
                   
                 WTDQDSKDSTYSMSSTLTLTKDEYERHNSYT 
                 a PD-L1 marker 
               
               
                   
                 CEATHKTSTSPIVKSFNRNEC 
                 and IFNα 
               
               
                   
                   
                 therapeutic domain 
               
               
                   
                   
                 and having an IFNα 
               
               
                   
                   
                 therapeutic activity 
               
               
                   
               
               
                 SEQ ID NO: 48 
                 CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKD 
                 ExemplarySeq_C_ 
               
               
                   
                 RHDFGFPQEEFGNQFQKAETIPVLHEMIQQIF 
                 Pep1 
               
               
                   
                 NLFSTKDSSAAWDETLLDKFCTELYQQLNDL 
                 Asymmetric DBA- 
               
               
                   
                 EACVMQEERVGETPLMNADSILAVKKYFRRI 
                 Cytokine Complex 
               
               
                   
                 TLYLTEKKYSPCAWEVVRAEIVRSLSLSTNLQ 
                 scFv-IgG format 
               
               
                   
                 ERLRRKEGGGGSGGGGSGGGGSGGGGSQVQ 
                   
               
               
                   
                 LVQSGAEVKKPGASVKVSCKASGGTFSSYAIS 
                   
               
               
                   
                 WVRQAPGQGLEWMGIIDPSVTYTRYAQKFQ 
                   
               
               
                   
                 GRVTMTRDTSTSTVYMELSSLRSEDTAVYYC 
                   
               
               
                   
                 ARSLFPTIFGVEVAFDIWGQGTLVTVSSASGG 
                   
               
               
                   
                 GGSGGGGSGGGGSHASDIQMTQSPSSLSASV 
                   
               
               
                   
                 GDRVTITCQASQDISNYLNWYQQKPGKAPKL 
                   
               
               
                   
                 LIYGASTLQSGVPSRFSGSGSGTDFTLTISSLQP 
                   
               
               
                   
                 EDFATYYCQQTYSTPITFGQGTKVEIKAKTTA 
                   
               
               
                   
                 PSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPV 
                   
               
               
                   
                 TLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVT 
                   
               
               
                   
                 VTSSTWPSQSITCNVAHPASSTKVDKKIEPRG 
                   
               
               
                   
                 PTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVL 
                   
               
               
                   
                 MISLSPIVTCVVVDVSEDDPDVQISWFVNNVE 
                   
               
               
                   
                 VHTAQTQTHREDYNSTLRVVSALPIQHQDW 
                   
               
               
                   
                 MSGKEFKCKVNNKDLGAPIERTISKPKGSVR 
                   
               
               
                   
                 APQVYVLPPCEEEMTKKQVTLSCAVTDFMPE 
                   
               
               
                   
                 DIYVEWTNNGKTELNYKNTEPVLDSDGSYFM 
                   
               
               
                   
                 VSKLRVEKKNWVERNSYSCSVVHEGLHNHH 
                   
               
               
                   
                 TTKSFSRTPGK 
                   
               
               
                   
               
               
                 SEQ ID NO: 49 
                 QVQLVQSGAEVKKPGASVKVSCKASGYTFSG 
                 ExemplarySeq_C_ 
               
               
                   
                 YYIHWVRQAPGQGLEWMGWMDSNSGGTGY 
                 Pep2 
               
               
                   
                 AQKFQGRVTMTRDTSTSTVYMELSSLRSEDT 
                 Asymmetric DBA- 
               
               
                   
                 AVYYCAKEVFSGWYDYWGQGTLVTVSSAKT 
                 Cytokine Complex 
               
               
                   
                 TAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPE 
                 scFv-IgG format 
               
               
                   
                 PVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSS 
                   
               
               
                   
                 VTVTSSTWPSQSITCNVAHPASSTKVDKKIEP 
                   
               
               
                   
                 RGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKD 
                   
               
               
                   
                 VLMISLSPIVTCVVVDVSEDDPDVQISWFVNN 
                   
               
               
                   
                 VEVHTAQTQTHREDYNSTLRVVSALPIQHQD 
                   
               
               
                   
                 WMSGKEFKCKVNNKDLGAPIERTISKPKGSV 
                   
               
               
                   
                 RAPQVCVLPPPEEEMTKKQVTLWCMVTDFM 
                   
               
               
                   
                 PEDIYVEWTNNGKTELNYKNTEPVLDSDGSY 
                   
               
               
                   
                 FMYSKLRVEKKNWVERNSYSCSVVHEGLHN 
                   
               
               
                   
                 HHTTKSFSRTPGK 
                   
               
               
                   
               
               
                 SEQ ID NO: 50 
                 DIQMTQSPSSLSASVGDRVTITCRASQSISSYL 
                 ExemplarySeq_C_ 
               
               
                   
                 NWYQQKPGKAPKLLIYAASSLQSGVPSRFSG 
                 Pep3 
               
               
                   
                 SGSGTDFTLTISSLQPEDFATYYCQQSYSTPYT 
                 Asymmetric DBA- 
               
               
                   
                 FGQGTKVEIKRADAAPTVSIFPPSSEQLTSGGA 
                 Cytokine Complex 
               
               
                   
                 SVVCFLNNFYPKDINVKWKIDGSERQNGVLN 
                 scFv-IgG format 
               
               
                   
                 SWTDQDSKDSTYSMSSTLTLTKDEYERHNSY 
                   
               
               
                   
                 TCEATHKTSTSPIVKSFNRNEC 
                   
               
               
                   
               
               
                 SEQ ID NO: 51 
                 QVQLVQSGAEVKKPGASVKVSCKASGYTFST 
                 PD1- 
               
               
                   
                 YYIHWVRQAPGQGLEWMGIINPSGGGTVYA 
                 IL2_6C12_N36T_ 
               
               
                   
                 QKFQGRVTMTRDTSTSTVYMELSSLRSEDTA 
                 Sym_L_Long_Pep1 
               
               
                   
                 VYYCAAGLFIWGQGTLVTVSSAKTTAPSVYP 
                 Symmetric DBA- 
               
               
                   
                 LAPVCGDTTGSSVTLGCLVKGYFPEPVTLTW 
                 Cytokine Complex 
               
               
                   
                 NSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSST 
                 IgG format 
               
               
                   
                 WPSQSITCNVAHPASSTKVDKKIEPRGPTIKPC 
                   
               
               
                   
                 PPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPI 
                   
               
               
                   
                 VTCVVVDVSEDDPDVQISWFVNNVEVHTAQ 
                   
               
               
                   
                 TQTHREDYNSTLRVVSALPIQHQDWMSGKEF 
                   
               
               
                   
                 KCKVNNKDLGAPIERTISKPKGSVRAPQVYV 
                   
               
               
                   
                 LPPPEEEMTKKQVTLTCMVTDFMPEDIYVEW 
                   
               
               
                   
                 TNNGKTELNYKNTEPVLDSDGSYFMYSKLRV 
                   
               
               
                   
                 EKKNWVERNSYSCSVVHEGLHNHHTTKSFSR 
                   
               
               
                   
                 TPGK 
                   
               
               
                   
               
               
                 SEQ ID NO: 52 
                 APTSSSTKKTQLQLEHLLLDLQMILNGINNYK 
                 PD1- 
               
               
                   
                 NPKLTRMLTFKFYMPKKATELKHLQCLEEEL 
                 IL2_6C12_N36T_ 
               
               
                   
                 KPLEEVLNLAQSKNFHLRPRDLISNINVIVLEL 
                 Sym_L_Long_Pep2 
               
               
                   
                 KGSETTFMCEYADETATIVEFLNRWITFCQSII 
                 Symmetric DBA- 
               
               
                   
                 STLTVPGVGVPGAGVPGVGVPGGGVPGVGV 
                 Cytokine Complex 
               
               
                   
                 PGGGVPGAGVPGGGVPGVGVPGAGVPGVGV 
                 IgG format 
               
               
                   
                 PGGGDIQMTQSPSSLSASVGDRVTITCRASQY 
                   
               
               
                   
                 ISSGLAWYQQKPGKAPKLLIYKASSLDNGVPS 
                   
               
               
                   
                 RFSGSGSGTDFTLTISSLQPEDFATYYCQQYE 
                   
               
               
                   
                 RLPLTFGGGTKVEIKRADAAPTVSIFPPSSEQL 
                   
               
               
                   
                 TSGGASVVCFLNNFYPKDINVKWKIDGSERQ 
                   
               
               
                   
                 NGVLNSWTDQDSKDSTYSMSSTLTLTKDEYE 
                   
               
               
                   
                 RHNSYTCEATHKTSTSPIVKSFNRNEC 
                   
               
               
                   
               
               
                 SEQ ID NO: 53 
                 APTSSSTKKTQLQLEHLLLDLQMILNGINNYK 
                 PD1- 
               
               
                   
                 NPKLTRMLTFKFYMPKKATELKHLQCLEEEL 
                 IL2_6C12_N36T_ 
               
               
                   
                 KPLEEVLNLAQSKNFHLRPRDLISNINVIVLEL 
                 D68E_Sym_H_ 
               
               
                   
                 KGSETTFMCEYADETATIVEFLNRWITFCQSII 
                 ShortPep1 
               
               
                   
                 STLTGGGGSGGGGSGGGGSGGGGSQVQLVQ 
                 Symmetric DBA- 
               
               
                   
                 SGAEVKKPGASVKVSCKASGYTFSTYYIHWV 
                 Cytokine Complex 
               
               
                   
                 RQAPGQGLEWMGIINPSGGGTVYAQKFQGR 
                 IgG format 
               
               
                   
                 VTMTRDTSTSTVYMELSSLRSEDTAVYYCAA 
                   
               
               
                   
                 GLFIWGQGTLVTVSSAKTTAPSVYPLAPVCG 
                   
               
               
                   
                 DTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSS 
                   
               
               
                   
                 GVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSIT 
                   
               
               
                   
                 CNVAHPASSTKVDKKIEPRGPTIKPCPPCKCP 
                   
               
               
                   
                 APNAAGGPSVFIFPPKIKDVLMISLSPIVTCVV 
                   
               
               
                   
                 VDVSEDDPDVQISWFVNNVEVHTAQTQTHRE 
                   
               
               
                   
                 DYNSTLRVVSALPIQHQDWMSGKEFKCKVN 
                   
               
               
                   
                 NKDLGAPIERTISKPKGSVRAPQVYVLPPPEEE 
                   
               
               
                   
                 MTKKQVTLTCMVTDFMPEDIYVEWTNNGKT 
                   
               
               
                   
                 ELNYKNTEPVLDSDGSYFMYSKLRVEKKNW 
                   
               
               
                   
                 VERNSYSCSVVHEGLHNHHTTKSFSRTPGK 
                   
               
               
                   
               
               
                 SEQ ID NO: 54 
                 DIQMTQSPSSLSASVGDRVTITCRASQYISSGL 
                 PD1- 
               
               
                   
                 AWYQQKPGKAPKLLIYKASSLENGVPSRFSG 
                 IL2_6C12_N36T_ 
               
               
                   
                 SGSGTDFTLTISSLQPEDFATYYCQQYERLPLT 
                 D68E_Sym_H_ 
               
               
                   
                 FGGGTKVEIKRADAAPTVSIFPPSSEQLTSGGA 
                 Short_Pep2 
               
               
                   
                 SVVCFLNNFYPKDINVKWKIDGSERQNGVLN 
                 Symmetric DBA- 
               
               
                   
                 SWTDQDSKDSTYSMSSTLTLTKDEYERHNSY 
                 Cytokine Complex 
               
               
                   
                 TCEATHKTSTSPIVKSFNRNEC 
                 IgG format 
               
               
                   
               
               
                 SEQ ID NO: 77 
                 QVQLVQSGAEVKKPGASVKVSCKASGYTFTT 
                 PD1- 
               
               
                   
                 YYVHWVRQAPGQGLEWMGIINPSGGSTSYA 
                 IL2_L_7A05scFv_ 
               
               
                   
                 QNFQGRVTMTRDTSTSTVYMELSSLRSEDTA 
                 PD1-R07- 
               
               
                   
                 VYYCASGWDVWGQGTTVTVSSAKTTAPSVY 
                 A05_Pep1 
               
               
                   
                 PLAPVCGDTTGSSVTLGCLVKGYFPEPVTLT 
                 Asymmetric DBA- 
               
               
                   
                 WNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTS 
                 Cytokine Complex 
               
               
                   
                 STWPSQSITCNVAHPASSTKVDKKIEPRGPTIK 
                 IgG-scFv format 
               
               
                   
                 PCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISL 
                   
               
               
                   
                 SPIVTCVVVDVSEDDPDVQISWFVNNVEVHT 
                   
               
               
                   
                 AQTQTHREDYNSTLRVVSALPIQHQDWMSG 
                   
               
               
                   
                 KEFKCKVNNKDLGAPIERTISKPKGSVRAPQV 
                   
               
               
                   
                 YVLPPPEEEMTKKQVTLTCMVTDFMPEDIYV 
                   
               
               
                   
                 EWTNNGKTELNYKNTEPVLDSDGSYFMYSD 
                   
               
               
                   
                 LRVEKKNWVERNSYSCSVVHEGLHNHHTTE 
                   
               
               
                   
                 SFSRTPGK 
                   
               
               
                   
               
               
                 SEQ ID NO: 78 
                 QVQLVQSGAEVKKPGASVKVSCKASGYTFTT 
                 PD1- 
               
               
                   
                 YYVHWVRQAPGQGLEWMGIINPSGGSTSYA 
                 IL2_L_7A05scFv_ 
               
               
                   
                 QNFQGRVTMTRDTSTSTVYMELSSLRSEDTA 
                 PD1-R07- 
               
               
                   
                 VYYCASGWDVWGQGTTVTVSSASGGGGSG 
                 A05_Pep2 
               
               
                   
                 GGGSGGGGSHASEIVMTQSPATESVSPGERAT 
                 Asymmetric DBA- 
               
               
                   
                 LSCRASQSVNTYLAWYQQKPGQAPRLLIYGA 
                 Cytokine Complex 
               
               
                   
                 STRATGIPARFSGSGSGTEFTLTISSLQSEDFAV 
                 IgG-scFv format 
               
               
                   
                 YYCQQYGSSPVTFGQGTRLEIKPRGPTIKPCPP 
                   
               
               
                   
                 CKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIV 
                   
               
               
                   
                 TCVVVDVSEDDPDVQISWFVNNVEVHTAQT 
                   
               
               
                   
                 QTHREDYNSTLRVVSALPIQHQDWMSGKEFK 
                   
               
               
                   
                 CKVNNKDLGAPIERTISKPKGSVRAPQVYVLP 
                   
               
               
                   
                 PPEKEMTKKQVSLTCLVKDFMPEDIYVEWTN 
                   
               
               
                   
                 NGKTELNYKNTEPVLKSDGSYFMYSKLTVEK 
                   
               
               
                   
                 KNWVERNSYSCSVVHEGLHNHHTTKSFSRTP 
                   
               
               
                   
                 GGGGSGGGSHHHHHH 
                   
               
               
                   
               
               
                 SEQ ID NO: 79 
                 APTSSSTKKTQLQLEHLLLDLQMILNGINNYK 
                 PD1- 
               
               
                   
                 NPKLTDMLTFKFYMPKKATELKHLQCLEEEL 
                 IL2_L_7A05scFv_ 
               
               
                   
                 KPLEEVLNLAQSKNFHLRPRDLISNINVIVLEL 
                 PD1-R07- 
               
               
                   
                 KGSETTFMCEYADETATIVEFLNRWITFCQSII 
                 A05_Pep3 
               
               
                   
                 STLTGGGGSGGGGSGGGGSGGGGSEIVMTQS 
                 Asymmetric DBA- 
               
               
                   
                 PATLSVSPGERATLSCRASQSVNTYLAWYQQ 
                 Cytokine Complex 
               
               
                   
                 KPGQAPRLLIYGASTRATGIPARFSGSGSGTEF 
                 IgG-scFv format 
               
               
                   
                 TLTISSLQSEDFAVYYCQQYGSSPVTFGQGTR 
                   
               
               
                   
                 LEIKRADAAPTVSIFPPSSEQLTSGGASVVCFL 
                   
               
               
                   
                 NNFYPKDINVKWKIDGSERQNGVLNSWTDQ 
                   
               
               
                   
                 DSKDSTYSMSSTLTLTKDEYERHNSYTCEAT 
                   
               
               
                   
                 HKTSTSPIVKSFNRNEC 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 13 
               
             
            
               
                   
               
               
                 Dual-Binding Antibodies (DBAs) 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
            
               
                   
                   
                   
                 HV* 
                 LV** 
                   
                   
                   
                   
                   
                   
               
               
                 Dual- 
                   
                   
                 SEQ 
                 SEQ 
                 HV_cdr1 
                 HV_cdr2 
                 HV_cdr3 
                 LV_cdr1 
                 LV_cdr2 
                 LV_cdr3 
               
               
                 Binding 
                   
                   
                 ID 
                 ID 
                 SEQ ID 
                 SEQ ID 
                 SEQ ID 
                 SEQ ID 
                 SEQ ID 
                 SEQ ID 
               
               
                 Antibody 
                 Marker 
                 Therapeutic 
                 NO: 
                 NO: 
                 NO: 
                 NO: 
                 NO: 
                 NO: 
                 NO: 
                 NO: 
               
               
                   
               
               
                 AB001718 
                 PD-1 
                 IL-2 
                 127 
                 135 
                 142 
                 148 
                 154 
                 157 
                 163 
                 168 
               
               
                 AB001744 
                 PD-1 
                 IL-2 
                 128 
                 136 
                 143 
                 148 
                 154 
                 158 
                 163 
                 169 
               
               
                 AB002022 
                 PD-1 
                 IL-2 
                 129 
                 137 
                 144 
                 149 
                 154 
                 159 
                 164 
                 170 
               
               
                 AB001609 
                 PD-L1 
                 IFNα 
                 130 
                 138 
                 145 
                 150 
                 155 
                 160 
                 165 
                 171 
               
               
                 AB001638 
                 PD-L1 
                 IFNα 
                 130 
                 139 
                 145 
                 150 
                 155 
                 161 
                 165 
                 172 
               
               
                 AB001843 
                 PD-L1 
                 IFNα 
                 131 
                 140 
                 146 
                 151 
                 156 
                 162 
                 166 
                 173 
               
               
                 AB001866 
                 PD-L1 
                 IFNα 
                 132 
                 140 
                 147 
                 152 
                 156 
                 162 
                 166 
                 173 
               
               
                 AB001875 
                 PD-L1 
                 IFNα 
                 133 
                 140 
                 143 
                 153 
                 156 
                 162 
                 166 
                 173 
               
               
                 AB001909 
                 PD-L1 
                 IFNα 
                 134 
                 141 
                 143 
                 151 
                 156 
                 162 
                 167 
                 173 
               
               
                   
               
               
                 * HV refers to the heavy chain variable region of the respective antibodies 
               
               
                 ** LV refers to the light chain variable region of the respective antibodies 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 14 
               
             
            
               
                   
               
               
                 Sequences of DBA Protein Components 
               
            
           
           
               
               
               
            
               
                 SEQ ID NO: 
                 DBA Protein Component 
                 Sequence 
               
               
                   
               
               
                 SEQ ID NO: 127 
                 AB001718_HV 
                 QVQLVQSGAEVKKPGASVKVSCKASG 
               
               
                   
                   
                 DTFSTYYVHWVRQAPGQGLEWMGIINP 
               
               
                   
                   
                 SGGGTVYAQKFQGRVTMTRDTSTSTVY 
               
               
                   
                   
                 MELSSLRSEDTAVYYCAAGLFIWGQGT 
               
               
                   
                   
                 LVTVSS 
               
               
                   
               
               
                 SEQ ID NO: 128 
                 AB001744_HV 
                 QVQLVQSGAEVKKPGASVKVSCKASG 
               
               
                   
                   
                 YTFSNYYIHWVRQAPGQGLEWMGIINP 
               
               
                   
                   
                 SGGGTVYAQKFQGRVTMTRDTSTSTVY 
               
               
                   
                   
                 MELSSLRSEDTAVYYCAAGLFIWGQGT 
               
               
                   
                   
                 LVTVSS 
               
               
                   
               
               
                 SEQ ID NO: 129 
                 AB002022_HV 
                 QVQLVQSGAEVKKPGASVKVSCKASG 
               
               
                   
                   
                 DTFTRHYVHWVRQAPGQGLEWMGIIN 
               
               
                   
                   
                 PSGGYASYAQKFQGRVTMTRDTSTSTV 
               
               
                   
                   
                 YMELSSLRSEDTAVYYCAAGLFIWGQG 
               
               
                   
                   
                 TLVTVSS 
               
               
                   
               
               
                 SEQ ID NO: 130 
                 AB001609_HV 
                 QVQLVQSGAEVKKPGASVKVSCKASG 
               
               
                   
                   
                 GTFSSYAISWVRQAPGQGLEWMGIIDPS 
               
               
                   
                   
                 VTYTRYAQKFQGRVTMTRDTSTSTVY 
               
               
                   
                   
                 MELSSLRSEDTAVYYCARSLFPTIFGVE 
               
               
                   
                   
                 VAFDIWGQGTLVTVSS 
               
               
                   
               
               
                 SEQ ID NO: 131 
                 AB001843_HV 
                 QVQLVQSGAEVKKPGASVKVSCKASG 
               
               
                   
                   
                 YTFSGYYIHWVRQAPGQGLEWMGWM 
               
               
                   
                   
                 DSNSGGTGYAQKFQGRVTMTRDTSTST 
               
               
                   
                   
                 VYMELSSLRSEDTAVYYCAKEVFSGW 
               
               
                   
                   
                 YDYWGQGTLVTVSS 
               
               
                   
               
               
                 SEQ ID NO: 132 
                 AB001866_HV 
                 QVQLVQSGAEVKKPGASVKVSCKASG 
               
               
                   
                   
                 YTFSNYYVHWVRQAPGQGLEWMGWM 
               
               
                   
                   
                 DPNSGGTGYAHQFQGRVTMTRDTSTST 
               
               
                   
                   
                 VYMELSSLRSEDTAVYYCAKEVFSGW 
               
               
                   
                   
                 YDYWGQGTLVTVSS 
               
               
                   
               
               
                 SEQ ID NO: 133 
                 AB001875_HV 
                 QVQLVQSGAEVKKPGASVKVSCKASG 
               
               
                   
                   
                 YTFSNYYIHWVRQAPGQGLEWMGWM 
               
               
                   
                   
                 DSNSGYTGYAQQFQGRVTMTRDTSTST 
               
               
                   
                   
                 VYMELSSLRSEDTAVYYCAKEVFSGW 
               
               
                   
                   
                 YDYWGQGTLVTVSS 
               
               
                   
               
               
                 SEQ ID NO: 134 
                 AB001909_HV 
                 QVQLVQSGAEVKKPGASVKVSCKASG 
               
               
                   
                   
                 YTFSNYYIHWVRQAPGQGLEWMGWM 
               
               
                   
                   
                 DSNSGGTGYAQKFQGRVTMTRDTSTST 
               
               
                   
                   
                 VYMELSSLRSEDTAVYYCAKEVFSGW 
               
               
                   
                   
                 YDYWGQGTLVTVSS 
               
               
                   
               
               
                 SEQ ID NO: 135 
                 AB001718_LV 
                 DIQMTQSPSSLSASVGDRVTITCRASQYI 
               
               
                   
                   
                 SSGLAWYQQKPGKAPKLLIYKASSLDN 
               
               
                   
                   
                 GVPSRFSGSGSGTDFTLTISSLQPEDFAT 
               
               
                   
                   
                 YYCQQYERLPLTFGGGTKVEIK 
               
               
                   
               
               
                 SEQ ID NO: 136 
                 AB001744_LV 
                 DIQMTQSPSSLSASVGDRVTITCRASQSI 
               
               
                   
                   
                 GTGLAWYQQKPGKAPKLLIYKASSLDN 
               
               
                   
                   
                 GVPSRFSGSGSGTDFTLTISSLQPEDFAT 
               
               
                   
                   
                 YYCQQYNRAPLTFGGGTKVEIK 
               
               
                   
               
               
                 SEQ ID NO: 137 
                 AB002022_LV 
                 DIQMTQSPSSLSASVGDRVTITCRASQSI 
               
               
                   
                   
                 GRWLAWYQQKPGKAPKLLIYSASNLET 
               
               
                   
                   
                 GVPSRFSGSGSGTDFTLTISSLQPEDFAT 
               
               
                   
                   
                 YYCQQYESFPVTFGPGTKVDIK 
               
               
                   
               
               
                 SEQ ID NO: 138 
                 AB001609_LV 
                 DIQMTQSPSSLSASVGDRVTITCRASQSI 
               
               
                   
                   
                 SNRLAWYQQKPGKAPKLLIYKASSLES 
               
               
                   
                   
                 GVPSRFSGSGSGTDFTLTISSLQPEDFAT 
               
               
                   
                   
                 YYCQQSNSTPFTFGQGTKVEIK 
               
               
                   
               
               
                 SEQ ID NO: 139 
                 AB001638_LV 
                 DIQMTQSPSSLSASVGDRVTITCQASQSI 
               
               
                   
                   
                 SNYLAWYQQKPGKAPKLLIYKASSLES 
               
               
                   
                   
                 GVPSRFSGSGSGTDFTLTISSLQPEDFAT 
               
               
                   
                   
                 YYCQQTYSTPITFGQGTKVEIK 
               
               
                   
               
               
                 SEQ ID NO: 140 
                 AB001843_LV 
                 DIQMTQSPSSLSASVGDRVTITCRASQSI 
               
               
                   
                   
                 SSYLNWYQQKPGKAPKLLIYAASSLQS 
               
               
                   
                   
                 GVPSRFSGSGSGTDFTLTISSLQPEDFAT 
               
               
                   
                   
                 YYCQQSYSTPYTFGQGTKVEIK 
               
               
                   
               
               
                 SEQ ID NO: 141 
                 AB001909LV 
                 DIQMTQSPSSLSASVGDRVTITCRASQSI 
               
               
                   
                   
                 SSYLNWYQQKPGKAPKLLIYAASSLES 
               
               
                   
                   
                 GVPSRFSGSGSGTDFTLTISSLQPEDFAT 
               
               
                   
                   
                 YYCQQSYSTPYTFGQGTKVDIK 
               
               
                 SEQ ID NO 142 
                 AB001718_HV_cdr1 
                 GDTFSTYYVH 
               
               
                   
               
               
                 SEQ ID NO 143 
                 AB001744_HV_cdr1 
                 GYTFSNYYIH 
               
               
                   
               
               
                 SEQ ID NO 144 
                 AB002022_HV_cdr1 
                 GDTFTRHYVH 
               
               
                   
               
               
                 SEQ ID NO 145 
                 AB001609_HV_cdr1 
                 GGTFSSYAIS 
               
               
                   
               
               
                 SEQ ID NO 146 
                 AB001843_HV_cdr1 
                 GYTFSGYYIH 
               
               
                   
               
               
                 SEQ ID NO 147 
                 AB001866_HV_cdr1 
                 GYTFSNYYVH 
               
               
                   
               
               
                 SEQ ID NO 148 
                 AB001718_HV_cdr2 
                 IINPSGGGTVYAQKFQG 
               
               
                   
               
               
                 SEQ ID NO 149 
                 AB002022_HV_cdr2 
                 IINPSGGYASYAQKFQG 
               
               
                   
               
               
                 SEQ ID NO 150 
                 AB001609_HV_cdr2 
                 IIDPSVTYTRYAQKFQG 
               
               
                   
               
               
                 SEQ ID NO 151 
                 AB001843_HV_cdr2 
                 WMDSNSGGTGYAQKFQG 
               
               
                   
               
               
                 SEQ ID NO 152 
                 AB001866_HV_cdr2 
                 WMDPNSGGTGYAHQFQG 
               
               
                   
               
               
                 SEQ ID NO 153 
                 AB001875_HV_cdr2 
                 WMDSNSGYTGYAQQFQG 
               
               
                   
               
               
                 SEQ ID NO 154 
                 AB001718_HV_cdr3 
                 AAGLFI 
               
               
                   
               
               
                 SEQ ID NO 155 
                 AB001609_HV_cdr3 
                 ARSLFPTIFGVEVAFDI 
               
               
                   
               
               
                 SEQ ID NO 156 
                 AB001843_HV_cdr3 
                 AKEVFSGWYDY 
               
               
                   
               
               
                 SEQ ID NO 157 
                 AB001718_LV_cdrl 
                 RASQYISSGLA 
               
               
                   
               
               
                 SEQ ID NO 158 
                 AB001744_LV_cdrl 
                 RASQSIGTGLA 
               
               
                   
               
               
                 SEQ ID NO 159 
                 AB002022_LV_cdrl 
                 RASQSIGRWLA 
               
               
                   
               
               
                 SEQ ID NO 160 
                 AB001609_LV_cdrl 
                 RASQSISNRLA 
               
               
                   
               
               
                 SEQ ID NO 161 
                 AB001638_LV_cdrl 
                 QASQSISNYLA 
               
               
                   
               
               
                 SEQ ID NO 162 
                 AB001843_LV_cdrl 
                 RASQSISSYLN 
               
               
                   
               
               
                 SEQ ID NO 163 
                 AB001718_LV_cdr2 
                 KASSLDN 
               
               
                   
               
               
                 SEQ ID NO 164 
                 AB002022_LV_cdr2 
                 SASNLET 
               
               
                   
               
               
                 SEQ ID NO 165 
                 AB001609_LV_cdr2 
                 KASSLES 
               
               
                   
               
               
                 SEQ ID NO 166 
                 AB001843_LV_cdr2 
                 AASSLQS 
               
               
                   
               
               
                 SEQ ID NO 167 
                 AB001909_LV_cdr2 
                 AASSLES 
               
               
                   
               
               
                 SEQ ID NO 168 
                 AB001718_LV_cdr3 
                 QQYERLPL 
               
               
                   
               
               
                 SEQ ID NO 169 
                 AB001744_LV_cdr3 
                 QQYNRAPL 
               
               
                   
               
               
                 SEQ ID NO 170 
                 AB002022_LV_cdr3 
                 QQYESFPV 
               
               
                   
               
               
                 SEQ ID NO 171 
                 AB001609_LV_cdr3 
                 QQSNSTPF 
               
               
                   
               
               
                 SEQ ID NO 172 
                 AB001638_LV_cdr3 
                 QQTYSTPI 
               
               
                   
               
               
                 SEQ ID NO 173 
                 AB001843_LV_cdr3 
                 QQSYSTPY 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 15 
               
             
            
               
                   
               
               
                 Exemplary DBA-Cytokine Protein Complexes 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                   
                   
                   
                 Heavy 
                 Heavy 
                 Heavy 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Chain 1 
                 Chain 2 
                 Chain 3 
               
               
                   
                 DBA/ 
                   
                   
                 DBA 
                 Therapeutic 
                 2nd Ab 
                 SEQID 
                 SEQID 
                 SEQID 
               
               
                 Name 
                 Therapeutic 
                 DBA 
                 Type 
                 domains 
                 domains 
                 domain 
                 NO: 
                 NO: 
                 NO: 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                 AF00 
                 PD-1/IL-2 
                 AB001 
                 FIG. 9b 
                 2 
                 1 
                 N/A 
                 80 
                 97 
                 114 
               
               
                 3229 
                   
                 718 
                   
                   
                   
                   
                   
                   
                   
               
               
                 AF00 
                 PD-1/IL-2 
                 AB001 
                 FIG. 9b 
                 2 
                 1 
                 N/A 
                 81 
                 98 
                 115 
               
               
                 3230 
                   
                 744 
                   
                   
                   
                   
                   
                   
                   
               
               
                 AF00 
                 PD-1/IL-2 
                 AB002 
                 FIG. 9b 
                 2 
                 1 
                 N/A 
                 82 
                 99 
                 116 
               
               
                 3232 
                   
                 022 
                   
                   
                   
                   
                   
                   
                   
               
               
                 AF00 
                 PD-1/IL-2 
                 AB001 
                 FIG. 9a 
                 1 
                 1 
                 anti-PD-1 
                 83 
                 100 
                 117 
               
               
                 3250 
                   
                 718 
                   
                   
                   
                   
                   
                   
                   
               
               
                 AF00 
                 PD-1/IL-2 
                 AB001 
                 FIG. 9a 
                 1 
                 1 
                 anti-PD-1 
                 84 
                 101 
                 118 
               
               
                 3251 
                   
                 744 
                   
                   
                   
                   
                   
                   
                   
               
               
                 AF00 
                 PD-1/IL-2 
                 AB002 
                 FIG. 9a 
                 1 
                 1 
                 anti-PD-1 
                 85 
                 102 
                 119 
               
               
                 3253 
                   
                 022 
                   
                   
                   
                   
                   
                   
                   
               
               
                 AF00 
                 PD-L1/ 
                 AB001 
                 FIG. 9b 
                 2 
                 1 
                 N/A 
                 86 
                 103 
                 120 
               
               
                 3103 
                 IFNα 
                 609 
                   
                   
                   
                   
                   
                   
                   
               
               
                 AF00 
                 PD-L1/ 
                 AB001 
                 FIG. 9b 
                 2 
                 1 
                 N/A 
                 87 
                 104 
                 126 
               
               
                 3104 
                 IFNα 
                 909 
                   
                   
                   
                   
                   
                   
                   
               
               
                 AF00 
                 PD-L1/ 
                 AB001 
                 FIG. 9b 
                 2 
                 1 
                 N/A 
                 88 
                 105 
                 122 
               
               
                 3105 
                 IFNα 
                 843 
                   
                   
                   
                   
                   
                   
                   
               
               
                 AF00 
                 PD-L1/ 
                 AB001 
                 FIG. 9b 
                 2 
                 1 
                 N/A 
                 89 
                 106 
                 123 
               
               
                 3106 
                 IFNα 
                 875 
                   
                   
                   
                   
                   
                   
                   
               
               
                 AF00 
                 PD-L1/ 
                 AB001 
                 FIG. 9a 
                 1 
                 1 
                 anti-PD- 
                 90 
                 107 
                 124 
               
               
                 3217 
                 IFNα 
                 609 
                   
                   
                   
                 L1 
                   
                   
                   
               
               
                 AF00 
                 PD-L1/ 
                 AB001 
                 FIG. 9a 
                 1 
                 1 
                 anti-PD- 
                 91 
                 108 
                 125 
               
               
                 3218 
                 IFNα 
                 843 
                   
                   
                   
                 L1 
                   
                   
                   
               
               
                 AF00 
                 PD-L1/ 
                 AB001 
                 FIG. 9a 
                 1 
                 1 
                 anti-PD- 
                 92 
                 109 
                 126 
               
               
                 3219 
                 IFNα 
                 909 
                   
                   
                   
                 L1 
                   
                   
                   
               
               
                 AF00 
                 PD-L1/ 
                 AB001 
                 FIG. 9d 
                 2 
                 2 
                 N/A 
                 93 
                   
                 110 
               
               
                 2618 
                 IFNα 
                 609 
                   
                   
                   
                   
                   
                   
                   
               
               
                 AF00 
                 PD-L1/ 
                 AB001 
                 FIG. 9d 
                 2 
                 2 
                 N/A 
                 94 
                   
                 111 
               
               
                 2639 
                 IFNα 
                 875 
                   
                   
                   
                   
                   
                   
                   
               
               
                 AF00 
                 PD-L1/ 
                 AB001 
                 FIG. 9e 
                 2 
                 2 
                 N/A 
                 95 
                   
                 112 
               
               
                 2645 
                 IFNα 
                 609 
                   
                   
                   
                   
                   
                   
                   
               
               
                 AF00 
                 PD-L1/ 
                 AB001 
                 FIG. 9e 
                 2 
                 2 
                 N/A 
                 96 
                   
                 277 
               
               
                 2666 
                 IFNα 
                 875 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 16 
               
             
            
               
                   
               
               
                 Sequences of Peptides in TABLE 15 
               
            
           
           
               
               
               
            
               
                 SEQ ID 
                   
                   
               
               
                 NO: 
                 DBA 
                 Sequence 
               
               
                   
               
               
                 SEQ ID 
                 AF003229_ 
                 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTF 
               
               
                 NO: 80 
                 Pep1 
                 KFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPR 
               
               
                   
                   
                 DLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQ 
               
               
                   
                   
                 SIISTLTGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKP 
               
               
                   
                   
                 GASVKVSCKASGDTFSTYYVHWVRQAPGQGLEWMGIINPS 
               
               
                   
                   
                 GGGTVYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVY 
               
               
                   
                   
                 YCAAGLFIWGQGTLVTVSSAKTTAPSVYPLAPVCGDTTGSS 
               
               
                   
                   
                 VTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYT 
               
               
                   
                   
                 LSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKP 
               
               
                   
                   
                 CPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDV 
               
               
                   
                   
                 SEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALP 
               
               
                   
                   
                 IQHQDWMSGKEFKCKVNNKDLGAPIERTISKPKGSVRAPQV 
               
               
                   
                   
                 YVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTE 
               
               
                   
                   
                 LNYKNTEPVLDSDGSYFMYSDLRVEKKNWVERNSYSCSVV 
               
               
                   
                   
                 HEGLHNHHTTESFSRTPGK 
               
               
                   
               
               
                 SEQ ID 
                 AF003230_ 
                 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTF 
               
               
                 NO: 81 
                 Pep1 
                 KFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPR 
               
               
                   
                   
                 DLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQ 
               
               
                   
                   
                 SIISTLTGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKP 
               
               
                   
                   
                 GASVKVSCKASGYTFSNYYIHWVRQAPGQGLEWMGIINPS 
               
               
                   
                   
                 GGGTVYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVY 
               
               
                   
                   
                 YCAAGLFIWGQGTLVTVSSAKTTAPSVYPLAPVCGDTTGSS 
               
               
                   
                   
                 VTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYT 
               
               
                   
                   
                 LSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKP 
               
               
                   
                   
                 CPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDV 
               
               
                   
                   
                 SEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALP 
               
               
                   
                   
                 IQHQDWMSGKEFKCKVNNKDLGAPIERTISKPKGSVRAPQV 
               
               
                   
                   
                 YVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTE 
               
               
                   
                   
                 LNYKNTEPVLDSDGSYFMYSDLRVEKKNWVERNSYSCSVV 
               
               
                   
                   
                 HEGLHNHHTTESFSRTPGK 
               
               
                   
               
               
                 SEQ ID 
                 AF003232_ 
                 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTF 
               
               
                 NO: 82 
                 Pep1 
                 KFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPR 
               
               
                   
                   
                 DLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQ 
               
               
                   
                   
                 SIISTLTGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKP 
               
               
                   
                   
                 GASVKVSCKASGDTFTRHYVHWVRQAPGQGLEWMGIINPS 
               
               
                   
                   
                 GGYASYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVY 
               
               
                   
                   
                 YCAAGLFIWGQGTLVTVSSAKTTAPSVYPLAPVCGDTTGSS 
               
               
                   
                   
                 VTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYT 
               
               
                   
                   
                 LSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKP 
               
               
                   
                   
                 CPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDV 
               
               
                   
                   
                 SEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALP 
               
               
                   
                   
                 IQHQDWMSGKEFKCKVNNKDLGAPIERTISKPKGSVRAPQV 
               
               
                   
                   
                 YVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTE 
               
               
                   
                   
                 LNYKNTEPVLDSDGSYFMYSDLRVEKKNWVERNSYSCSVV 
               
               
                   
                   
                 HEGLHNHHTTESFSRTPGK 
               
               
                   
               
               
                 SEQ ID 
                 AF003250_ 
                 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTF 
               
               
                 NO: 83 
                 Pep1 
                 KFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPR 
               
               
                   
                   
                 DLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQ 
               
               
                   
                   
                 SIISTLTGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKP 
               
               
                   
                   
                 GASVKVSCKASGDTFSTYYVHWVRQAPGQGLEWMGIINPS 
               
               
                   
                   
                 GGGTVYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVY 
               
               
                   
                   
                 YCAAGLFIWGQGTLVTVSSAKTTAPSVYPLAPVCGDTTGSS 
               
               
                   
                   
                 VTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYT 
               
               
                   
                   
                 LSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKP 
               
               
                   
                   
                 CPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDV 
               
               
                   
                   
                 SEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALP 
               
               
                   
                   
                 IQHQDWMSGKEFKCKVNNKDLGAPIERTISKPKGSVRAPQV 
               
               
                   
                   
                 YVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTE 
               
               
                   
                   
                 LNYKNTEPVLDSDGSYFMYSDLRVEKKNWVERNSYSCSVV 
               
               
                   
                   
                 HEGLHNHHTTESFSRTPGK 
               
               
                   
               
               
                 SEQ ID 
                 AF003251_ 
                 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTF 
               
               
                 NO: 84 
                 Pep1 
                 KFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPR 
               
               
                   
                   
                 DLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQ 
               
               
                   
                   
                 SIISTLTGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKP 
               
               
                   
                   
                 GASVKVSCKASGYTFSNYYIHWVRQAPGQGLEWMGIINPS 
               
               
                   
                   
                 GGGTVYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVY 
               
               
                   
                   
                 YCAAGLFIWGQGTLVTVSSAKTTAPSVYPLAPVCGDTTGSS 
               
               
                   
                   
                 VTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYT 
               
               
                   
                   
                 LSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKP 
               
               
                   
                   
                 CPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDV 
               
               
                   
                   
                 SEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALP 
               
               
                   
                   
                 IQHQDWMSGKEFKCKVNNKDLGAPIERTISKPKGSVRAPQV 
               
               
                   
                   
                 YVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTE 
               
               
                   
                   
                 LNYKNTEPVLDSDGSYFMYSDLRVEKKNWVERNSYSCSVV 
               
               
                   
                   
                 HEGLHNHHTTESFSRTPGK 
               
               
                   
               
               
                 SEQ ID 
                 AF003253_ 
                 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTF 
               
               
                 NO: 85 
                 Pep1 
                 KFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPR 
               
               
                   
                   
                 DLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQ 
               
               
                   
                   
                 SIISTLTGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKP 
               
               
                   
                   
                 GASVKVSCKASGDTFTRHYVHWVRQAPGQGLEWMGIINPS 
               
               
                   
                   
                 GGYASYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVY 
               
               
                   
                   
                 YCAAGLFIWGQGTLVTVSSAKTTAPSVYPLAPVCGDTTGSS 
               
               
                   
                   
                 VTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYT 
               
               
                   
                   
                 LSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKP 
               
               
                   
                   
                 CPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDV 
               
               
                   
                   
                 SEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALP 
               
               
                   
                   
                 IQHQDWMSGKEFKCKVNNKDLGAPIERTISKPKGSVRAPQV 
               
               
                   
                   
                 YVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTE 
               
               
                   
                   
                 LNYKNTEPVLDSDGSYFMYSDLRVEKKNWVERNSYSCSVV 
               
               
                   
                   
                 HEGLHNHHTTESFSRTPGK 
               
               
                   
               
               
                 SEQ ID 
                 AF003103_ 
                 CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEE 
               
               
                 NO: 86 
                 Pep1 
                 FGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKF 
               
               
                   
                   
                 CTELYQQLNDLEACVMQEERVGETPLMNADSILAVKKYFR 
               
               
                   
                   
                 RITLYLTEKKYSPCAWEVVRAEIVRSLSLSTNLQERLRRKEG 
               
               
                   
                   
                 GGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKV 
               
               
                   
                   
                 SCKASGGTFSSYAISWVRQAPGQGLEWMGIIDPSVTYTRYA 
               
               
                   
                   
                 QKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSLFP 
               
               
                   
                   
                 TIFGVEVAFDIWGQGTLVTVSSAKTTAPSVYPLAPVCGDTT 
               
               
                   
                   
                 GSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSD 
               
               
                   
                   
                 LYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPT 
               
               
                   
                   
                 IKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVV 
               
               
                   
                   
                 DVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVS 
               
               
                   
                   
                 ALPIQHQDWMSGKEFKCKVNNKDLGAPIERTISKPKGSVRA 
               
               
                   
                   
                 PQVCVLPPPEEEMTKKQVTLWCMVTDFMPEDIYVEWTNNG 
               
               
                   
                   
                 KTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSC 
               
               
                   
                   
                 SVVHEGLHNHHTTKSFSRTPGK 
               
               
                   
               
               
                 SEQ ID 
                 AF003104_ 
                 CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEE 
               
               
                 NO: 87 
                 Pep1 
                 FGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKF 
               
               
                   
                   
                 CTELYQQLNDLEACVMQEERVGETPLMNADSILAVKKYFR 
               
               
                   
                   
                 RITLYLTEKKYSPCAWEVVRAEIVRSLSLSTNLQERLRRKEG 
               
               
                   
                   
                 GGGSGGGGSGGGGSGGGGS 
               
               
                   
                   
                 QVQLVQSGAEVKKPGASVKVSCKASGYTFSNYYIHWVRQA 
               
               
                   
                   
                 PGQGLEWMGWMDSNSGGTGYAQKFQGRVTMTRDTSTSTV 
               
               
                   
                   
                 YMELSSLRSEDTAVYYCAKEVFSGWYDYWGQGTLVTVSS 
               
               
                   
                   
                 AKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTW 
               
               
                   
                   
                 NSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNV 
               
               
                   
                   
                 AHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFP 
               
               
                   
                   
                 PKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHT 
               
               
                   
                   
                 AQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNK 
               
               
                   
                   
                 DLGAPIERTISKPKGSVRAPQVCVLPPPEEEMTKKQVTLWC 
               
               
                   
                   
                 MVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMY 
               
               
                   
                   
                 SKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK 
               
               
                   
               
               
                 SEQ ID 
                 AF003105_ 
                 CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEE 
               
               
                 NO: 88 
                 Pep1 
                 FGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKF 
               
               
                   
                   
                 CTELYQQLNDLEACVMQEERVGETPLMNADSILAVKKYFR 
               
               
                   
                   
                 RITLYLTEKKYSPCAWEVVRAEIVRSLSLSTNLQERLRRKEG 
               
               
                   
                   
                 GGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKV 
               
               
                   
                   
                 SCKASGYTFSGYYIHWVRQAPGQGLEWMGWMDSNSGGTG 
               
               
                   
                   
                 YAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAKE 
               
               
                   
                   
                 VFSGWYDYWGQGTLVTVSSAKTTAPSVYPLAPVCGDTTGS 
               
               
                   
                   
                 SVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLY 
               
               
                   
                   
                 TLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIK 
               
               
                   
                   
                 PCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVD 
               
               
                   
                   
                 VSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSA 
               
               
                   
                   
                 LPIQHQDWMSGKEFKCKVNNKDLGAPIERTISKPKGSVRAP 
               
               
                   
                   
                 QVCVLPPPEEEMTKKQVTLWCMVTDFMPEDIYVEWTNNG 
               
               
                   
                   
                 KTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSC 
               
               
                   
                   
                 SVVHEGLHNHHTTKSFSRTPGK 
               
               
                   
               
               
                 SEQ ID 
                 AF003106_ 
                 CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEE 
               
               
                 NO: 89 
                 Pep1 
                 FGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKF 
               
               
                   
                   
                 CTELYQQLNDLEACVMQEERVGETPLMNADSILAVKKYFR 
               
               
                   
                   
                 RITLYLTEKKYSPCAWEVVRAEIVRSLSLSTNLQERLRRKEG 
               
               
                   
                   
                 GGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKV 
               
               
                   
                   
                 SCKASGYTFSNYYIHWVRQAPGQGLEWMGWMDSNSGYTG 
               
               
                   
                   
                 YAQQFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAKE 
               
               
                   
                   
                 VFSGWYDYWGQGTLVTVSSAKTTAPSVYPLAPVCGDTTGS 
               
               
                   
                   
                 SVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLY 
               
               
                   
                   
                 TLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIK 
               
               
                   
                   
                 PCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVD 
               
               
                   
                   
                 VSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSA 
               
               
                   
                   
                 LPIQHQDWMSGKEFKCKVNNKDLGAPIERTISKPKGSVRAP 
               
               
                   
                   
                 QVCVLPPPEEEMTKKQVTLWCMVTDFMPEDIYVEWTNNG 
               
               
                   
                   
                 KTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSC 
               
               
                   
                   
                 SVVHEGLHNHHTTKSFSRTPGK 
               
               
                   
               
               
                 SEQ ID 
                 AF003217_ 
                 CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEE 
               
               
                 NO: 90 
                 Pep1 
                 FGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKF 
               
               
                   
                   
                 CTELYQQLNDLEACVMQEERVGETPLMNADSILAVKKYFR 
               
               
                   
                   
                 RITLYLTEKKYSPCAWEVVRAEIVRSLSLSTNLQERLRRKEG 
               
               
                   
                   
                 GGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKV 
               
               
                   
                   
                 SCKASGGTFSSYAISWVRQAPGQGLEWMGIIDPSVTYTRYA 
               
               
                   
                   
                 QKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSLFP 
               
               
                   
                   
                 TIFGVEVAFDIWGQGTLVTVSSAKTTAPSVYPLAPVCGDTT 
               
               
                   
                   
                 GSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSD 
               
               
                   
                   
                 LYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPT 
               
               
                   
                   
                 IKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVV 
               
               
                   
                   
                 DVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVS 
               
               
                   
                   
                 ALPIQHQDWMSGKEFKCKVNNKDLGAPIERTISKPKGSVRA 
               
               
                   
                   
                 PQVCVLPPPEEEMTKKQVTLWCMVTDFMPEDIYVEWTNNG 
               
               
                   
                   
                 KTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSC 
               
               
                   
                   
                 SVVHEGLHNHHTTKSFSRTPGK 
               
               
                   
               
               
                 SEQ ID 
                 AF003218_ 
                 CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEE 
               
               
                 NO: 91 
                 Pep1 
                 FGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKF 
               
               
                   
                   
                 CTELYQQLNDLEACVMQEERVGETPLMNADSILAVKKYFR 
               
               
                   
                   
                 RITLYLTEKKYSPCAWEVVRAEIVRSLSLSTNLQERLRRKEG 
               
               
                   
                   
                 GGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKV 
               
               
                   
                   
                 SCKASGYTFSGYYIHWVRQAPGQGLEWMGWMDSNSGGTG 
               
               
                   
                   
                 YAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAKE 
               
               
                   
                   
                 VFSGWYDYWGQGTLVTVSSAKTTAPSVYPLAPVCGDTTGS 
               
               
                   
                   
                 SVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLY 
               
               
                   
                   
                 TLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIK 
               
               
                   
                   
                 PCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVD 
               
               
                   
                   
                 VSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSA 
               
               
                   
                   
                 LPIQHQDWMSGKEFKCKVNNKDLGAPIERTISKPKGSVRAP 
               
               
                   
                   
                 QVCVLPPPEEEMTKKQVTLWCMVTDFMPEDIYVEWTNNG 
               
               
                   
                   
                 KTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSC 
               
               
                   
                   
                 SVVHEGLHNHHTTKSFSRTPGK 
               
               
                   
               
               
                 SEQ ID 
                 AF003219_ 
                 CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEE 
               
               
                 NO: 92 
                 Pep1 
                 FGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKF 
               
               
                   
                   
                 CTELYQQLNDLEACVMQEERVGETPLMNADSILAVKKYFR 
               
               
                   
                   
                 RITLYLTEKKYSPCAWEVVRAEIVRSLSLSTNLQERLRRKEG 
               
               
                   
                   
                 GGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKV 
               
               
                   
                   
                 SCKASGYTFSNYYIHWVRQAPGQGLEWMGWMDSNSGGTG 
               
               
                   
                   
                 YAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAKE 
               
               
                   
                   
                 VFSGWYDYWGQGTLVTVSSAKTTAPSVYPLAPVCGDTTGS 
               
               
                   
                   
                 SVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLY 
               
               
                   
                   
                 TLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIK 
               
               
                   
                   
                 PCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVD 
               
               
                   
                   
                 VSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSA 
               
               
                   
                   
                 LPIQHQDWMSGKEFKCKVNNKDLGAPIERTISKPKGSVRAP 
               
               
                   
                   
                 QVCVLPPPEEEMTKKQVTLWCMVTDFMPEDIYVEWTNNG 
               
               
                   
                   
                 KTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSC 
               
               
                   
                   
                 SVVHEGLHNHHTTKSFSRTPGK 
               
               
                   
               
               
                 SEQ ID 
                 AF002618_ 
                 QVQLVQSGAEVKKPGASVKVSCKASGGTFSSYAISWVRQA 
               
               
                 NO: 93 
                 Pep1 
                 PGQGLEWMGIIDPSVTYTRYAQKFQGRVTMTRDTSTSTVY 
               
               
                   
                   
                 MELSSLRSEDTAVYYCARSLFPTIFGVEVAFDIWGQGTLVTV 
               
               
                   
                   
                 SSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLT 
               
               
                   
                   
                 WNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITC 
               
               
                   
                   
                 NVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFI 
               
               
                   
                   
                 FPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEV 
               
               
                   
                   
                 HTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVN 
               
               
                   
                   
                 NKDLGAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLT 
               
               
                   
                   
                 CMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFM 
               
               
                   
                   
                 YSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPG 
               
               
                   
                   
                 K 
               
               
                   
               
               
                 SEQ ID 
                 AF002639_ 
                 QVQLVQSGAEVKKPGASVKVSCKASGYTFSNYYIHWVRQA 
               
               
                 NO: 94 
                 Pep1 
                 PGQGLEWMGWMDSNSGYTGYAQQFQGRVTMTRDTSTSTV 
               
               
                   
                   
                 YMELSSLRSEDTAVYYCAKEVFSGWYDYWGQGTLVTVSSA 
               
               
                   
                   
                 KTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWN 
               
               
                   
                   
                 SGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVA 
               
               
                   
                   
                 HPASSTKVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPP 
               
               
                   
                   
                 KIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHT 
               
               
                   
                   
                 AQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNK 
               
               
                   
                   
                 DLGAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCM 
               
               
                   
                   
                 VTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYS 
               
               
                   
                   
                 KLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK 
               
               
                   
               
               
                 SEQ ID 
                 AF002645_ 
                 CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEE 
               
               
                 NO: 95 
                 Pep1 
                 FGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKF 
               
               
                   
                   
                 YTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQR 
               
               
                   
                   
                 ITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEVP 
               
               
                   
                   
                 GVGVPGAGVPGVGVPGGGVPGVGVPGGGVPGAGVPGGGV 
               
               
                   
                   
                 PGVGVPGAGVPGVGVPGGGQVQLVQSGAEVKKPGASVKV 
               
               
                   
                   
                 SCKASGGTFSSYAISWVRQAPGQGLEWMGIIDPSVTYTRYA 
               
               
                   
                   
                 QKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSLFP 
               
               
                   
                   
                 TIFGVEVAFDIWGQGTLVTVSSAKTTAPSVYPLAPVCGDTT 
               
               
                   
                   
                 GSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSD 
               
               
                   
                   
                 LYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPT 
               
               
                   
                   
                 IKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVV 
               
               
                   
                   
                 DVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVS 
               
               
                   
                   
                 ALPIQHQDWMSGKEFKCKVNNKDLGAPIERTISKPKGSVRA 
               
               
                   
                   
                 PQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNG 
               
               
                   
                   
                 KTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSC 
               
               
                   
                   
                 SVVHEGLHNHHTTKSFSRTPGK 
               
               
                   
               
               
                 SEQ ID 
                 AF002666_ 
                 CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEE 
               
               
                 NO: 96 
                 Pep1 
                 FGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKF 
               
               
                   
                   
                 YTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQR 
               
               
                   
                   
                 ITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEVP 
               
               
                   
                   
                 GVGVPGAGVPGVGVPGGGVPGVGVPGGGVPGAGVPGGGV 
               
               
                   
                   
                 PGVGVPGAGVPGVGVPGGGQVQLVQSGAEVKKPGASVKV 
               
               
                   
                   
                 SCKASGYTFSNYYIHWVRQAPGQGLEWMGWMDSNSGYTG 
               
               
                   
                   
                 YAQQFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAKE 
               
               
                   
                   
                 VFSGWYDYWGQGTLVTVSSAKTTAPSVYPLAPVCGDTTGS 
               
               
                   
                   
                 SVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLY 
               
               
                   
                   
                 TLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIK 
               
               
                   
                   
                 PCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVD 
               
               
                   
                   
                 VSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSA 
               
               
                   
                   
                 LPIQHQDWMSGKEFKCKVNNKDLGAPIERTISKPKGSVRAP 
               
               
                   
                   
                 QVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGK 
               
               
                   
                   
                 TELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCS 
               
               
                   
                   
                 VVHEGLHNHHTTKSFSRTPGK 
               
               
                   
               
               
                 SEQ ID 
                 AF003229_ 
                 QVQLVQSGAEVKKPGASVKVSCKASGDTFSTYYVHWVRQ 
               
               
                 NO: 97 
                 Pep2 
                 APGQGLEWMGIINPSGGGTVYAQKFQGRVTMTRDTSTSTV 
               
               
                   
                   
                 YMELSSLRSEDTAVYYCAAGLFIWGQGTLVTVSSAKTTAPS 
               
               
                   
                   
                 VYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSS 
               
               
                   
                   
                 GVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASS 
               
               
                   
                   
                 TKVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDV 
               
               
                   
                   
                 LMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQT 
               
               
                   
                   
                 HREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLGAP 
               
               
                   
                   
                 IERTISKPKGSVRAPQVYVLPPPEKEMTKKQVSLTCLVKDFM 
               
               
                   
                   
                 PEDIYVEWTNNGKTELNYKNTEPVLKSDGSYFMYSKLTVE 
               
               
                   
                   
                 KKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGGGGSGGG 
               
               
                   
                   
                 SHHHHHH 
               
               
                   
               
               
                 SEQ ID 
                 AF003230_ 
                 QVQLVQSGAEVKKPGASVKVSCKASGYTFSNYYIHWVRQA 
               
               
                 NO: 98 
                 Pep2 
                 PGQGLEWMGIINPSGGGTVYAQKFQGRVTMTRDTSTSTVY 
               
               
                   
                   
                 MELSSLRSEDTAVYYCAAGLFIWGQGTLVTVSSAKTTAPSV 
               
               
                   
                   
                 YPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSG 
               
               
                   
                   
                 VHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASST 
               
               
                   
                   
                 KVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVL 
               
               
                   
                   
                 MISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTH 
               
               
                   
                   
                 REDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLGAPI 
               
               
                   
                   
                 ERTISKPKGSVRAPQVYVLPPPEKEMTKKQVSLTCLVKDFM 
               
               
                   
                   
                 PEDIYVEWTNNGKTELNYKNTEPVLKSDGSYFMYSKLTVE 
               
               
                   
                   
                 KKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGGGGSGGG 
               
               
                   
                   
                 SHHHHHH 
               
               
                   
               
               
                 SEQ ID 
                 AF003232_ 
                 QVQLVQSGAEVKKPGASVKVSCKASGDTFTRHYVHWVRQ 
               
               
                 NO: 99 
                 Pep2 
                 APGQGLEWMGIINPSGGYASYAQKFQGRVTMTRDTSTSTV 
               
               
                   
                   
                 YMELSSLRSEDTAVYYCAAGLFIWGQGTLVTVSSAKTTAPS 
               
               
                   
                   
                 VYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSS 
               
               
                   
                   
                 GVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASS 
               
               
                   
                   
                 TKVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDV 
               
               
                   
                   
                 LMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQT 
               
               
                   
                   
                 HREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLGAP 
               
               
                   
                   
                 IERTISKPKGSVRAPQVYVLPPPEKEMTKKQVSLTCLVKDFM 
               
               
                   
                   
                 PEDIYVEWTNNGKTELNYKNTEPVLKSDGSYFMYSKLTVE 
               
               
                   
                   
                 KKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGGGGSGGG 
               
               
                   
                   
                 SHHHHHH 
               
               
                   
               
               
                 SEQ ID 
                 AF003250_ 
                 QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYYVHWVRQ 
               
               
                 NO: 100 
                 Pep2 
                 APGQGLEWMGIINPSGGSTSYAQNFQGRVTMTRDTSTSTVY 
               
               
                   
                   
                 MELSSLRSEDTAVYYCASGWDVWGQGTTVTVSSASGGGGS 
               
               
                   
                   
                 GGGGSGGGGSHASEIVMTQSPATLSVSPGERATLSCRASQS 
               
               
                   
                   
                 VNTYLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGT 
               
               
                   
                   
                 EFTLTISSLQSEDFAVYYCQQYGSSPVTFGQGTRLEIKPRGPT 
               
               
                   
                   
                 IKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVV 
               
               
                   
                   
                 DVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVS 
               
               
                   
                   
                 ALPIQHQDWMSGKEFKCKVNNKDLGAPIERTISKPKGSVRA 
               
               
                   
                   
                 PQVYVLPPPEKEMTKKQVSLTCLVKDFMPEDIYVEWTNNG 
               
               
                   
                   
                 KTELNYKNTEPVLKSDGSYFMYSKLTVEKKNWVERNSYSC 
               
               
                   
                   
                 SVVHEGLHNHHTTKSFSRTPGGGGSGGGSHHHHHH 
               
               
                   
               
               
                 SEQ ID 
                 AF003251_ 
                 QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYYVHWVRQ 
               
               
                 NO: 101 
                 Pep2 
                 APGQGLEWMGIINPSGGSTSYAQNFQGRVTMTRDTSTSTVY 
               
               
                   
                   
                 MELSSLRSEDTAVYYCASGWDVWGQGTTVTVSSASGGGGS 
               
               
                   
                   
                 GGGGSGGGGSHASEIVMTQSPATLSVSPGERATLSCRASQS 
               
               
                   
                   
                 VNTYLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGT 
               
               
                   
                   
                 EFTLTISSLQSEDFAVYYCQQYGSSPVTFGQGTRLEIKPRGPT 
               
               
                   
                   
                 IKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVV 
               
               
                   
                   
                 DVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVS 
               
               
                   
                   
                 ALPIQHQDWMSGKEFKCKVNNKDLGAPIERTISKPKGSVRA 
               
               
                   
                   
                 PQVYVLPPPEKEMTKKQVSLTCLVKDFMPEDIYVEWTNNG 
               
               
                   
                   
                 KTELNYKNTEPVLKSDGSYFMYSKLTVEKKNWVERNSYSC 
               
               
                   
                   
                 SVVHEGLHNHHTTKSFSRTPGGGGSGGGSHHHHHH 
               
               
                   
               
               
                 SEQ ID 
                 AF003253_ 
                 QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYYVHWVRQ 
               
               
                 NO: 102 
                 Pep2 
                 APGQGLEWMGIINPSGGSTSYAQNFQGRVTMTRDTSTSTVY 
               
               
                   
                   
                 MELSSLRSEDTAVYYCASGWDVWGQGTTVTVSSASGGGGS 
               
               
                   
                   
                 GGGGSGGGGSHASEIVMTQSPATLSVSPGERATLSCRASQS 
               
               
                   
                   
                 VNTYLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGT 
               
               
                   
                   
                 EFTLTISSLQSEDFAVYYCQQYGSSPVTFGQGTRLEIKPRGPT 
               
               
                   
                   
                 IKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVV 
               
               
                   
                   
                 DVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVS 
               
               
                   
                   
                 ALPIQHQDWMSGKEFKCKVNNKDLGAPIERTISKPKGSVRA 
               
               
                   
                   
                 PQVYVLPPPEKEMTKKQVSLTCLVKDFMPEDIYVEWTNNG 
               
               
                   
                   
                 KTELNYKNTEPVLKSDGSYFMYSKLTVEKKNWVERNSYSC 
               
               
                   
                   
                 SVVHEGLHNHHTTKSFSRTPGGGGSGGGSHHHHHH 
               
               
                   
               
               
                 SEQ ID 
                 AF003103_ 
                 QVQLVQSGAEVKKPGASVKVSCKASGGTFSSYAISWVRQA 
               
               
                 NO: 103 
                 Pep2 
                 PGQGLEWMGIIDPSVTYTRYAQKFQGRVTMTRDTSTSTVY 
               
               
                   
                   
                 MELSSLRSEDTAVYYCARSLFPTIFGVEVAFDIWGQGTLVTV 
               
               
                   
                   
                 SSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLT 
               
               
                   
                   
                 WNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITC 
               
               
                   
                   
                 NVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFI 
               
               
                   
                   
                 FPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEV 
               
               
                   
                   
                 HTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVN 
               
               
                   
                   
                 NKDLGAPIERTISKPKGSVRAPQVYVLPPCEEEMTKKQVTLS 
               
               
                   
                   
                 CAVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFM 
               
               
                   
                   
                 VSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPG 
               
               
                   
                   
                 KHHHHHH 
               
               
                   
               
               
                 SEQ ID 
                 AF003104_ 
                 QVQLVQSGAEVKKPGASVKVSCKASGYTFSNYYIHWVRQA 
               
               
                 NO: 104 
                 Pep2 
                 PGQGLEWMGWMDSNSGGTGYAQKFQGRVTMTRDTSTSTV 
               
               
                   
                   
                 YMELSSLRSEDTAVYYCAKEVFSGWYDYWGQGTLVTVSSA 
               
               
                   
                   
                 KTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWN 
               
               
                   
                   
                 SGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVA 
               
               
                   
                   
                 HPASSTKVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPP 
               
               
                   
                   
                 KIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHT 
               
               
                   
                   
                 AQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNK 
               
               
                   
                   
                 DLGAPIERTISKPKGSVRAPQVYVLPPCEEEMTKKQVTLSCA 
               
               
                   
                   
                 VTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMVS 
               
               
                   
                   
                 KLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGKH 
               
               
                   
                   
                 HHHHH 
               
               
                   
               
               
                 SEQ ID 
                 AF003105_ 
                 QVQLVQSGAEVKKPGASVKVSCKASGYTFSGYYIHWVRQA 
               
               
                 NO: 105 
                 Pep2 
                 PGQGLEWMGWMDSNSGGTGYAQKFQGRVTMTRDTSTSTV 
               
               
                   
                   
                 YMELSSLRSEDTAVYYCAKEVFSGWYDYWGQGTLVTVSSA 
               
               
                   
                   
                 KTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWN 
               
               
                   
                   
                 SGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVA 
               
               
                   
                   
                 HPASSTKVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPP 
               
               
                   
                   
                 KIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHT 
               
               
                   
                   
                 AQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNK 
               
               
                   
                   
                 DLGAPIERTISKPKGSVRAPQVYVLPPCEEEMTKKQVTLSCA 
               
               
                   
                   
                 VTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMVS 
               
               
                   
                   
                 KLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGKH 
               
               
                   
                   
                 HHHHH 
               
               
                   
               
               
                 SEQ ID 
                 AF003106_ 
                 QVQLVQSGAEVKKPGASVKVSCKASGYTFSNYYIHWVRQA 
               
               
                 NO: 106 
                 Pep2 
                 PGQGLEWMGWMDSNSGYTGYAQQFQGRVTMTRDTSTSTV 
               
               
                   
                   
                 YMELSSLRSEDTAVYYCAKEVFSGWYDYWGQGTLVTVSSA 
               
               
                   
                   
                 KTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWN 
               
               
                   
                   
                 SGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVA 
               
               
                   
                   
                 HPASSTKVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPP 
               
               
                   
                   
                 KIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHT 
               
               
                   
                   
                 AQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNK 
               
               
                   
                   
                 DLGAPIERTISKPKGSVRAPQVYVLPPCEEEMTKKQVTLSCA 
               
               
                   
                   
                 VTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMVS 
               
               
                   
                   
                 KLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGKH 
               
               
                   
                   
                 HHHHH 
               
               
                   
               
               
                 SEQ ID 
                 AF003217_ 
                 QVQLVQSGAEVKKPGASVKVSCKASGYTFTKNYMHWVRQ 
               
               
                 NO: 107 
                 Pep2 
                 APGQGLEWLGWVSPDSGYTGYAQKFQGRVTMTRDTSTSTV 
               
               
                   
                   
                 YMELSSLRSEDTAVYYCTTDLLSLELDDAFDIWGQGTMVT 
               
               
                   
                   
                 VSSASGGGGSGGGGSGGGGSHASDIQMTQSPSSLSASVGDR 
               
               
                   
                   
                 VTITCRASQSISSWLAWYQQKPGKAPKLLIYAASTLQSGVPS 
               
               
                   
                   
                 RFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTK 
               
               
                   
                   
                 LEIKPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISL 
               
               
                   
                   
                 SPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDY 
               
               
                   
                   
                 NSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLGAPIERTIS 
               
               
                   
                   
                 KPKGSVRAPQVYVLPPCEEEMTKKQVTLSCAVTDFMPEDIY 
               
               
                   
                   
                 VEWTNNGKTELNYKNTEPVLDSDGSYFMVSKLRVEKKNW 
               
               
                   
                   
                 VERNSYSCSVVHEGLHNHHTTKSFSRTPGK 
               
               
                   
               
               
                 SEQ ID 
                 AF003218_ 
                 QVQLVQSGAEVKKPGASVKVSCKASGYTFTKNYMHWVRQ 
               
               
                 NO: 108 
                 Pep2 
                 APGQGLEWLGWVSPDSGYTGYAQKFQGRVTMTRDTSTSTV 
               
               
                   
                   
                 YMELSSLRSEDTAVYYCTTDLLSLELDDAFDIWGQGTMVT 
               
               
                   
                   
                 VSSASGGGGSGGGGSGGGGSHASDIQMTQSPSSLSASVGDR 
               
               
                   
                   
                 VTITCRASQSISSWLAWYQQKPGKAPKLLIYAASTLQSGVPS 
               
               
                   
                   
                 RFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTK 
               
               
                   
                   
                 LEIKPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISL 
               
               
                   
                   
                 SPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDY 
               
               
                   
                   
                 NSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLGAPIERTIS 
               
               
                   
                   
                 KPKGSVRAPQVYVLPPCEEEMTKKQVTLSCAVTDFMPEDIY 
               
               
                   
                   
                 VEWTNNGKTELNYKNTEPVLDSDGSYFMVSKLRVEKKNW 
               
               
                   
                   
                 VERNSYSCSVVHEGLHNHHTTKSFSRTPGK 
               
               
                   
               
               
                 SEQ ID 
                 AF003219_ 
                 QVQLVQSGAEVKKPGASVKVSCKASGYTFTKNYMHWVRQ 
               
               
                 NO: 109 
                 Pep2 
                 APGQGLEWLGWVSPDSGYTGYAQKFQGRVTMTRDTSTSTV 
               
               
                   
                   
                 YMELSSLRSEDTAVYYCTTDLLSLELDDAFDIWGQGTMVT 
               
               
                   
                   
                 VSSASGGGGSGGGGSGGGGSHASDIQMTQSPSSLSASVGDR 
               
               
                   
                   
                 VTITCRASQSISSWLAWYQQKPGKAPKLLIYAASTLQSGVPS 
               
               
                   
                   
                 RFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTK 
               
               
                   
                   
                 LEIKPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISL 
               
               
                   
                   
                 SPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDY 
               
               
                   
                   
                 NSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLGAPIERTIS 
               
               
                   
                   
                 KPKGSVRAPQVYVLPPCEEEMTKKQVTLSCAVTDFMPEDIY 
               
               
                   
                   
                 VEWTNNGKTELNYKNTEPVLDSDGSYFMVSKLRVEKKNW 
               
               
                   
                   
                 VERNSYSCSVVHEGLHNHHTTKSFSRTPGK 
               
               
                   
               
               
                 SEQ ID 
                 AF002618_ 
                 CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEE 
               
               
                 NO: 110 
                 Pep2 
                 FGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKF 
               
               
                   
                   
                 YTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQR 
               
               
                   
                   
                 ITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEGG 
               
               
                   
                   
                 GGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITC 
               
               
                   
                   
                 RASQSISNRLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGS 
               
               
                   
                   
                 GSGTDFTLTISSLQPEDFATYYCQQSNSTPFTFGQGTKVEIKR 
               
               
                   
                   
                 ADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKI 
               
               
                   
                   
                 DGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHN 
               
               
                   
                   
                 SYTCEATHKTSTSPIVKSFNRNEC 
               
               
                   
               
               
                 SEQ ID 
                 AF002639_ 
                 CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEE 
               
               
                 NO: 111 
                 Pep2 
                 FGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKF 
               
               
                   
                   
                 YTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQR 
               
               
                   
                   
                 ITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEGG 
               
               
                   
                   
                 GGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITC 
               
               
                   
                   
                 RASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGS 
               
               
                   
                   
                 GSGTDFTLTISSLQPEDFATYYCQQSYSTPYTFGQGTKVEIK 
               
               
                   
                   
                 RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWK 
               
               
                   
                   
                 IDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHN 
               
               
                   
                   
                 SYTCEATHKTSTSPIVKSFNRNEC 
               
               
                   
               
               
                 SEQ ID 
                 AF002645_ 
                 DIQMTQSPSSLSASVGDRVTITCRASQSISNRLAWYQQKPGK 
               
               
                 NO: 112 
                 Pep2 
                 APKLLIYKASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATY 
               
               
                   
                   
                 YCQQSNSTPFTFGQGTKVEIKRADAAPTVSIFPPSSEQLTSGG 
               
               
                   
                   
                 ASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSK 
               
               
                   
                   
                 DSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNR 
               
               
                   
                   
                 NEC 
               
               
                   
               
               
                 SEQ ID 
                 AF003229_ 
                 DIQMTQSPSSLSASVGDRVTITCRASQYISSGLAWYQQKPGK 
               
               
                 NO: 114 
                 Pep3 
                 APKLLIYKASSLDNGVPSRFSGSGSGTDFTLTISSLQPEDFAT 
               
               
                   
                   
                 YYCQQYERLPLTFGGGTKVEIKRADAAPTVSIFPPSSEQLTS 
               
               
                   
                   
                 GGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQD 
               
               
                   
                   
                 SKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSF 
               
               
                   
                   
                 NRNEC 
               
               
                   
               
               
                 SEQ ID 
                 AF003230_ 
                 DIQMTQSPSSLSASVGDRVTITCRASQSIGTGLAWYQQKPGK 
               
               
                 NO: 115 
                 Pep3 
                 APKLLIYKASSLDNGVPSRFSGSGSGTDFTLTISSLQPEDFAT 
               
               
                   
                   
                 YYCQQYNRAPLTFGGGTKVEIKRADAAPTVSIFPPSSEQLTS 
               
               
                   
                   
                 GGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQD 
               
               
                   
                   
                 SKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSF 
               
               
                   
                   
                 NRNEC 
               
               
                   
               
               
                 SEQ ID 
                 AF003232_ 
                 DIQMTQSPSSLSASVGDRVTITCRASQSIGRWLAWYQQKPG 
               
               
                 NO: 116 
                 Pep3 
                 KAPKLLIYSASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFA 
               
               
                   
                   
                 TYYCQQYESFPVTFGPGTKVDIKRADAAPTVSIFPPSSEQLTS 
               
               
                   
                   
                 GGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQD 
               
               
                   
                   
                 SKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSF 
               
               
                   
                   
                 NRNEC 
               
               
                   
               
               
                 SEQ ID 
                 AF003219_ 
                 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGK 
               
               
                 NO: 126 
                 Pep3 
                 APKLLIYAASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATY 
               
               
                   
                   
                 YCQQSYSTPYTFGQGTKVDIKRADAAPTVSIFPPSSEQLTSG 
               
               
                   
                   
                 GASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDS 
               
               
                   
                   
                 KDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFN 
               
               
                   
                   
                 RNEC 
               
               
                   
               
            
           
         
       
     
     Example 7 
     Reduced Type I IFNα Reporter Activation by a PD-L1/IFNα Protein Complex 
     This example demonstrates reduced Type I IFNα reporter activation by a PD-L1/IFNα protein complex of the present disclosure, specifically a PD-L1/IFNα DBA/cytokine complexes, relative to unregulated antibody-IFNα immune cytokines. The DBA-cytokine protein complexes and control immune cytokines used in this experiment were IgG proteins with IFNα fused to the N-terminus of the heavy chain through a linker composed of 4 repeats of “GGGGS,” as exemplified in  FIG.  9 E . The genes for two DBA-cytokine complexes, C08 IFNα 
                        (CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEM                   IQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAV               RKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEGGGGSGGGGSGGG               GSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGII               DPSVTYTRYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSLFPTIFGVEVA               FDIWGQGTLVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGS               LSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKP               CPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVE               VHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLGAPIERTISKPKG               SVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLD               SDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK SEQ ID NO: 57;               and               DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYGASTLQSGVPS               RFSGSGSGTDFTLTISSLQPEDFATYYCQQTYSTPITFGQGTKVEIKRADAAPTVSIFPPSS               EQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLT               LTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC; SEQ ID NO: 58)       and               B09 IFNα       (CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEM               IQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAV               RKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEGGGGSGGGGSGGG               GSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGGTFTGYYMHWVRQAPGQGLEWM               GWVNPNSGNTGYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSLFPTIFG               VEVAFDIWGQGTLVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTW               NSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGP               TIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVN               NVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLGAPIERTISK               PKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEP               VLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK; SEQ ID               NO: 59       and               DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYAASSLQSGVPS               RFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSPPPTFGQGTKLEIKRADAAPTVSIFPPSS               EQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLT               LTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC; SEQ ID NO: 60),       and               two control immune-cytokines, anti-Her2 IFNα       (CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEM               IQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAV               RKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEGGGGSGGGGSGGG               GSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGGTFSSYAISWVRQAPGQGLEWMG               WINPNSGGTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSLFPTIFGVE               VAFDIWGQGTTVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNS               GSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTI               KPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNN               VEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLGAPIERTISKP               KGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEP               VLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK; SEQ ID               NO: 61       and               DIQMTQSPSSLSASVGDRVTITCRASQSIIDRLAWYQQKPGKAPKLLIYKASSLESGVPSR               FSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPFTFGPGTKVDIKRADAAPTVSIFPPSSE               QLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTL               TKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC; SEQ ID NO: 62)       and               anti-PD-L1 IFNα       (CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEM               IQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAV               RKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEGGGGSGGGGSGGG               GSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARI               YPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMD               YWGQGTLVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSL               SSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPC               PPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEV               HTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLGAPIERTISKPKGS               VRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDS               DGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK; SEQ ID NO: 63       and               DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVP               SRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRADAAPTVSIFPPS               SEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTL               TLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC; SEQ ID NO: 64)            
were synthesized, expressed in HEK293 cells and Protein A purified (GenScript). Titrated concentrations of the DBA-cytokine complexes and antibody-cytokine controls were added to a 96-well plate along with 50,000 type I IFNα reporter cells (InvivoGen) in complete DMEM (+10% FBS, 2 mM L-glutamine, sodium pyruvate). Plates were incubated at 37° C. overnight and developed by adding 20 uL of culture supernatant to 180 uL QUANTI-Blue Solution (InvivoGen). After a 30 minute incubation at room temperature, plates were read on an Envision (Perkin Elmer) at 630 nm. In the absence of PD-L1, the IFNα tethered PD-L1-IFNα DBA complexes C08 and B09 show decreased reporter activation compared to equimolar amounts of the control anti-PD-L1 or anti-HER2 IFNα immunocytokines ( FIG.  10   ).
 
     Example 8 
     Reduced CD8 +  T Cell STAT5 Phosphorylation by a PD-1/IL-2 Dual Binding Antibody (DBA) Cytokine Complexes 
     This example describes CD8+ T-cell STAT5 phosphorylation by PD-1/IL-2 DBA-cytokine complexes of the present disclosure. Genes for the PD-1/IL-2 DBAs shown in TABLE 17 were synthesized and expressed in HEK293 as IgG proteins with IL-2 fused to the N-terminus of the heavy or light chain through a linker (Genscript). Although only two of the antibodies blocked IL-2 binding to IL-2RB as scFvs, over 30 of the antibodies were able to reduce IL-2 signaling by a linked IL-2 domain. An exemplary set of these DBAs were chosen for analysis and compared to a control anti-HER2-IL-2 immunocytokine (TABLE 17). 
     
       
         
           
               
             
               
                 TABLE 17 
               
             
            
               
                   
               
               
                 IgG PD-1/IL-2 DBA protein complexes 
               
            
           
           
               
               
               
            
               
                   
                 Heavy Chain Sequence 
                 Light Chain Sequence 
               
               
                 Name 
                 SEQ ID NO 
                 SEQ ID NO 
               
               
                   
               
               
                 Anti-HER2 
                 (SEQ ID NO: 65) 
                 (SEQ ID NO: 66) 
               
               
                   
                 EVQLVESGGGLVQPGGSLRLSCAA 
                 APTSSSTKKTQLQLEHLLLDLQ 
               
               
                   
                 SGFNIKDTYIHWVRQAPGKGLEWV 
                 MILNGINNYKNPKLTRMLTFKF 
               
               
                   
                 ARIYPTNGYTRYADSVKGRFTISAD 
                 YMPKKATELKHLQCLEEELKPL 
               
               
                   
                 TSKNTAYLQMNSLRAEDTAVYYCS 
                 EEVLNLAQSKNFHLRPRDLISNI 
               
               
                   
                 RWGGDGFYAMDYWGQGTLVTVSS 
                 NVIVLELKGSETTFMCEYADET 
               
               
                   
                 AKTTAPSVYPLAPVCGDTTGSSVTL 
                 ATIVEFLNRWITFCQSIISTLTVP 
               
               
                   
                 GCLVKGYFPEPVTLTWNSGSLSSG 
                 GVGVPGAGVPGVGVPGGGVPG 
               
               
                   
                 VHTFPAVLQSDLYTLSSSVTVTSST 
                 VGVPGGGVPGAGVPGGGVPGV 
               
               
                   
                 WPSQSITCNVAHPASSTKVDKKIEP 
                 GVPGAGVPGVGVPGGGDIQMT 
               
               
                   
                 RGPTIKPCPPCKCPAPNAAGGPSVFI 
                 QSPSSLSASVGDRVTITCRASQD 
               
               
                   
                 FPPKIKDVLMISLSPIVTCVVVDVSE 
                 VNTAVAWYQQKPGKAPKLLIY 
               
               
                   
                 DDPDVQISWFVNNVEVHTAQTQTH 
                 SASFLYSGVPSRFSGSRSGTDFT 
               
               
                   
                 REDYNSTLRVVSALPIQHQDWMSG 
                 LTISSLQPEDFATYYCQQHYTTP 
               
               
                   
                 KEFKCKVNNKDLGAPIERTISKPKG 
                 PTFGQGTKVEIKRADAAPTVSIF 
               
               
                   
                 SVRAPQVYVLPPPEEEMTKKQVTL 
                 PPSSEQLTSGGASVVCFLNNFYP 
               
               
                   
                 TCMVTDFMPEDIYVEWTNNGKTEL 
                 KDINVKWKIDGSERQNGVLNS 
               
               
                   
                 NYKNTEPVLDSDGSYFMYSKLRVE 
                 WTDQDSKDSTYSMSSTLTLTKD 
               
               
                   
                 KKNWVERNSYSCSVVHEGLHNHH 
                 EYERHNSYTCEATHKTSTSPIVK 
               
               
                   
                 TTKSFSRTPGK 
                 SFNRNEC 
               
               
                   
               
               
                 2-A08 
                 (SEQ ID NO: 67) 
                 (SEQ ID NO: 68) 
               
               
                   
                 QVQLVQSGAEVKKPGASVKVSCK 
                 APTSSSTKKTQLQLEHLLLDLQ 
               
               
                   
                 VSGYTFTSYDINWVRQAPGQGLEW 
                 MILNGINNYKNPKLTRMLTFKF 
               
               
                   
                 MGWINPNSGDTGYAQKFQGRVTM 
                 YMPKKATELKHLQCLEEELKPL 
               
               
                   
                 TRDTSTSTVYMELSSLRSEDTAVYY 
                 EEVLNLAQSKNFHLRPRDLISNI 
               
               
                   
                 CARDTGLGYYYGSGDFDYWGQGT 
                 NVIVLELKGSETTFMCEYADET 
               
               
                   
                 LVTVSSAKTTAPSVYPLAPVCGDTT 
                 ATIVEFLNRWITFCQSIISTLTVP 
               
               
                   
                 GSSVTLGCLVKGYFPEPVTLTWNS 
                 GVGVPGAGVPGVGVPGGGVPG 
               
               
                   
                 GSLSSGVHTFPAVLQSDLYTLSSSV 
                 VGVPGGGVPGAGVPGGGVPGV 
               
               
                   
                 TVTSSTWPSQSITCNVAHPASSTKV 
                 GVPGAGVPGVGVPGGGDIQMT 
               
               
                   
                 DKKIEPRGPTIKPCPPCKCPAPNAA 
                 QSPSSLSASVGDRVTITCQASQD 
               
               
                   
                 GGPSVFIFPPKIKDVLMISLSPIVTCV 
                 IHNYLNWYQQKPGKAPKLLIYD 
               
               
                   
                 VVDVSEDDPDVQISWFVNNVEVHT 
                 VSNLETGVPSRFSGSGSGTDFTL 
               
               
                   
                 AQTQTHREDYNSTLRVVSALPIQH 
                 TISSLQPEDFATYYCQQAISFPLT 
               
               
                   
                 QDWMSGKEFKCKVNNKDLGAPIE 
                 FGGGTKVEIKRADAAPTVSIFPP 
               
               
                   
                 RTISKPKGSVRAPQVYVLPPPEEEM 
                 SSEQLTSGGASVVCFLNNFYPK 
               
               
                   
                 TKKQVTLTCMVTDFMPEDIYVEWT 
                 DINVKWKIDGSERQNGVLNSW 
               
               
                   
                 NNGKTELNYKNTEPVLDSDGSYFM 
                 TDQDSKDSTYSMSSTLTLTKDE 
               
               
                   
                 YSKLRVEKKNWVERNSYSCSVVHE 
                 YERHNSYTCEATHKTSTSPIVKS 
               
               
                   
                 GLHNHHTTKSFSRTPGK 
                 FNRNEC 
               
               
                   
               
               
                 2-A11 
                 (SEQ ID NO: 69) 
                 (SEQ ID NO: 70) 
               
               
                   
                 QVQLVQSGAEVKKPGASVKVSCK 
                 APTSSSTKKTQLQLEHLLLDLQ 
               
               
                   
                 ASGHTFTRYYMHWVRQAPGQGLE 
                 MILNGINNYKNPKLTRMLTFKF 
               
               
                   
                 WMGIINPSGGYATYAQKFQGRVTM 
                 YMPKKATELKHLQCLEEELKPL 
               
               
                   
                 TRDTSTSTVYMELSSLRSEDTAVYY 
                 EEVLNLAQSKNFHLRPRDLISNI 
               
               
                   
                 CASGWDVWGQGTLVTVSSAKTTA 
                 NVIVLELKGSETTFMCEYADET 
               
               
                   
                 PSVYPLAPVCGDTTGSSVTLGCLVK 
                 ATIVEFLNRWITFCQSIISTLTVP 
               
               
                   
                 GYFPEPVTLTWNSGSLSSGVHTFPA 
                 GVGVPGAGVPGVGVPGGGVPG 
               
               
                   
                 VLQSDLYTLSSSVTVTSSTWPSQSIT 
                 VGVPGGGVPGAGVPGGGVPGV 
               
               
                   
                 CNVAHPASSTKVDKKIEPRGPTIKP 
                 GVPGAGVPGVGVPGGGDIQMT 
               
               
                   
                 CPPCKCPAPNAAGGPSVFIFPPKIKD 
                 QSPSSLSASVGDRVTITCRASQSI 
               
               
                   
                 VLMISLSPIVTCVVVDVSEDDPDVQ 
                 NSWLAWYQQKPGKAPKLLIYA 
               
               
                   
                 ISWFVNNVEVHTAQTQTHREDYNS 
                 TSTLESGVPSRFSGSGSGTDFTL 
               
               
                   
                 TLRVVSALPIQHQDWMSGKEFKCK 
                 TISSLQPEDFATYYCQQSYSFPP 
               
               
                   
                 VNNKDLGAPIERTISKPKGSVRAPQ 
                 TFGQGTKVEIKRADAAPTVSIFP 
               
               
                   
                 VYVLPPPEEEMTKKQVTLTCMVTD 
                 PSSEQLTSGGASVVCFLNNFYPK 
               
               
                   
                 FMPEDIYVEWTNNGKTELNYKNTE 
                 DINVKWKIDGSERQNGVLNSW 
               
               
                   
                 PVLDSDGSYFMYSKLRVEKKNWV 
                 TDQDSKDSTYSMSSTLTLTKDE 
               
               
                   
                 ERNSYSCSVVHEGLHNHHTTKSFSR 
                 YERHNSYTCEATHKTSTSPIVKS 
               
               
                   
                 TPGK 
                 FNRNEC 
               
               
                   
               
               
                 2-B05 
                 (SEQ ID NO: 71) 
                 (SEQ ID NO: 72) 
               
               
                   
                 QVQLVQSGAEVKKPGASVKVSCK 
                 APTSSSTKKTQLQLEHLLLDLQ 
               
               
                   
                 ASGYTFTNYYIHWVRQAPGQGLE 
                 MILNGINNYKNPKLTRMLTFKF 
               
               
                   
                 WMGIINPRAGYTSYALKFQGRVTM 
                 YMPKKATELKHLQCLEEELKPL 
               
               
                   
                 TRDTSTSTVYMELSSLRSEDTAVYY 
                 EEVLNLAQSKNFHLRPRDLISNI 
               
               
                   
                 CAGGWLDWGQGTLVTVSSAKTTA 
                 NVIVLELKGSETTFMCEYADET 
               
               
                   
                 PSVYPLAPVCGDTTGSSVTLGCLVK 
                 ATIVEFLNRWITFCQSIISTLTVP 
               
               
                   
                 GYFPEPVTLTWNSGSLSSGVHTFPA 
                 GVGVPGAGVPGVGVPGGGVPG 
               
               
                   
                 VLQSDLYTLSSSVTVTSSTWPSQSIT 
                 VGVPGGGVPGAGVPGGGVPGV 
               
               
                   
                 CNVAHPASSTKVDKKIEPRGPTIKP 
                 GVPGAGVPGVGVPGGGDIQMT 
               
               
                   
                 CPPCKCPAPNAAGGPSVFIFPPKIKD 
                 QSPSSLSASVGDRVTITCRASQSI 
               
               
                   
                 VLMISLSPIVTCVVVDVSEDDPDVQ 
                 SSWLAWYQQKPGKAPKLLIYA 
               
               
                   
                 ISWFVNNVEVHTAQTQTHREDYNS 
                 ASSLQSGVPSRFSGSGSGTDFTL 
               
               
                   
                 TLRVVSALPIQHQDWMSGKEFKCK 
                 TISSLQPEDFATYYCQQSFTMPI 
               
               
                   
                 VNNKDLGAPIERTISKPKGSVRAPQ 
                 TFGQGTRLEIKRADAAPTVSIFP 
               
               
                   
                 VYVLPPPEEEMTKKQVTLTCMVTD 
                 PSSEQLTSGGASVVCFLNNFYPK 
               
               
                   
                 FMPEDIYVEWTNNGKTELNYKNTE 
                 DINVKWKIDGSERQNGVLNSW 
               
               
                   
                 PVLDSDGSYFMYSKLRVEKKNWV 
                 TDQDSKDSTYSMSSTLTLTKDE 
               
               
                   
                 ERNSYSCSVVHEGLHNHHTTKSFSR 
                 YERHNSYTCEATHKTSTSPIVKS 
               
               
                   
                 TPGK 
                 FNRNEC 
               
               
                   
               
               
                 2-B07 
                 (SEQ ID NO: 73) 
                 (SEQ ID NO: 74) 
               
               
                   
                 QVQLVQSGAEVKKPGASVKVSCK 
                 APTSSSTKKTQLQLEHLLLDLQ 
               
               
                   
                 ASGDTFTRHYVHWVRQAPGQGLE 
                 MILNGINNYKNPKLTRMLTFKF 
               
               
                   
                 WMGIINPSGGYASYAQKFQGRVTM 
                 YMPKKATELKHLQCLEEELKPL 
               
               
                   
                 TRDTSTSTVYMELSSLRSEDTAVYY 
                 EEVLNLAQSKNFHLRPRDLISNI 
               
               
                   
                 CAAGLFIWGQGTLVTVSSAKTTAPS 
                 NVIVLELKGSETTFMCEYADET 
               
               
                   
                 VYPLAPVCGDTTGSSVTLGCLVKG 
                 ATIVEFLNRWITFCQSIISTLTVP 
               
               
                   
                 YFPEPVTLTWNSGSLSSGVHTFPAV 
                 GVGVPGAGVPGVGVPGGGVPG 
               
               
                   
                 LQSDLYTLSSSVTVTSSTWPSQSITC 
                 VGVPGGGVPGAGVPGGGVPGV 
               
               
                   
                 NVAHPASSTKVDKKIEPRGPTIKPC 
                 GVPGAGVPGVGVPGGGDIQMT 
               
               
                   
                 PPCKCPAPNAAGGPSVFIFPPKIKDV 
                 QSPSSLSASVGDRVTITCRASQSI 
               
               
                   
                 LMISLSPIVTCVVVDVSEDDPDVQIS 
                 GRWLAWYQQKPGKAPKLLIYS 
               
               
                   
                 WFVNNVEVHTAQTQTHREDYNST 
                 ASNLETGVPSRFSGSGSGTDFTL 
               
               
                   
                 LRVVSALPIQHQDWMSGKEFKCKV 
                 TISSLQPEDFATYYCQQANSFPV 
               
               
                   
                 NNKDLGAPIERTISKPKGSVRAPQV 
                 TFGPGTKVDIKRADAAPTVSIFP 
               
               
                   
                 YVLPPPEEEMTKKQVTLTCMVTDF 
                 PSSEQLTSGGASVVCFLNNFYPK 
               
               
                   
                 MPEDIYVEWTNNGKTELNYKNTEP 
                 DINVKWKIDGSERQNGVLNSW 
               
               
                   
                 VLDSDGSYFMYSKLRVEKKNWVE 
                 TDQDSKDSTYSMSSTLTLTKDE 
               
               
                   
                 RNSYSCSVVHEGLHNHHTTKSFSRT 
                 YERHNSYTCEATHKTSTSPIVKS 
               
               
                   
                 PGK 
                 FNRNEC 
               
               
                   
               
               
                 7-A04 
                 (SEQ ID NO: 75) 
                 (SEQ ID NO: 76) 
               
               
                   
                 QVQLVQSGAEVKKPGASVKVSCK 
                 APTSSSTKKTQLQLEHLLLDLQ 
               
               
                   
                 ASGYTFTDYYMHWVRQAPGQGLE 
                 MILNGINNYKNPKLTRMLTFKF 
               
               
                   
                 WMGIINPRAGYTSYALKFQGRVTM 
                 YMPKKATELKHLQCLEEELKPL 
               
               
                   
                 TRDTSTSTVYMELSSLRSEDTAVYY 
                 EEVLNLAQSKNFHLRPRDLISNI 
               
               
                   
                 CTSGMDVWGQGTLVTVSSAKTTAP 
                 NVIVLELKGSETTFMCEYADET 
               
               
                   
                 SVYPLAPVCGDTTGSSVTLGCLVK 
                 ATIVEFLNRWITFCQSIISTLTVP 
               
               
                   
                 GYFPEPVTLTWNSGSLSSGVHTFPA 
                 GVGVPGAGVPGVGVPGGGVPG 
               
               
                   
                 VLQSDLYTLSSSVTVTSSTWPSQSIT 
                 VGVPGGGVPGAGVPGGGVPGV 
               
               
                   
                 CNVAHPASSTKVDKKIEPRGPTIKP 
                 GVPGAGVPGVGVPGGGDIQMT 
               
               
                   
                 CPPCKCPAPNAAGGPSVFIFPPKIKD 
                 QSPSSLSASVGDRVTITCRASQSI 
               
               
                   
                 VLMISLSPIVTCVVVDVSEDDPDVQ 
                 STWLAWYQQKPGKAPKLLIYA 
               
               
                   
                 ISWFVNNVEVHTAQTQTHREDYNS 
                 ASSLQSGVPSRFSGSGSGTDFTL 
               
               
                   
                 TLRVVSALPIQHQDWMSGKEFKCK 
                 TISSLQPEDFATYYCQQSYSFPV 
               
               
                   
                 VNNKDLGAPIERTISKPKGSVRAPQ 
                 TFGQGTKVEIKRADAAPTVSIFP 
               
               
                   
                 VYVLPPPEEEMTKKQVTLTCMVTD 
                 PSSEQLTSGGASVVCFLNNFYPK 
               
               
                   
                 FMPEDIYVEWTNNGKTELNYKNTE 
                 DINVKWKIDGSERQNGVLNSW 
               
               
                   
                 PVLDSDGSYFMYSKLRVEKKNWV 
                 TDQDSKDSTYSMSSTLTLTKDE 
               
               
                   
                 ERNSYSCSVVHEGLHNHHTTKSFSR 
                 YERHNSYTCEATHKTSTSPIVKS 
               
               
                   
                 TPGK 
                 FNRNEC 
               
               
                   
               
            
           
         
       
     
     The PD-1/IL-2 DBA-cytokine complexes were serially diluted in complete RPMI (+1000 FBS, 2 mM L-glutamine, sodium pyruvate) and added to a 96-well plate. 2×10 5  human PBMCs were added to each well and plates were incubated at 37° C. for 20 minutes. An equal volume prewarmed fixation buffer (Biolegend) was then added to each well and plates were incubated at 37° C. for 10 minutes. Cells were then fixed in pre-chilled Perm Buffer III (BD Biosciences) for 30 minutes at 4′° C. Cells were washed with FACS wash buffer (PBS+2% FBS, 2 mM EDTA) and stained with fluorophore labeled antibodies directed against CD3, CD4, CD8, (BioLegend) and phospho-STAT5 (BD Biosciences) diluted 1:20 in FACS wash buffer. Cells were incubated 1 hour at 4° C., washed with FACS wash buffer, and analyzed on a SA3800 Spectral Analyzer. In the absence of PD-1, the PD-1/IL-2 DBA/cytokine complexes induced less STAT5 phosphorylation in T cells compared to the monospecific control anti-HER2 IL-2 immunocytokine ( FIG.  11   ). 
     Example 9 
     Regulated Interferon Receptor Binding by a PD-L1/IFNα Dual Binding Antibody (DBA) Cytokine Complex 
     This example describes regulated interferon receptor binding by a PD-L1/IFNα DBA-cytokine complex. DBA-cytokine complexes of SEQ ID NO: 41 and SEQ ID NO: 55 (MSTSTCDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIP VLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKE DSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEGGGGSGG GGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGNTFTDYYMHWVRQAPGQ GLEWMGWMNPNSGNTGYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARS LFPTIFGVEVAFDIWGQGTLVTVSSASGGGGSGGGGSGGGGSHASDIQMTQSPSSLSAS VGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYAASSLESGVPSRFSGSGSGTDFTLT ISSLQPEDFATYYCQQSYSTPPTFGQGTRLEIKGKPIPNPLLGLDST) were chosen for analysis with a negative control with a similar structure based on a HER2 binding scFv (SEQ ID NO: 56, MSTSTCDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPV LHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKED SILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSL STNLQESLRSKEGGGGSGGG GSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLE WVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDG FYAMDYWGQGTLVTVSSASGGGGSGGGGSGGGGSHASDIQMTQSPSSLSASVGDRVTI TCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPE DFATYYCQQHYTTPPTFGQGTKVEIKGKPIPNPLLGLDST). The proteins were expressed using a cell-free transcription/translation system (Cosmo Bio USA, Inc., PUREfrex2.1, Product #GFK-PF213 with DS Supplement, Prod. #GFK-PF005). 96-well ELISA plates were coated with anti-V5 antibody (SV5-pk1) at 50 ng/well overnight at 4° C. The plates were washed twice by adding 200 ul/well of SuperBlock with 0.05% Tween 20 (SBT), and the final SBT wash was incubated for 15 min at room temperature before aspiration. A dilution series of the protein for each construct in SBT was then added to the anti-V5-coated plates at 50 μl/well and incubated for 1 hour at room temperature. Each plate was then washed three times with PBS with 0.05% Tween 20 (PBST). Bound constructs were then probed with either anti-IFNα, IFNAR2-Biotin, PDL1-hFc-Avi or the combination of IFNAR2-Biotin and PDL1-hFc-Avi in SBT at 50 ul/well for 1 hour at room temperature. Plates were washed 3× with PBST. Goat anti-mIgG-TRP or Streptavidin-TRP was added at 50 ul/well and incubated for 30 min at room temperature followed by 3× wash with PBST. Plates were developed by adding 50 ul/well of TMB and the reaction was terminated with an equal volume of ELISA stop solution. As shown in  FIG.  12    (top left), IFNAR2 binding to the DBA-IFNα complex (SEQ ID NO: 41) increased in a dose dependent manner with the addition of PD-L1. IFNAR2 binding to a control HER2-specific antibody-IFNα complex (SEQ ID NO: 56) was unaffected by the addition of PD-L1 ( FIG.  12    top right). IFNAR2 binding to a DBA-IFNα complex containing SEQ ID NO: 55, was not affected by addition of PD-L1 at these concentrations ( FIG.  12    bottom). The protein complex of SEQ ID NO: 55 is similar to the protein complex of SEQ ID NO: 41, except that the sensor domain of SEQ ID NO: 55 has a higher affinity for IFNα than the sensor domain of SEQ ID NO: 41. Protein complexes of the present invention may need the correct balance between their affinity for the marker and their affinity for the therapeutic domain. 
     Example 10 
     PD-L1/IFNα Protein Complexes for PD-L1 Dependent IFNα Activity In Vitro 
     This example describes PD-L1/IFNα protein complexes for PD-L1 dependent IFNα activity in vitro. PD-L1/IFNα protein complexes comprise a DBA capable of binding PD-L1 marker and an IFNα therapeutic domain where the protein complex is linked to the IFNα therapeutic cytokine via a linker. In the absence of PD-L1, the PD-L1 sensor domain binds the IFNα therapeutic domain, rendering the IFNα therapeutic inert. In the presence of PD-L1 (e.g., PD-L1 is expressed on a cell, such as a tumor cell or immune cell), the PD-L1 sensor domain binds PD-L1, thereby unbinding the IFNα therapeutic domain and allowing for IFNα to exhibit therapeutic activity. 
     PD-L1/IFNα protein complexes are designed and recombinantly expressed or chemically synthesized. PD-L1/IFNα protein complexes are administered in vitro to a cell (e.g., in cell culture). In the absence of the PD-L1 marker, the IFNα domain remains bound to the PD-L1 sensor domain and no therapeutic effect is observed. The cell may express PD-L1 endogenously or after activation, or following introduction of a gene encoding PD-L1. If the cell is a tumor cell expressing a PD-L1 marker, the therapeutic effect may be inhibition of cell growth or induction of IFNα-responsive genes. Where the cell is an immune cell, the therapeutic effect may be cell growth, activation or induction of IFN-responsive genes. Where the cell is part of a mixture of cell types, any of these changes may be monitored for a responding cell population in the mixture. 
     Example 11 
     PD-L1 Dependent IFNα Activity In Vivo 
     This example describes PD-L1/IFNα protein complexes for PD-L1 dependent IFNα activity in vivo. PD-L1/IFNα protein complexes comprise a PD-L1 sensor domain (e.g., an anti-PD-L1 antibody or an anti-PD-L1 scFv) linked to an IFNα cytokine via a linker, where the IFNα cytokine is a therapeutic. In the absence of PD-L1, the PD-L1 sensor domain binds the IFNα therapeutic domain, rendering the IFNα therapeutic inert. In the presence of PD-L1 (e.g., PD-L1 is expressed on a cell, such as a tumor cell or immune cell), the PD-L1 sensor domain binds PD-L1, thereby unbinding the IFNα therapeutic domain and allowing for IFNα to exhibit therapeutic activity. 
     PD-L1/IFNα protein complexes are recombinantly expressed or chemically synthesized. PD-L1/IFNα protein complexes are administered in vivo to a subject in need thereof. Administration is performed intravenously, intramuscularly, subcutaneously, intradermally, intraperitoneally, or mucosally. In the absence of PD-L1, the IFNα therapeutic domain remains bound to the PD-L1 sensor domain and no therapeutic efficacy is observed (e.g., cell proliferation in the subject is uninhibited). In the presence of PD-L1, the PD-L1 sensor domain binds PD-L1 and unbinds the IFNα therapeutic domain. Therapeutic efficacy is observed (e.g., cell proliferation is inhibited or immune cell activation occurs). The cell is a tumor cell expressing PD-L1. The subject is a human or non-human animal in need thereof. The subject has a disease. The disease is cancer. 
     Example 12 
     PD-1 Dependent IL-2 Activity In Human Cells 
     This example describes PD-1/IL-2 protein complexes for PD-1 dependent IL-2 activity in human cells, in vitro and in vivo. PD-1/IL-2 protein complexes comprise a PD-1 sensor domain (e.g., an anti-PD-1 antibody or an anti-PD-1 scFv) linked to an IL-2 cytokine therapeutic domain via a linker, where the IL-2 cytokine is a therapeutic. In the absence of PD-1, the PD-1 sensor domain binds the IL-2 therapeutic domain, rendering the IL-2 therapeutic inert. In the presence of PD-1 (e.g., PD-1 is expressed on a cell, such as an immune cell), the PD-1 sensor domain binds PD-1, thereby unbinding the IL-2 therapeutic domain and allowing for IL-2 to exhibit therapeutic activity. 
     PD-1/IL-2 protein complexes are recombinantly expressed or chemically synthesized. PD-1/IL-2 protein complexes are administered in vitro to a human cell or in vivo to a mouse or to a human subject in need thereof. The human cell is a cell expressing PD-1. Administration to a mouse or to a human subject is performed intravenously, intramuscularly, subcutaneously, intradermally, intraperitoneally, or mucosally. In the absence of PD-1, the IL-2 therapeutic domain remains bound to the PD-1 sensor domain and no therapeutic efficacy is observed (e.g., cell activation in vitro and in the subject is unaltered). In the presence of PD-1, the PD-1 sensor domain binds PD-1 and unbinds the IL-2 therapeutic domain. Therapeutic efficacy is observed (e.g., cell activation is observed in vitro and, in the subject, in vivo). The subject has a disease. The disease is cancer. The cell may express PD-1 endogenously or after activation, or following introduction of a gene encoding PD-1. The therapeutic effect may be cell growth, differentiation, activation or induction of IL2-responsive genes. In vitro, if the cell is part of a mixture of cell types, any of these changes may be monitored for a responding cell population in the mixture. 
     Example 13 
     Bioactivity in Tumor Tissues 
     This example describes bioactivity in tumor tissues. A protein complex of the present disclosure is recombinantly expressed or chemically synthesized. The protein complex includes a sensor domain linked to a therapeutic domain. The linker is a peptide linker. The sensor domain is capable of binding to the therapeutic and a marker. In the absence if the marker, the sensor domain binds the therapeutic domain, rendering the therapeutic domain unable to bind to its target and unable to exert therapeutic activity. In the presence of the marker, the sensor domain binds the marker rendering the therapeutic domain free to bind to its target and able to exert therapeutic activity. The protein complex is administered in vitro to a cell or in vivo to a subject in need thereof. The cell expresses the marker to which the sensor domain binds. The cell is a tumor cell or immune cell. The subject is a human or non-human animal. The subject has a disease. The disease is cancer. Administration to a subject is performed intravenously, intramuscularly, subcutaneously, intradermally, intraperitoneally, or mucosally. 
     Upon administration of the protein-complex, the sensor domain binds the marker in vitro or in vivo in the subject and unbinds the therapeutic domain. Therapeutic efficacy is observed in vitro or in vivo in the subject, for example, cell proliferation of tumor cells is slowed down or completely halted and tumor cells are eliminated. 
     Example 14 
     Activity in Tumor Models of Protein Complexes 
     This example describes activity in tumor models. The tumor models assess the efficacy and systemic on-target effects of the protein complexes. The tumor model assesses the ability of a protein complex of the present disclosure to exhibit sensor domain regulated activity of the therapeutic domain. 
     A protein complex of the present disclosure is recombinantly expressed or chemically synthesized. The protein complex includes a sensor domain linked to a therapeutic domain. The linker is a peptide linker. The sensor domain is capable of binding to the therapeutic domain and a marker. In the absence of the marker, the sensor domain binds the therapeutic domain rendering the therapeutic domain unable to bind to its target and unable to exert therapeutic activity. In the presence of the marker, the sensor domain binds the marker rendering the therapeutic domain free to bind to its target and able to exert therapeutic activity. The protein complex is administered in a tumor model, in vitro or in vivo. The tumor cells or the immune cells in the tumor model express the marker to which the sensor domain binds. Administration in vivo is performed intravenously, intramuscularly, subcutaneously, intradermally, intraperitoneally, or mucosally. 
     Tumor measurement. Prior to administration of the protein-complex, or in a control sample where the protein-complex is not administered, measurements of the tumor indicate that the tumor is continuing to proliferate. For example, a solid tumor in a tumor model (e.g., a mouse model of a tumor) is measured using calipers. Prior to administration of the protein-complex, or in a control sample where the protein-complex is not administered, the solid tumor continues to grow in size, as indicated by increasing measurements of tumor size with calipers. Upon administration of the protein-complex, over time, the sensor domain binds the marker expressed on the tumor and the therapeutic domain is unbound, thus, resulting in therapeutic efficacy. Therapeutic efficacy is validated by observing decreasing measurements of tumor size with calipers over time. 
     Immune activation. Prior to administration of the protein-complex, or in a control sample where the protein-complex is not administered, the solid tumor continues to grow in size, as indicated by increasing measurements of tumor size with calipers. Upon administration of the protein-complex, over time, the sensor domain binds the marker and the therapeutic domain is unbound, thus, resulting in therapeutic efficacy. Therapeutic efficacy is validated by observing differences in the immune cells in the tumor, lymph node or systemically, when compared to the control sample. The differences may be immune cell phenotypes, activation state, differentiation state or specificity. 
     Systemic induction of cytokine markers. No induction of cytokine markers or reduced systemic induction of cytokine markers, when compared to the control, is observed upon administration of the protein complex. 
     Weight loss. No weight loss or reduced weight loss, when compared to the control, is observed upon administration of the protein complex. 
     Example 15 
     PD-1/IL-2 DBA Cytokine Complex Induction of STAT5 Phosphorylation in a Lymphocyte Cell Line 
     This example describes PD-1/IL-2 DBA-cytokine complex induction of STAT5 phosphorylation in a lymphocytic cell line. To assess the dependence of PD-1/IL-2 DBA-cytokine complex activity on binding to PD-1, a PD-1-expressing variant is generated of an IL-2R+ T cell line such as Hut78 or Jurkat E6.1. The PD-1+ and PD-1− variant cell lines are treated with titrating concentrations of a PD-1/IL-2 DBA-cytokine complex of this disclosure, and STAT5 phosphorylation is assessed by phospho-flow, TR-FRET, or other assays for measuring IL-2 signaling. 
     A HEK 293 IL-2 reporter cell line is engineered to express PD-1. The PD-1+ and PD-1-variant cell lines are treated with titrating concentrations of PD-1/IL-2 DBA-cytokine complexes, and reporter activity is assessed as a measurement of IL-2 signaling. The PD-1/IL-2 DBA-cytokine complex exhibits increased potency on PD-1+ variant cell lines. 
     Example 16 
     PD-1/IL-2 DBA Cytokine Complex Induction of STAT5 Phosphorylation and Other Markers of Activation, and Proliferation in Primary Lymphocytes 
     This example describes PD-1/IL-2 DBA-cytokine complex induction of STAT5 phosphorylation and other markers of activation and proliferation in primary lymphocytes. PBMCs are labeled with cell proliferation dye and incubated for 4 days with titrating concentrations of a PD-1/IL-2 DBA-cytokine complex of the present disclosure. PBMCs are stained with antibodies directed against immune cell phenotyping markers to distinguish CD4+ and CD8+ T cells, Treg cells, and natural killer (NK) cells and markers of cell activation, such as CD25. Dye dilution on immune cell subsets is examined by flow cytometry as a measurement of proliferation. 
     Total T cells are isolated from PBMCs using immunomagnetic negative selection (STEMCELL) and stimulated with plate-bound anti-CD3 and soluble anti-CD28 for 72 hours to induce expression of PD-1. The PD-1+ T cells are incubated for 20 minutes with titrating concentrations of PD-1/IL-2 DBA-cytokine complexes. STAT5 phosphorylation is measured in fixed and permeabilized T cells by flow cytometry. In some experiments, PD-1 may be blocked on T cells with anti-PD-1 prior to treatment with PD-1/IL-2 DBA-cytokine complexes to assess the dependence of PD-1/IL-2 DBA-cytokine complex activity on binding to PD-1. The PD-1/IL-2 DBA-cytokine complex induces minimal STAT5 phosphorylation when PD-1 is blocked, showing activity that is conditional on its ability to bind PD-1. 
     Example 17 
     In Vivo PD-1/IL-2 DBA Cytokine Complex Signaling in Non-Tumor Peripheral Tissues 
     This example describes PD-1/IL-2 DBA-cytokine complex pharmacokinetics in the blood of wild-type mice and the signaling of the complex in non-tumor peripheral tissue. The serum half-lives and peripheral tissue activities of PD-1/IL-2 DBA-cytokine complexes and suitable non-regulated controls such as anti-PD-1, anti-HER2-IL-2, or anti-PD-1-IL-2 were measured in mice dosed intravenously (i.v.) with the complexes. Blood, spleens, or both were collected at various timepoints after treatment and stained to identify CD8+ T cells and NK cells. 
     To examine the half-life of PD-1/IL-2 DBA-cytokine complex in circulation, wild-type C57BL/6 mice received a single 2.5 milligrams per kilogram intravenous dose of a PD-1/IL-2 DBA-cytokine complex (2B07 IL-2 mut; SEQ ID NO: 205-206), anti-HER2/IL-2-cytokine complex (Always-on IL-2 mut; SEQ ID NO: 64 and SEQ ID NO: 207), or anti-IL-2/IL-2-cytokine complex (Always-off IL-2 mut; SEQ ID SEQ ID NO: 208-209), as outlined in TABLE 20. Mice were bled via retro-orbital sinus at 30 minutes, 4, 24, 48, 72, 96, and 168 hours post-dosing. The blood was collected into serum separator tubes, and the isolated serum was frozen at −80° C. until analysis. To determine serum levels of the cytokine complexes, 96-well high-binding ELISA plates were coated with 1 ug/mL rabbit anti-hu IL-2 capture antibody (clone ab9618, Abcam) in carbonate-bicarbonate buffer overnight at 4 C. Plates were washed three times and blocked for 1 hour with SuperBlock blocking buffer (Thermo Scientific). Serum samples from the various timepoints and treatment groups were diluted in SuperBlock, added to the plates, and incubated 1 hr. To detect cytokine complexes, plates were incubated with goat anti-mouse Fc-HRP (Jackson ImmunoResearch) at 1:5000 in SuperBlock for 1 hour. The plates were then washed and developed with TMB substrate. Absorbance (GD) was measured using an EnVision 2105 microplate reader (PerkinElmer) at 450 nm. As shown in  FIG.  18   , at all timepoints examined the PD-1/IL-2 DBA-cytokine complex was detected at similar serum concentrations as the anti-IL-2/IL-2-cytokine complex. In contrast, the serum concentration of the non-regulated anti-HER2/IL-2-cytokine complex showed a greater decrease in serum concentration over time. 
     
       
         
           
               
             
               
                 TABLE 20 
               
             
            
               
                   
               
               
                 IgG PD-1/IL-2 DBA and control protein complexes 
               
            
           
           
               
               
               
            
               
                 Protein 
                 SEQ ID 
                   
               
               
                 Complexes 
                 NO: 
                 Sequence 
               
               
                   
               
               
                 2B07 IL-2 
                 SEQ ID 
                 APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFAMPKKATELKHL 
               
               
                 mut 
                 NO: 205 
                 QCLEEELKPLEEVLNGAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIV 
               
               
                   
                   
                 EFLNRWITFAQSIISTLTGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGAS 
               
               
                   
                   
                 VKVSCKASGDTFTRHYVHWVRQAPGQGLEWMGIINPSGGYASYAQKFQGRVTM 
               
               
                   
                   
                 TRDTSTSTVYMELSSLRSEDTAVYYCAAGLFIWGQGTLVTVSSAKTTAPSVYPLAPV 
               
               
                   
                   
                 CGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVT 
               
               
                   
                   
                 SSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPK 
               
               
                   
                   
                 IKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRV 
               
               
                   
                   
                 VSALPIQHQDWMSGKEFKCKVNNKDLGAPIERTISKPKGSVRAPQVYVLPPPEEE 
               
               
                   
                   
                 MTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLR 
               
               
                   
                   
                 VEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK 
               
               
                   
                 SEQ ID 
                 DIQMTQSPSSLSASVGDRVTITCRASQSIGRWLAWYQQKPGKAPKLLIYSASNLET 
               
               
                   
                 NO: 206 
                 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYESFPVTFGPGTKVDIKRADAAPT 
               
               
                   
                   
                 VSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSK 
               
               
                   
                   
                 DSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC 
               
               
                   
               
               
                 anti- 
                 SEQ ID 
                 DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYS 
               
               
                 HER2/IL-2- 
                 NO: 64 
                 GVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRADAAP 
               
               
                 cytokine 
                   
                 TVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSK 
               
               
                 complex 
                   
                 DSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC 
               
               
                   
                 SEQ ID 
                 APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFAMPKKATELKHL 
               
               
                   
                 NO: 207 
                 QCLEEELKPLEEVLNGAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIV 
               
               
                   
                   
                 EFLNRWITFAQSIISTLTGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGS 
               
               
                   
                   
                 LRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISAD 
               
               
                   
                   
                 TSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSAKTTAP 
               
               
                   
                   
                 SVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYT 
               
               
                   
                   
                 LSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNAAGG 
               
               
                   
                   
                 PSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHRE 
               
               
                   
                   
                 DYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLGAPIERTISKPKGSVRAPQVY 
               
               
                   
                   
                 VLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSY 
               
               
                   
                   
                 FMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK 
               
               
                   
               
               
                 Always-off 
                 SEQ ID 
                 APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFAMPKKATELKHL 
               
               
                 IL-2 mut 
                 NO: 208 
                 QCLEEELKPLEEVLNGAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIV 
               
               
                   
                   
                 EFLNRWITFAQSIISTLTGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVKPGGS 
               
               
                   
                   
                 LRLSCAASGFTFSSYTLAWVRQAPGKGLEWVAAIDSSSYTYSPDTVRGRFTISRDNA 
               
               
                   
                   
                 KNSLYLQMNSLRAEDTAVYYCARDSNWDALDYWGQGTLVTVSSAKTTAPSVYPL 
               
               
                   
                   
                 APVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSV 
               
               
                   
                   
                 TVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIF 
               
               
                   
                   
                 PPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNST 
               
               
                   
                   
                 LRVVSALPIQHQDWMSGKEFKCKVNNKDLGAPIERTISKPKGSVRAPQVYVLPPPE 
               
               
                   
                   
                 EEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKL 
               
               
                   
                   
                 RVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK 
               
               
                   
                 SEQ ID 
                 DIQMTQSPSSLSASVGDRVSITCKASQNVGTNVGWYQQKPGKAPKALIYSASFRYS 
               
               
                   
                 NO: 209 
                 GVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYYTYPYTFGGGTKLEIKRADAAPT 
               
               
                   
                   
                 VSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSK 
               
               
                   
                   
                 DSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC 
               
               
                   
               
            
           
         
       
     
     To examine the activity of PD-1/IL-2 DBA-cytokine complexes in peripheral tissues, wild-type C57BL/6 mice received a single 2.5 milligrams per kilogram intravenous dose of PD-1/IL-2 DBA-cytokine complex (2B07 IL-2 mut; SEQ ID NO: 205-206), anti-HER2/IL-2-cytokine complex (Always-on IL-2 mut; SEQ ID NO: 64 and SEQ ID NO: 207), anti-IL-2/IL-2-cytokine complex (Always-off IL-2 mut; SEQ ID NO: 208-209), as shown in TABLE 20 or PBS. Prior to dosing, the presence of intact IL-2 within each IL-2 cytokine complex was confirmed by ELISA as a means of verifying their potential for biological activity. Blood and spleens were collected 5 days following treatment and analyzed by flow cytometry to quantify the number of CD8+ T cells and NK cells per spleen and per microliter of blood. The PD-1/IL-2 DBA-cytokine complex did not induce expansion of CD8 T cells or NK cells, whereas the HER2/IL-2-cytokine complex induced expansion of peripheral CD8+ T cells and NK cells ( FIG.  19 A-D ). 
     Example 18 
     PD-1/IL-2 DBA Cytokine Complex Modulation of Anti-Tumor Immunity in Syngeneic Tumor Models 
     This example describes PD-1/IL-2 DBA-cytokine complex modulation of anti-tumor immunity in a MC38 syngeneic mouse tumor model. A PD-1/IL-2 DBA-cytokine complex was assessed for the ability to drive anti-tumor immunity in vivo. 500,000 MC38 tumor cells were implanted subcutaneously in human PD-1 knock-in mice (GenOway). Tumors were measured twice weekly, and volumes calculated as (Length×Width×Width/2). Mice were randomized into treatment groups, and treatments were initiated when tumors reached a volume of ˜100 mm 3 . Mice were treated intravenously with PD-1/IL-2 DBA-cytokine complex (2B07 IL-2 mut; SEQ ID NO: 210-212), PD-1/IL-2 DBA lacking IL-2 (2B07; SEQ ID NO: 212-213), or an isotype control (SEQ ID NO: 214-215), as shown in TABLE 21 below, at the indicated doses of 5 or 0.5 milligrams per kilogram on days 7, 10, and 13 post tumor implantation. The PD-1/IL-2 DBA-cytokine complex showed increased tumor growth inhibition compared to either the PD-1/IL-2 DBA lacking IL-2 or the isotype control ( FIG.  20   ). 
     
       
         
           
               
             
               
                 TABLE 21 
               
             
            
               
                   
               
               
                 IgG PD-1/IL-2 DBA and control protein complexes 
               
            
           
           
               
               
               
            
               
                 Protein 
                 SEQ ID 
                   
               
               
                 Complex 
                 NO: 
                 Sequence 
               
               
                   
               
               
                 2B07 IL-2 
                 SEQ ID 
                 APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFAMPKKATELKHL 
               
               
                 mut 
                 NO: 210 
                 QCLEEELKPLEEVLNGAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIV 
               
               
                   
                   
                 EFLNRWITFAQSIISTLTGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGAS 
               
               
                   
                   
                 VKVSCKASGDTFTRYYVHWVRQAPGQGLEWMGIINPSGGYASYAQKFQGRVTM 
               
               
                   
                   
                 TRDTSTSTVYMELSSLRSEDTAVYYCAAGLFIWGQGTLVTVSSAKTTAPSVYPLAPV 
               
               
                   
                   
                 CGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVT 
               
               
                   
                   
                 SSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPK 
               
               
                   
                   
                 IKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRV 
               
               
                   
                   
                 VSALPIQHQDWMSGKEFKCKVNNKDLGAPIERTISKPKGSVRAPQVYVLPPPEEE 
               
               
                   
                   
                 MTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSDLR 
               
               
                   
                   
                 VEKKNWVERNSYSCSVVHEGLHNHHTTESFSRTPGK 
               
               
                   
                 SEQ ID 
                 QVQLVQSGAEVKKPGASVKVSCKASGDTFTRYYVHWVRQAPGQGLEWMGIINPS 
               
               
                   
                 NO: 211 
                 GGYASYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAAGLFIWGQGTLV 
               
               
                   
                   
                 TVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTF 
               
               
                   
                   
                 PAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCK 
               
               
                   
                   
                 CPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEV 
               
               
                   
                   
                 HTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLGAPIERTISKP 
               
               
                   
                   
                 KGSVRAPQVYVLPPPEKEMTKKQVSLTCLVKDFMPEDIYVEWTNNGKTELNYKNT 
               
               
                   
                   
                 EPVLKSDGSYFMYSKLTVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK 
               
               
                   
                 SEQ ID 
                 DIQMTQSPSSLSASVGDRVTITCRASQSIGRYLAWYQQKPGKAPKLLIYSASNLETG 
               
               
                   
                 NO: 212 
                 VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSFPVTFGPGTKVDIKRADAAPT 
               
               
                   
                   
                 VSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSK 
               
               
                   
                   
                 DSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC 
               
               
                   
               
               
                 PD-1/IL-2 
                 SEQ ID 
                 DIQMTQSPSSLSASVGDRVTITCRASQSIGRYLAWYQQKPGKAPKLLIYSASNLETG 
               
               
                 DBA 
                 NO: 212 
                 VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSFPVTFGPGTKVDIKRADAAPT 
               
               
                 lacking IL-2 
                   
                 VSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSK 
               
               
                   
                   
                 DSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC 
               
               
                   
                 SEQ ID 
                 QVQLVQSGAEVKKPGASVKVSCKASGDTFTRYYVHWVRQAPGQGLEWMGIINPS 
               
               
                   
                 NO: 213 
                 GGYASYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAAGLFIWGQGTLV 
               
               
                   
                   
                 TVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTF 
               
               
                   
                   
                 PAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCK 
               
               
                   
                   
                 CPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEV 
               
               
                   
                   
                 HTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLGAPIERTISKP 
               
               
                   
                   
                 KGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKN 
               
               
                   
                   
                 TEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK 
               
               
                   
               
               
                 isotype 
                 SEQ ID 
                 EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTN 
               
               
                 control 
                 NO: 214 
                 GYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDY 
               
               
                   
                   
                 WGQGTLVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGS 
               
               
                   
                   
                 LSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPT 
               
               
                   
                   
                 IKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWF 
               
               
                   
                   
                 VNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLGAPI 
               
               
                   
                   
                 ERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKT 
               
               
                   
                   
                 ELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSR 
               
               
                   
                   
                 TPGK 
               
               
                   
                 SEQ ID 
                 DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYS 
               
               
                   
                 NO: 215 
                 GVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRADAAP 
               
               
                   
                   
                 TVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSK 
               
               
                   
                   
                 DSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC 
               
               
                   
               
            
           
         
       
     
     Example 19 
     PD-1/IL-2 DBA Cytokine Complex Modulation of Anti-Tumor Immunity in Xenograft/Human Immune Cell Admixture Models 
     This example describes PD-1/IL-2 DBA-cytokine complex modulation of anti-tumor immunity in xenograft/human immune cell admixture models. To examine the ability of PD-1/IL-2 DBA-cytokine complexes to drive anti-tumor immunity in an in vivo setting, an admixture system is used. Total human PBMCs or a combination of human T cells and monocyte-derived dendritic cells (moDCs) are mixed with human tumor cells (e.g., HPAC, A375, H441) at a 1:4 ratio and co-implanted subcutaneously into the flanks of NSG mice. One day later, treatment with a PD-1/IL-2 DBA-cytokine complex of the present disclosure, or suitable non-regulated controls such as anti-PD-1, anti-HER2-IL-2, or anti-PD-1-IL-2, is initiated. Tumors are measured at least twice weekly and volumes calculated as (Length×Width×Height/2). PD-1/IL-2 DBA-cytokine complexes exhibit increased anti-tumor efficacy compared to anti-PD-1 and anti-HER2-IL-2 and decreased off-tumor activity compared to anti-PD-1-IL-2. 
     Example 20 
     PD-L1/IFN-α DBA Cytokine Complex Induction of Myeloid Cell Maturation in the Presence of Plate-Bound PD-L1 or PD-L1-Expressing Tumor Cells 
     This example describes PD-L1/IFN-α DBA-cytokine complex induction of myeloid cell maturation in the presence of plate-bound PD-L1 or PD-L1-expressing tumor cells. CD14+ monocytes are purified from fresh human PBMCs by immunomagnetic negative selection (STEMCELL). Monocyte-derived dendritic cells (moDCs) are generated by culturing purified monocytes with hGM-CSF and hIL-4 in RPMI-1640 medium containing 10% FBS for 5 days. To examine the conditional activity of PD-L1/IFN-α DBA-cytokine complex, monocytes or moDCs are added to plates coated with either PD-L1 or HER-2 along with titrating concentrations of a PD-L1/IFN-α DBA-cytokine complex of the present disclosure. In some experiments, human monocytes or moDCs are co-cultured with tumor cell lines expressing varying levels of PD-L1 and titrating concentrations of PD-L1/IFN-α DBA-cytokine complex. Cultures are incubated overnight at 37° C., and expression of CD80, CD83, CD86, and HLA-DR is assessed by flow cytometry as a measurement of myeloid cell activation. PD-L1/IFN-α DBA-cytokine complex is expected to induce monocyte and moDC activation solely in the presence of PD-L1. 
     Example 21 
     PD-L1/IFN-α DBA Cytokine Complex Induction of T cell Activation in a Mixed Lymphocyte Reaction 
     This example describes PD-L1/IFN-α DBA-cytokine complex induction of T cell activation in a mixed lymphocyte reaction. To assess the direct and indirect effects of PD-L1/IFN-α DBA-cytokine complex on T cell function, CD14+ monocytes are isolated from human PBMCs using immunomagnetic negative selection (STEMCELL) and cultured for 5 days in the presence of hGM-CSF and hIL-4 to induce moDCs. CD8+ T cells are purified from human PBMCs of a different healthy donor and labeled with cell proliferation dye. The two cell types are combined in plates coated with PD-L1 or HER2 along with titrating concentrations of a PD-L1/IFN-α DBA-cytokine complex of the present disclosure. In other experiments, the two cell types are cultured with titrating concentrations of PD-L1/IFN-α DBA-cytokine complex and tumor cell lines expressing varying levels of PD-L1. In other experiments, the cells may be of mouse origin. Cultures are incubated for 5 days, and T cell dye dilution is assessed by flow cytometry as a measurement of proliferation. The concentration of cytokines (e.g., IFN-γ) in culture supernatants is assessed by ELISA. The PD-L1/IFN-α DBA-cytokine complex increases T cell activation and proliferation solely in the presence of PD-L1. 
     Example 22 
     In Vivo PD-L1/IFN-α DBA Cytokine Complex Signaling in Peripheral Tissues 
     This example describes in vivo PD-L1/IFN-α DBA-cytokine complex signaling in peripheral tissues. To examine PD-L1/IFN-α DBA-cytokine complex activity in non-tumor tissue, wild-type C57BL/6 mice are injected intravenously (i.v.) with 100 ug of either a PD-L1/IFN-α DBA-cytokine complex of the present disclosure or a nonregulated immunocytokine of a comparable structure consisting of anti-PD-L1 and IFN-α (PD-L1-IFNα immunocytokine). Animals are weighed daily to monitor IFN-α induced toxicity. Serum is collected at 6 and 24 hours post dosing, and MCP-1, IL-6, IL-10, TNF-α, and IFN-γ levels are quantified by ELISA. In some groups, RNA is isolated from the spleen and liver 6 and 24 hours post dosing. Induction of IFN-stimulated genes including ISG15, IRF7, and MX2 is assessed by qPCR. Mice that received unregulated anti-PD-L1-IFN-α immunocytokine experience weight loss, increased serum cytokine levels, and IFN target gene induction, whereas those dosed with the PD-L1/IFN-α DBA-cytokine complex display minimal evidence of peripheral IFN-α signaling. 
     Example 23 
     PD-L1-IFN-α DBA-Cytokine Complex Modulation of Anti-Tumor Immunity in Syngeneic Tumor Models 
     This example describes PD-L1/IFN-α DBA-cytokine complex modulation of anti-tumor immunity in syngeneic tumor models. PD-L1/IFN-α DBA cytokine complex proteins are assessed for their ability to drive anti-tumor immunity in vivo. Wild-type or human PD-L1-expressing syngeneic mouse tumor cells (e.g., MC38, CT26, 4T1, or A20) are implanted subcutaneously into wild-type or human PD-L1 knock-in mice (Genoway). Tumors are measured at least twice weekly and volumes are calculated as (Length×Width×Height/2). Mice are randomized into different groups and therapy is initiated when tumors reached a volume of ˜100 mm 3 . Mice are treated i.v. or intratumorally with a PD-L1/IFN-α DBA-cytokine complex of the present disclosure or suitable nonregulated controls such as anti-PD-L1, anti-HER2-IFN-α immunocytokine, or anti-PD-L1-IFN-α immunocytokine. In some experiments, mice are sacrificed 5 days post treatment, and tumors are harvested and enzymatically dissociated for immunophenotyping. The frequency and phenotype of tumor-infiltrating immune cell subsets, including CD4+ and CD8+ T cells, Treg cells, NK cells, and DCs, is determined by flow cytometry. The PD-L1/IFN-α DBA-cytokine complex inhibits tumor growth to an equal or greater extent than anti-HER2-IFN-α, but with less off-tumor activity. The PD-L1/IFNα DBA-cytokine complex increases an anti-tumor immune response as indicated by the amount and phenotype of immune infiltrates to an equal or greater extent than anti-HER2-IFNα immunocytokine, but with less off-tumor activity. 
     Example 24 
     PD-L1/IFN-α DBA Modulation of Anti-Tumor Immunity in Xenograft/Human Immune Cell Admixture Models 
     This example describes PD-L1/IFN-α DBA-cytokine complex modulation of anti-tumor immunity in xenograft/human immune cell admixture models. To examine the ability of PD-L1/IFN-α DBA-cytokine complexes to drive anti-tumor immunity in an in vivo setting, an admixture system is used. Total human PBMCs or a combination of human T cells and moDCs are mixed with human tumor cells (e.g., HPAC, A375, H441) at a 1:4 ratio and co-implanted subcutaneously into the flanks of NSG mice. One day later, i.v. treatment with a PD-L1/IFN-α DBA-cytokine complex of the present disclosure or suitable non-regulated controls such as anti-PD-L1, anti-HER2-IFN-α immunocytokine, or anti-PD-L1-IFN-α immunocytokine is initiated. Tumors are measured at least twice weekly and volumes are calculated as (Length×Width×Height/2). The PD-L1/IFN-α DBA-cytokine complex inhibits tumor growth to an equal or greater extent than anti-HER2-IFN-α, but with less off-tumor activity. 
     Example 25 
     In Vitro and In Vivo Characterization of Protein Complexes 
     This example describes the evaluation of DBA-cytokine complexes for in vitro and in vivo stability. A protein complex of the present disclosure is recombinantly expressed or chemically synthesized. The protein complex includes a sensor domain linked to a therapeutic domain. The linker is a peptide linker. The sensor domain is capable of binding to the therapeutic domain and a marker. In the absence if the marker, the sensor domain binds the therapeutic domain rendering the therapeutic domain unable to bind to its target and unable to exert therapeutic activity. In the presence of the marker, the sensor domain binds the marker rendering the therapeutic domain free to bind to its target and able to exert therapeutic activity. 
     In vitro, the protein complexes are tested for stability and functionality at baseline or after incubation in conditions of stress, such as elevated temperature, pH changes, oxidative buffers, or serum/plasma, using methods of biophysical characterization to measure fragmentation, unfolding, or aggregation, and/or using methods to test for changes in functional activity. In vivo, the pharmacokinetic properties of the proteins are measured following dosing in a mammal, such as a mouse, rat, or non-human primate, and properties of distribution, clearance and degradation are measured. These measurements are used to engineer or select the optimal therapeutic form of the DBA-protein complex. 
     Example 26 
     Regulated IL-2 Receptor Signaling by a PD-1/IL-2 Dual Binding Antibody (DBA) Cytokine Complex 
     This example describes PD-1 regulated IL-2 activity in a HEK-Blue™ IL-2 reporter cell by PD-1/IL-2 DBA-cytokine complexes. The DBA-cytokine complexes and control antibody-cytokine complexes were produced in three formats shown in  FIGS.  14 A-C  by expression in mammalian cells using standard protocols. The wells of a 384-well ELISA plate were coated with constant concentration of PD-1-Fc or an IgG1 control protein captured with an anti-Fc antibody (Jackson ImmunoResearch, Prod. #109-005-098). The cytokine complexes were serially diluted 1:4 for 8 points in growth media from a starting concentration of 6 nM and incubated briefly before addition of the HEK-Blue™ IL-2 reporter cells. 
     Results with a protein complexes comprising the structure shown in  FIG.  14 A  are shown in  FIG.  15 A-D . As depicted in  FIG.  14 A , this symmetric format is comprised of one IL-2 linked to each antibody variable domain. The IL-2 activity of the PD-1/IL-2 DBA-IL-2 complex AF4379 comprising SEQ ID NO: 174-175 had an EC50 of 31 pM in the PD-1 coated wells versus 62 pM in the IgG1 coated wells, as shown in  FIG.  15 A , demonstrating PD-1 dependence. The IL-2 activity of antibody-cytokine complexes AF4377 comprising SEQ ID NO: 64 and 176 (anti-Her2 antibody) and AF4378 comprising SEQ ID NO: 177-178 (anti-IL-2 antibody) was unchanged in the presence of PD-1 (as shown in  FIG.  15 B  and  FIG.  15 C , respectively), while the IL-2 activity of the anti-PD-1 antibody AF4376 comprising SEQ ID NO: 179-180 is reduced in the presence of PD-1, as shown in  FIG.  15 D . Sequences of the protein complexes are summarized in TABLE 22 below. 
     
       
         
           
               
             
               
                 TABLE 22 
               
             
            
               
                   
               
               
                 IgG PD-1/IL-2 DBA with heavy chain IL-2 therapeutic domains, and control 
               
               
                 protein complexes 
               
            
           
           
               
               
               
            
               
                 Protein 
                 SEQ ID 
                   
               
               
                 Complex 
                 NO: 
                 Sequence 
               
               
                   
               
               
                 AF4379 
                 SEQ ID 
                 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTFEFYM 
               
               
                   
                 NO: 174 
                 PKKATELKHLQCLERELKPLEEVLNLAQSKNFHLRPRDLISNINVIV 
               
               
                   
                   
                 LELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTGGGGSGG 
               
               
                   
                   
                 GGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGDTFTR 
               
               
                   
                   
                 YYVHWVRQAPGQGLEWMGIINPSGGYASYAQKFQGRVTMTRDTS 
               
               
                   
                   
                 TSTVYMELSSLRSEDTAVYYCAAGLFIWGQGTLVTVSSAKTTAPSV 
               
               
                   
                   
                 YPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFP 
               
               
                   
                   
                 AVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRG 
               
               
                   
                   
                 PTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDV 
               
               
                   
                   
                 SEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQ 
               
               
                   
                   
                 DWMSGKEFKCKVNNKDLGAPIERTISKPKGSVRAPQVYVLPPPEEE 
               
               
                   
                   
                 MTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSD 
               
               
                   
                   
                 GSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPG 
               
               
                   
                   
                 K 
               
               
                   
                 SEQ ID 
                 DIQMTQSPSSLSASVGDRVTITCRASQSIGRYLAWYQQKPGKAPKL 
               
               
                   
                 NO: 175 
                 LIYSASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSF 
               
               
                   
                   
                 PVTFGPGTKVDIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYP 
               
               
                   
                   
                 KDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEY 
               
               
                   
                   
                 ERHNSYTCEATHKTSTSPIVKSFNRNEC 
               
               
                   
               
               
                 AF4377 
                 SEQ ID 
                 DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPK 
               
               
                   
                 NO: 64 
                 LLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYT 
               
               
                   
                   
                 TPPTFGQGTKVEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFY 
               
               
                   
                   
                 PKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDE 
               
               
                   
                   
                 YERHNSYTCEATHKTSTSPIVKSFNRNEC 
               
               
                   
                 SEQ ID 
                 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTFEFYM 
               
               
                   
                 NO: 176 
                 PKKATELKHLQCLERELKPLEEVLNLAQSKNFHLRPRDLISNINVIV 
               
               
                   
                   
                 LELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTGGGGSGG 
               
               
                   
                   
                 GGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDT 
               
               
                   
                   
                 YIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKN 
               
               
                   
                   
                 TAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS 
               
               
                   
                   
                 AKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSL 
               
               
                   
                   
                 SSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKV 
               
               
                   
                   
                 DKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIV 
               
               
                   
                   
                 TCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVS 
               
               
                   
                   
                 ALPIQHQDWMSGKEFKCKVNNKDLGAPIERTISKPKGSVRAPQVY 
               
               
                   
                   
                 VLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNT 
               
               
                   
                   
                 EPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTT 
               
               
                   
                   
                 KSFSRTPGK 
               
               
                   
               
               
                 AF4378 
                 SEQ ID 
                 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTFEFYM 
               
               
                   
                 NO: 177 
                 PKKATELKHLQCLERELKPLEEVLNLAQSKNFHLRPRDLISNINVIV 
               
               
                   
                   
                 LELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTGGGGSGG 
               
               
                   
                   
                 GGSGGGGSGGGGSEVQLVESGGGLVKPGGSLRLSCAASGFTFSSYT 
               
               
                   
                   
                 LAWVRQAPGKGLEWVAAIDSSSYTYSPDTVRGRFTISRDNAKNSL 
               
               
                   
                   
                 YLQMNSLRAEDTAVYYCARDSNWDALDYWGQGTLVTVSSAKTT 
               
               
                   
                   
                 APSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGV 
               
               
                   
                   
                 HTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKI 
               
               
                   
                   
                 EPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVV 
               
               
                   
                   
                 VDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPI 
               
               
                   
                   
                 QHQDWMSGKEFKCKVNNKDLGAPIERTISKPKGSVRAPQVYVLPP 
               
               
                   
                   
                 PEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVL 
               
               
                   
                   
                 DSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSR 
               
               
                   
                   
                 TPGK 
               
               
                   
                 SEQ ID 
                 DIQMTQSPSSLSASVGDRVSITCKASQNVGTNVGWYQQKPGKAPK 
               
               
                   
                 NO: 178 
                 ALIYSASFRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYYT 
               
               
                   
                   
                 YPYTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFY 
               
               
                   
                   
                 PKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDE 
               
               
                   
                   
                 YERHNSYTCEATHKTSTSPIVKSFNRNEC 
               
               
                   
               
               
                 AF4376 
                 SEQ ID 
                 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTFEFYM 
               
               
                   
                 NO: 179 
                 PKKATELKHLQCLERELKPLEEVLNLAQSKNFHLRPRDLISNINVIV 
               
               
                   
                   
                 LELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTGGGGSGG 
               
               
                   
                   
                 GGSGGGGSGGGGSQVQLVESGGGVVQPGRSLRLDCKASGITFSNS 
               
               
                   
                   
                 GMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNS 
               
               
                   
                   
                 KNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSSAKTTAP 
               
               
                   
                   
                 SVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHT 
               
               
                   
                   
                 FPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEP 
               
               
                   
                   
                 RGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVV 
               
               
                   
                   
                 DVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQ 
               
               
                   
                   
                 HQDWMSGKEFKCKVNNKDLGAPIERTISKPKGSVRAPQVYVLPPP 
               
               
                   
                   
                 EEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVL 
               
               
                   
                   
                 DSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSR 
               
               
                   
                   
                 TPGK 
               
               
                   
                 SEQ ID 
                 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLL 
               
               
                   
                 NO: 180 
                 IYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNW 
               
               
                   
                   
                 PRTFGQGTKVEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYP 
               
               
                   
                   
                 KDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEY 
               
               
                   
                   
                 ERHNSYTCEATHKTSTSPIVKSFNRNEC 
               
               
                   
               
            
           
         
       
     
     Results with protein complexes comprising the structures depicted in  FIG.  14 B , are shown in  FIGS.  16 A-F . This format is composed of an asymmetric complex comprised of two antibody domains with a single IL-2 linked to one of the domains. The IL-2 activity of the PD-1/IL-2 DBA-IL-2 complexes AF4386 (comprising SEQ ID NO: 212 and 181-182, results shown in  FIG.  16 A ), AF4387 (comprising SEQ ID NO: 183-185, results shown in  FIG.  16 B ) and AF4389 (comprising SEQ ID NO: 186-188, results shown in  FIG.  16 C ) had an EC50 of 50 pM, 57 pM and 118 pM respectively in the PD-1 coated wells and 1.79 nM, 419 pM and 1.67 nM respectively in the IgG1 coated wells, demonstrating PD-1 dependence. The IL-2 activity of the anti-PD1 control protein AF4380 (comprising SEQ ID NO: 180, 189-190, results shown in  FIG.  16 D ), the anti-Her2 control protein AF4383 (comprising SEQ ID NO: 64, 191-192, results shown in  FIG.  16 E ), and the anti-IL-2 control protein AF4384 (comprising SEQ ID NO: 178, 193-194, results shown in  FIG.  16 F ) were unchanged. Sequences of the protein complexes are summarized in TABLE 23 below. 
     
       
         
           
               
             
               
                 TABLE 23 
               
             
            
               
                   
               
               
                 IgG PD-1/IL-2 PDA with single IL-2, and control protein complexes 
               
            
           
           
               
               
               
            
               
                 Protein 
                 SEQ ID 
                   
               
               
                 Complex 
                 NO: 
                 Sequence 
               
               
                   
               
               
                 AF4386 
                 SEQ ID 
                 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTFEFYMPKKATELKHLQ 
               
               
                   
                 NO: 181 
                 CLERELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVE 
               
               
                   
                   
                 FLN RWITFCQSIISTLTGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASV 
               
               
                   
                   
                 KVSCKASGDTFTRYYVHWVRQAPGQGLEWMGIINPSGGYASYAQKFQGRVTMT 
               
               
                   
                   
                 RDTSTSTVYMELSSLRSEDTAVYYCAAGLFIWGQGTLVTVSSAKTTAPSVYPLAPVC 
               
               
                   
                   
                 GDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSS 
               
               
                   
                   
                 TWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIK 
               
               
                   
                   
                 DVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVV 
               
               
                   
                   
                 SALPIQHQDWMSGKEFKCKVNNKDLGAPIERTISKPKGSVRAPQVYVLPPPEEEMT 
               
               
                   
                   
                 KKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSDLRVEK 
               
               
                   
                   
                 KNWVERNSYSCSVVHEGLHNHHTTESFSRTPGK 
               
               
                   
                 SEQ ID 
                 QVQLVQSGAEVKKPGASVKVSCKASGDTFTRYYVHWVRQAPGQGLEWMGIINPS 
               
               
                   
                 NO: 182 
                 GGYASYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAAGLFIWGQGTLV 
               
               
                   
                   
                 TVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTF 
               
               
                   
                   
                 PAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCK 
               
               
                   
                   
                 CPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEV 
               
               
                   
                   
                 HTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLGAPIERTISKP 
               
               
                   
                   
                 KGSVRAPQVYVLPPPEKEMTKKQVSLTCLVKDFMPEDIYVEWTNNGKTELNYKNT 
               
               
                   
                   
                 EPVLKSDGSYFMYSKLTVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGGGG 
               
               
                   
                   
                 SGGGSHHHHHH 
               
               
                   
                 SEQ ID 
                 DIQMTQSPSSLSASVGDRVTITCRASQSIGRYLAWYQQKPGKAPKLLIYSASNLETG 
               
               
                   
                 NO: 212 
                 VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSFPVTFGPGTKVDIKRADAAPT 
               
               
                   
                   
                 VSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSK 
               
               
                   
                   
                 DSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC 
               
               
                   
               
               
                 AF4387 
                 SEQ ID 
                 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTFEFYMPKKATELKHLQ 
               
               
                   
                 NO: 183 
                 CLERELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVE 
               
               
                   
                   
                 FLNRWITFCQSIISTLTGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASV 
               
               
                   
                   
                 KVSCKASGYTFTDYYMHWVRQAPGQGLEWMGIINPRAGYTSYALKFQGRVTMT 
               
               
                   
                   
                 RDTSTSTVYMELSSLRSEDTAVYYCTSGWDVWGQGTLVTVSSAKTTAPSVYPLAPV 
               
               
                   
                   
                 CGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVT 
               
               
                   
                   
                 SSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPK 
               
               
                   
                   
                 IKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRV 
               
               
                   
                   
                 VSALPIQHQDWMSGKEFKCKVNNKDLGAPIERTISKPKGSVRAPQVYVLPPPEEE 
               
               
                   
                   
                 MTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFIVIYSDLR 
               
               
                   
                   
                 VEKKNWVERNSYSCSVVHEGLHNHHTTESFSRTPGK 
               
               
                   
                 SEQ ID 
                 QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMHWVRQAPGQGLEWMGIINP 
               
               
                   
                 NO: 184 
                 RAGYTSYALKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCTSGWDVWGQGTL 
               
               
                   
                   
                 VTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHT 
               
               
                   
                   
                 FPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPC 
               
               
                   
                   
                 KCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEV 
               
               
                   
                   
                 HTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLGAPIERTISKP 
               
               
                   
                   
                 KGSVRAPQVYVLPPPEKEMTKKQVSLTCLVKDFMPEDIYVEWTNNGKTELNYKNT 
               
               
                   
                   
                 EPVLKSDGSYFMYSKLTVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGGGG 
               
               
                   
                   
                 SGGGSHHHHHH 
               
               
                   
                 SEQ ID 
                 DIQMTQSPSSLSASVGDRVTITCRASQSISTWLAWYQQKPGKAPKLLIYAASSLDSG 
               
               
                   
                 NO: 185 
                 VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSFPVTFGQGTKVEIKRADAAPTV 
               
               
                   
                   
                 SIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDS 
               
               
                   
                   
                 TYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC 
               
               
                   
               
               
                 AF4389 
                 SEQ ID 
                 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTFEFYMPKKATELKHLQ 
               
               
                   
                 NO: 186 
                 CLERELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVE 
               
               
                   
                   
                 FLNRWITFCQSIISTLTGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASV 
               
               
                   
                   
                 KVSCKASGHTFTRYYMHWVRQAPGQGLEWMG11NPSGGYATYAQKFQGRVTMT 
               
               
                   
                   
                 RDTSTSTVYMELSSLRSEDTAVYYCASGLFIWGQGTLVTVSSAKTTAPSVYPLAPVC 
               
               
                   
                   
                 GDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSS 
               
               
                   
                   
                 TWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIK 
               
               
                   
                   
                 DVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVV 
               
               
                   
                   
                 SALPIQHQDWMSGKEFKCKVNNKDLGAPIERTISKPKGSVRAPQVYVLPPPEEEMT 
               
               
                   
                   
                 KKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSDLRVEK 
               
               
                   
                   
                 KNWVERNSYSCSVVHEGLHNHHTTESFSRTPGK 
               
               
                   
                 SEQ ID 
                 QVQLVQSGAEVKKPGASVKVSCKASGHTFTRYYMHWVRQAPGQGLEWMGIINP 
               
               
                   
                 NO: 187 
                 SGGYATYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCASGLFIWGQGTL 
               
               
                   
                   
                 VTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHT 
               
               
                   
                   
                 FPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPC 
               
               
                   
                   
                 KCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEV 
               
               
                   
                   
                 HTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLGAPIERTISKP 
               
               
                   
                   
                 KGSVRAPQVYVLPPPEKEMTKKQVSLTCLVKDFMPEDIYVEWTNNGKTELNYKNT 
               
               
                   
                   
                 EPVLKSDGSYFMYSKLTVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGGGG 
               
               
                   
                   
                 SGGGSHHHHHH 
               
               
                   
                 SEQ ID 
                 DIQMTQSPSSLSASVGDRVTITCRASQSINSWLAWYQQKPGKAPKLLIYATSTLESG 
               
               
                   
                 NO: 188 
                 VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYRFPVTFGQGTKVEIKRADAAPT 
               
               
                   
                   
                 VSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSK 
               
               
                   
                   
                 DSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC 
               
               
                   
               
               
                 AF4380 
                 SEQ ID 
                 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLL 
               
               
                   
                 NO: 180 
                 IYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNW 
               
               
                   
                   
                 PRTFGQGTKVEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYP 
               
               
                   
                   
                 KDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEY 
               
               
                   
                   
                 ERHNSYTCEATHKTSTSPIVKSFNRNEC 
               
               
                   
                 SEQ ID 
                 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTFEFYMPKKATELKHLQ 
               
               
                   
                 NO: 189 
                 CLERELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVE 
               
               
                   
                   
                 FLNRWITFCQSIISTLTGGGGSGGGGSGGGGSGGGGSQVQLVESGGGVVQPGRSL 
               
               
                   
                   
                 RLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISR 
               
               
                   
                   
                 DNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSSAKTTAPSVYPLAPV 
               
               
                   
                   
                 CGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVT 
               
               
                   
                   
                 SSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPK 
               
               
                   
                   
                 IKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRV 
               
               
                   
                   
                 VSALPIQHQDWMSGKEFKCKVNNKDLGAPIERTISKPKGSVRAPQVYVLPPPEEE 
               
               
                   
                   
                 MTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSDLR 
               
               
                   
                   
                 VEKKNWVERNSYSCSVVHEGLHNHHTTESFSRTPGK 
               
               
                   
                 SEQ ID 
                 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWY 
               
               
                   
                 NO: 190 
                 DGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTL 
               
               
                   
                   
                 VTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHT 
               
               
                   
                   
                 FPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPC 
               
               
                   
                   
                 KCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEV 
               
               
                   
                   
                 HTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLGAPIERTISKP 
               
               
                   
                   
                 KGSVRAPQVYVLPPPEKEMTKKQVSLTCLVKDFMPEDIYVEWTNNGKTELNYKNT 
               
               
                   
                   
                 EPVLKSDGSYFMYSKLTVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGGGG 
               
               
                   
                   
                 SGGGSHHHHHH 
               
               
                   
               
               
                 AF4383 
                 SEQ ID 
                 DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPK 
               
               
                   
                 NO: 64 
                 LLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYT 
               
               
                   
                   
                 TPPTFGQGTKVEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFY 
               
               
                   
                   
                 PKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDE 
               
               
                   
                   
                 YERHNSYTCEATHKTSTSPIVKSFNRNEC 
               
               
                   
                 SEQ ID 
                 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTFEFYMPKKATELKHLQ 
               
               
                   
                 NO: 191 
                 CLERELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVE 
               
               
                   
                   
                 FLNRWITFCQSIISTLTGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSL 
               
               
                   
                   
                 RLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADT 
               
               
                   
                   
                 SKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSAKTTAPS 
               
               
                   
                   
                 VYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTL 
               
               
                   
                   
                 SSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNAAGGP 
               
               
                   
                   
                 SVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHRED 
               
               
                   
                   
                 YNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLGAPIERTISKPKGSVRAPQVYVL 
               
               
                   
                   
                 PPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYF 
               
               
                   
                   
                 MYSDLRVEKKNWVERNSYSCSVVHEGLHNHHTTESFSRTPGK 
               
               
                   
                 SEQ ID 
                 EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRY 
               
               
                   
                 NO: 192 
                 ADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVS 
               
               
                   
                   
                 SAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDL 
               
               
                   
                   
                 YTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVF 
               
               
                   
                   
                 IFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQSWFVNNVEVHTAQTQTHREDYNSTLRVV 
               
               
                   
                   
                 SALPIQHQDWMSGKEFKCKVNNKDLGAPIERTISKPKGSVRAPQVYVLPPPEKEMTKKQV 
               
               
                   
                   
                 SLTCLVKDFMPEDIYVEWTNNGKTELNYKNTEPVLKSDGSYFMYSKLTVEKKNWVERNSYS 
               
               
                   
                   
                 CSVVHEGLHNHHTTKSFSRTPGGGGSGGGSHHHHHH 
               
               
                   
               
               
                 AF4384 
                 SEQ ID 
                 DIQMTQSPSSLSASVGDRVSITCKASQNVGTNVGWYQQKPGKAPKALIYSASFRYS 
               
               
                   
                 NO: 178 
                 GVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYYTYPYTFGGGTKLEIKRADAAPT 
               
               
                   
                   
                 VSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSK 
               
               
                   
                   
                 DSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC 
               
               
                   
                 SEQ ID 
                 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTFEFYMPKKATELKHLQ 
               
               
                   
                 NO: 193 
                 CLERELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVE 
               
               
                   
                   
                 FLNRWITFCQSIISTLTGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVKPGGSL 
               
               
                   
                   
                 RLSCAASGFTFSSYTLAWVRQAPGKGLEWVAAIDSSSYTYSPDTVRGRFTISRDNAK 
               
               
                   
                   
                 NSLYLQMNSLRAEDTAVYYCARDSNWDALDYWGQGTLVTVSSAKTTAPSVYPLA 
               
               
                   
                   
                 PVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVT 
               
               
                   
                   
                 VTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFP 
               
               
                   
                   
                 PKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTL 
               
               
                   
                   
                 RVVSALPIQHQDWMSGKEFKCKVNNKDLGAPIERTISKPKGSVRAPQVYVLPPPEE 
               
               
                   
                   
                 EMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSDL 
               
               
                   
                   
                 RVEKKNWVERNSYSCSVVHEGLHNHHTTESFSRTPGK 
               
               
                   
                 SEQ ID 
                 EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYTLAWVRQAPGKGLEWVAAIDSSSY 
               
               
                   
                 NO: 194 
                 TYSPDTVRGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDSNWDALDYWGQG 
               
               
                   
                   
                 TLVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGV 
               
               
                   
                   
                 HTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCP 
               
               
                   
                   
                 PCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNV 
               
               
                   
                   
                 EVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLGAPIERTIS 
               
               
                   
                   
                 KPKGSVRAPQVYVLPPPEKEMTKKQVSLTCLVKDFMPEDIYVEWTNNGKTELNYK 
               
               
                   
                   
                 NTEPVLKSDGSYFMYSKLTVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGG 
               
               
                   
                   
                 GGSGGGSHHHHHH 
               
               
                   
               
            
           
         
       
     
     Results with protein complexes comprising the structures depicted in  FIG.  14 C  are shown in  FIGS.  17 A-H . As depicted in  FIG.  14 C , these complexes are asymmetric and comprised of two identical monospecific Fab arms with a single IL-2 attached to one Fc domain by flexible linker and a single scFv attached to the other Fc domain by a flexible linker. The active PD-1/IL-2 DBA complexes, AF4403 comprising SEQ ID NO: 180, 195, 199 and AF4404 comprising SEQ ID NO: 180, 196, 199, are composed of anti-PD-1 domains in the Fab arms and a PD-1/IL-2 DBA scFv on the Fc arm. The control antibody-cytokine complexes are composed of a) antibody-cytokine complexes with an irrelevant antibody on the Fab arms with the DBA scFv on the Fc (AF4395 comprising SEQ ID NO: 64, 197, 202 and AF4396 comprising SEQ ID NO: 64, 198, 202), b) antibody-cytokine complexes with a non-DBA scFv on the Fc arm (AF4400 comprising SEQ ID NO: 180, 199-200 and AF4401 comprising SEQ ID NO: 180, 199, 201), and c) antibody-cytokine complexes with non-DBA antibodies in both the Fab and scFv domains (AF4392 comprising SEQ ID NO: 64, 202-203 and AF4393 comprising SEQ ID NO: 64, 202, 204). As shown in  FIGS.  17 B and  17 D , the IL-2 activity of the DBA-cytokine complexes AF4403 and AF4404 had an EC50 of 31 pM and 26 pM respectively in the PD-1 coated wells and 62 pM and 64 pM respectively in the control wells, demonstrating PD-1 dependence of the IL-2 activity. None of the control proteins AF4395, AF4396, AF4400, AF4401, AF4392 and AF4393 described above showed a lower EC50 on PD-1 coated wells than on wells coated with the IgG1 protein, as shown in  FIGS.  17 A,  17 C and  17 E -H. Sequences of the protein complexes are summarized in TABLE 24 below. 
     
       
         
           
               
             
               
                 TABLE 24 
               
             
            
               
                   
               
               
                 IgG PD-1 with C-terminal scFv and IL-2, and control protein complexes 
               
            
           
           
               
               
               
            
               
                 Protein 
                 SEQ ID 
                   
               
               
                 Complex 
                 NO: 
                 Sequence 
               
               
                   
               
               
                 AF4403 
                 SEQ ID 
                 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLL 
               
               
                   
                 NO: 180 
                 IYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNW 
               
               
                   
                   
                 PRTFGQGTKVEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYP 
               
               
                   
                   
                 KDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEY 
               
               
                   
                   
                 ERHNSYTCEATHKTSTSPIVKSFNRNEC 
               
               
                   
                 SEQ ID 
                 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG 
               
               
                   
                 NO: 195 
                 LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE 
               
               
                   
                   
                 DTAVYYCATNDDYWGQGTLVTVSSAKTTAPSVYPLAPVCGDTTG 
               
               
                   
                   
                 SSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSS 
               
               
                   
                   
                 VTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAP 
               
               
                   
                   
                 NAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFV 
               
               
                   
                   
                 NNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKV 
               
               
                   
                   
                 NNKDLGAPIERTISKPKGSVRAPQVYVLPPPEKEMTKKQVSLTCLV 
               
               
                   
                   
                 KDFMPEDIYVEWTNNGKTELNYKNTEPVLKSDGSYFMYSKLTVEK 
               
               
                   
                   
                 KNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGGGGGSGGGGSGG 
               
               
                   
                   
                 GGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGDTFTRYYVHW 
               
               
                   
                   
                 VRQAPGQGLEWMGIINPSGGYASYAQKFQGRVTMTRDTSTSTVY 
               
               
                   
                   
                 MELSSLRSEDTAVYYCAAGLFIWGQGTLVTVSSASGGGGSGGGGS 
               
               
                   
                   
                 GGGGSHASDIQMTQSPSSLSASVGDRVTITCRASQSIGRYLAWYQQ 
               
               
                   
                   
                 KPGKAPKLLIYSASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATY 
               
               
                   
                   
                 YCQQYNSFPVTFGPGTKVDIKGGGSGGGSHHHHHH 
               
               
                   
                 SEQ ID 
                 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG 
               
               
                   
                 NO: 199 
                 LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE 
               
               
                   
                   
                 DTAVYYCATNDDYWGQGTLVTVSSAKTTAPSVYPLAPVCGDTTG 
               
               
                   
                   
                 SSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSS 
               
               
                   
                   
                 VTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAP 
               
               
                   
                   
                 NAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFV 
               
               
                   
                   
                 NNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKV 
               
               
                   
                   
                 NNKDLGAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMV 
               
               
                   
                   
                 TDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSDLRVEK 
               
               
                   
                   
                 KNWVERNSYSCSVVHEGLHNHHTTESFSRTPGGGGGSGGGGSGG 
               
               
                   
                   
                 GGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTFE 
               
               
                   
                   
                 FYMPKKATELKHLQCLERELKPLEEVLNLAQSKNFHLRPRDLISNI 
               
               
                   
                   
                 NVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT 
               
               
                   
               
               
                 AF4404 
                 SEQ ID 
                 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLL 
               
               
                   
                 NO: 180 
                 IYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNW 
               
               
                   
                   
                 PRTFGQGTKVEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYP 
               
               
                   
                   
                 KDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEY 
               
               
                   
                   
                 ERHNSYTCEATHKTSTSPIVKSFNRNEC 
               
               
                   
                 SEQ ID 
                 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG 
               
               
                   
                 NO: 196 
                 LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE 
               
               
                   
                   
                 DTAVYYCATNDDYWGQGTLVTVSSAKTTAPSVYPLAPVCGDTTG 
               
               
                   
                   
                 SSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSS 
               
               
                   
                   
                 VTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAP 
               
               
                   
                   
                 NAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFV 
               
               
                   
                   
                 NNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKV 
               
               
                   
                   
                 NNKDLGAPIERTISKPKGSVRAPQVYVLPPPEKEMTKKQVSLTCLV 
               
               
                   
                   
                 KDFMPEDIYVEWTNNGKTELNYKNTEPVLKSDGSYFMYSKLTVEK 
               
               
                   
                   
                 KNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGGGGGSGGGGSGG 
               
               
                   
                   
                 GGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMH 
               
               
                   
                   
                 WVRQAPGQGLEWMGIINPRAGYTSYALKFQGRVTMTRDTSTSTV 
               
               
                   
                   
                 YMELSSLRSEDTAVYYCTSGWDVWGQGTLVTVSSASGGGGSGGG 
               
               
                   
                   
                 GSGGGGSHASDIQMTQSPSSLSASVGDRVTITCRASQSISTWLAWY 
               
               
                   
                   
                 QQKPGKAPKLLIYAASSLDSGVPSRFSGSGSGTDFTLTISSLQPEDFA 
               
               
                   
                   
                 TYYCQQSYSFPVTFGQGTKVEIKGGGSGGGSHHHHHH 
               
               
                   
                 SEQ ID 
                 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG 
               
               
                   
                 NO: 199 
                 LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE 
               
               
                   
                   
                 DTAVYYCATNDDYWGQGTLVTVSSAKTTAPSVYPLAPVCGDTTG 
               
               
                   
                   
                 SSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSS 
               
               
                   
                   
                 VTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAP 
               
               
                   
                   
                 NAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFV 
               
               
                   
                   
                 NNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKV 
               
               
                   
                   
                 NNKDLGAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMV 
               
               
                   
                   
                 TDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSDLRVEK 
               
               
                   
                   
                 KNWVERNSYSCSVVHEGLHNHHTTESFSRTPGGGGGSGGGGSGG 
               
               
                   
                   
                 GGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTFE 
               
               
                   
                   
                 FYMPKKATELKHLQCLERELKPLEEVLNLAQSKNFHLRPRDLISNI 
               
               
                   
                   
                 NVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT 
               
               
                   
               
               
                 AF4395 
                 SEQ ID 
                 DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPK 
               
               
                   
                 NO: 64 
                 LLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYT 
               
               
                   
                   
                 TPPTFGQGTKVEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFY 
               
               
                   
                   
                 PKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDE 
               
               
                   
                   
                 YERHNSYTCEATHKTSTSPIVKSFNRNEC 
               
               
                   
                 SEQ ID 
                 EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGL 
               
               
                   
                 NO: 197 
                 EWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAED 
               
               
                   
                   
                 TAVYYCSRWGGDGFYAMDYWGQGTLVTVSSAKTTAPSVYPLAPV 
               
               
                   
                   
                 CGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSD 
               
               
                   
                   
                 LYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCP 
               
               
                   
                   
                 PCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPD 
               
               
                   
                   
                 VQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSG 
               
               
                   
                   
                 KEFKCKVNNKDLGAPIERTISKPKGSVRAPQVYVLPPPEKEMTKKQ 
               
               
                   
                   
                 VSLTCLVKDFMPEDIYVEWTNNGKTELNYKNTEPVLKSDGSYFMY 
               
               
                   
                   
                 SKLTVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGGGGGSG 
               
               
                   
                   
                 GGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGDTFT 
               
               
                   
                   
                 RYYVHWVRQAPGQGLEWMGIINPSGGYASYAQKFQGRVTMTRDT 
               
               
                   
                   
                 STSTVYMELSSLRSEDTAVYYCAAGLFIWGQGTLVTVSSASGGGGS 
               
               
                   
                   
                 GGGGSGGGGSHASDIQMTQSPSSLSASVGDRVTITCRASQSIGRYL 
               
               
                   
                   
                 AWYQQKPGKAPKLLIYSASNLETGVPSRFSGSGSGTDFTLTISSLQP 
               
               
                   
                   
                 EDFATYYCQQYNSFPVTFGPGTKVDIKGGGSGGGSHHHHHH 
               
               
                   
                 SEQ ID 
                 EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGL 
               
               
                   
                 NO: 202 
                 EWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAED 
               
               
                   
                   
                 TAVYYCSRWGGDGFYAMDYWGQGTLVTVSSAKTTAPSVYPLAPV 
               
               
                   
                   
                 CGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSD 
               
               
                   
                   
                 LYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCP 
               
               
                   
                   
                 PCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPD 
               
               
                   
                   
                 VQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSG 
               
               
                   
                   
                 KEFKCKVNNKDLGAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQ 
               
               
                   
                   
                 VTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFM 
               
               
                   
                   
                 YSDLRVEKKNWVERNSYSCSVVHEGLHNHHTTESFSRTPGGGGGS 
               
               
                   
                   
                 GGGGSGGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKL 
               
               
                   
                   
                 TDMLTFEFYMPKKATELKHLQCLERELKPLEEVLNLAQSKNFHLRP 
               
               
                   
                   
                 RDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIIST 
               
               
                   
                   
                 LT 
               
               
                   
               
               
                 AF4396 
                 SEQ ID 
                 DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPK 
               
               
                   
                 NO: 64 
                 LLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYT 
               
               
                   
                   
                 TPPTFGQGTKVEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFY 
               
               
                   
                   
                 PKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDE 
               
               
                   
                   
                 YERHNSYTCEATHKTSTSPIVKSFNRNEC 
               
               
                   
                 SEQ ID 
                 EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGL 
               
               
                   
                 NO: 198 
                 EWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAED 
               
               
                   
                   
                 TAVYYCSRWGGDGFYAMDYWGQGTLVTVSSAKTTAPSVYPLAPV 
               
               
                   
                   
                 CGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSD 
               
               
                   
                   
                 LYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCP 
               
               
                   
                   
                 PCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPD 
               
               
                   
                   
                 VQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSG 
               
               
                   
                   
                 KEFKCKVNNKDLGAPIERTISKPKGSVRAPQVYVLPPPEKEMTKKQ 
               
               
                   
                   
                 VSLTCLVKDFMPEDIYVEWTNNGKTELNYKNTEPVLKSDGSYFMY 
               
               
                   
                   
                 SKLTVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGGGGGSG 
               
               
                   
                   
                 GGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFT 
               
               
                   
                   
                 DYYMHWVRQAPGQGLEWMGIINPRAGYTSYALKFQGRVTMTRD 
               
               
                   
                   
                 TSTSTVYMELSSLRSEDTAVYYCTSGWDVWGQGTLVTVSSASGGG 
               
               
                   
                   
                 GSGGGGSGGGGSHASDIQMTQSPSSLSASVGDRVTITCRASQSIST 
               
               
                   
                   
                 WLAWYQQKPGKAPKLLIYAASSLDSGVPSRFSGSGSGTDFTLTISSL 
               
               
                   
                   
                 QPEDFATYYCQQSYSFPVTFGQGTKVEIKGGGSGGGSHHHHHH 
               
               
                   
                 SEQ ID 
                 EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGL 
               
               
                   
                 NO: 202 
                 EWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAED 
               
               
                   
                   
                 TAVYYCSRWGGDGFYAMDYWGQGTLVTVSSAKTTAPSVYPLAPV 
               
               
                   
                   
                 CGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSD 
               
               
                   
                   
                 LYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCP 
               
               
                   
                   
                 PCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPD 
               
               
                   
                   
                 VQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSG 
               
               
                   
                   
                 KEFKCKVNNKDLGAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQ 
               
               
                   
                   
                 VTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFM 
               
               
                   
                   
                 YSDLRVEKKNWVERNSYSCSVVHEGLHNHHTTESFSRTPGGGGGS 
               
               
                   
                   
                 GGGGSGGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKL 
               
               
                   
                   
                 TDMLTFEFYMPKKATELKHLQCLERELKPLEEVLNLAQSKNFHLRP 
               
               
                   
                   
                 RDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIIST 
               
               
                   
                   
                 LT 
               
               
                   
               
               
                 AF4400 
                 SEQ ID 
                 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLL 
               
               
                   
                 NO: 180 
                 IYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNW 
               
               
                   
                   
                 PRTFGQGTKVEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYP 
               
               
                   
                   
                 KDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEY 
               
               
                   
                   
                 ERHNSYTCEATHKTSTSPIVKSFNRNEC 
               
               
                   
                 SEQ ID 
                 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG 
               
               
                   
                 NO: 199 
                 LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE 
               
               
                   
                   
                 DTAVYYCATNDDYWGQGTLVTVSSAKTTAPSVYPLAPVCGDTTG 
               
               
                   
                   
                 SSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSS 
               
               
                   
                   
                 VTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAP 
               
               
                   
                   
                 NAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFV 
               
               
                   
                   
                 NNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKV 
               
               
                   
                   
                 NNKDLGAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMV 
               
               
                   
                   
                 TDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSDLRVEK 
               
               
                   
                   
                 KNWVERNSYSCSVVHEGLHNHHTTESFSRTPGGGGGSGGGGSGG 
               
               
                   
                   
                 GGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTFE 
               
               
                   
                   
                 FYMPKKATELKHLQCLERELKPLEEVLNLAQSKNFHLRPRDLISNI 
               
               
                   
                   
                 NVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT 
               
               
                   
                 SEQ ID 
                 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG 
               
               
                   
                 NO: 200 
                 LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE 
               
               
                   
                   
                 DTAVYYCATNDDYWGQGTLVTVSSAKTTAPSVYPLAPVCGDTTG 
               
               
                   
                   
                 SSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSS 
               
               
                   
                   
                 VTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAP 
               
               
                   
                   
                 NAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFV 
               
               
                   
                   
                 NNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKV 
               
               
                   
                   
                 NNKDLGAPIERTISKPKGSVRAPQVYVLPPPEKEMTKKQVSLTCLV 
               
               
                   
                   
                 KDFMPEDIYVEWTNNGKTELNYKNTEPVLKSDGSYFMYSKLTVEK 
               
               
                   
                   
                 KNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGGGGGSGGGGSGG 
               
               
                   
                   
                 GGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWV 
               
               
                   
                   
                 RQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQ 
               
               
                   
                   
                 MNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASGGG 
               
               
                   
                   
                 GSGGGGSGGGGSHASDIQMTQSPSSLSASVGDRVTITCRASQDVNT 
               
               
                   
                   
                 AVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSL 
               
               
                   
                   
                 QPEDFATYYCQQHYTTPPTFGQGTKVEIKGGGSGGGSHHHHHH 
               
               
                   
               
               
                 AF4401 
                 SEQ ID 
                 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLL 
               
               
                   
                 NO: 180 
                 lYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNW 
               
               
                   
                   
                 PRTFGQGTKVEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYP 
               
               
                   
                   
                 KDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEY 
               
               
                   
                   
                 ERHNSYTCEATHKTSTSPIVKSFNRNEC 
               
               
                   
                 SEQ ID 
                 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG 
               
               
                   
                 NO: 199 
                 LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE 
               
               
                   
                   
                 DTAVYYCATNDDYWGQGTLVTVSSAKTTAPSVYPLAPVCGDTTG 
               
               
                   
                   
                 SSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSS 
               
               
                   
                   
                 VTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAP 
               
               
                   
                   
                 NAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFV 
               
               
                   
                   
                 NNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKV 
               
               
                   
                   
                 NNKDLGAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMV 
               
               
                   
                   
                 TDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSDLRVEK 
               
               
                   
                   
                 KNWVERNSYSCSVVHEGLHNHHTTESFSRTPGGGGGSGGGGSGG 
               
               
                   
                   
                 GGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTFE 
               
               
                   
                   
                 FYMPKKATELKHLQCLERELKPLEEVLNLAQSKNFHLRPRDLISNI 
               
               
                   
                   
                 NVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT 
               
               
                   
                 SEQ ID 
                 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG 
               
               
                   
                 NO: 201 
                 LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE 
               
               
                   
                   
                 DTAVYYCATNDDYWGQGTLVTVSSAKTTAPSVYPLAPVCGDTTG 
               
               
                   
                   
                 SSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSS 
               
               
                   
                   
                 VTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAP 
               
               
                   
                   
                 NAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFV 
               
               
                   
                   
                 NNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKV 
               
               
                   
                   
                 NNKDLGAPIERTISKPKGSVRAPQVYVLPPPEKEMTKKQVSLTCLV 
               
               
                   
                   
                 KDFMPEDIYVEWTNNGKTELNYKNTEPVLKSDGSYFMYSKLTVEK 
               
               
                   
                   
                 KNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGGGGGSGGGGSGG 
               
               
                   
                   
                 GGSGGGGSEVQLVESGGGLVKPGGSLRLSCAASGFTFSSYTLAWV 
               
               
                   
                   
                 RQAPGKGLEWVAAIDSSSYTYSPDTVRGRFTISRDNAKNSLYLQM 
               
               
                   
                   
                 NSLRAEDTAVYYCARDSNWDALDYWGQGTLVTVSSASGGGGSG 
               
               
                   
                   
                 GGGSGGGGSHASDIQMTQSPSSLSASVGDRVSITCKASQNVGTNVG 
               
               
                   
                   
                 WYQQKPGKAPKALIYSASFRYSGVPSRFSGSGSGTDFTLTISSLQPE 
               
               
                   
                   
                 DFATYFCQQYYTYPYTFGGGTKLEIKGGGSGGGSHHHHHH 
               
               
                   
               
               
                 AF4392 
                 SEQ ID 
                 DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPK 
               
               
                   
                 NO: 64 
                 LLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYT 
               
               
                   
                   
                 TPPTFGQGTKVEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFY 
               
               
                   
                   
                 PKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDE 
               
               
                   
                   
                 YERHNSYTCEATHKTSTSPIVKSFNRNEC 
               
               
                   
                 SEQ ID 
                 EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGL 
               
               
                   
                 NO: 202 
                 EWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAED 
               
               
                   
                   
                 TAVYYCSRWGGDGFYAMDYWGQGTLVTVSSAKTTAPSVYPLAPV 
               
               
                   
                   
                 CGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSD 
               
               
                   
                   
                 LYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCP 
               
               
                   
                   
                 PCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPD 
               
               
                   
                   
                 VQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSG 
               
               
                   
                   
                 KEFKCKVNNKDLGAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQ 
               
               
                   
                   
                 VTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFM 
               
               
                   
                   
                 YSDLRVEKKNWVERNSYSCSVVHEGLHNHHTTESFSRTPGGGGGS 
               
               
                   
                   
                 GGGGSGGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKL 
               
               
                   
                   
                 TDMLTFEFYMPKKATELKHLQCLERELKPLEEVLNLAQSKNFHLRP 
               
               
                   
                   
                 RDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIIST 
               
               
                   
                   
                 LT 
               
               
                   
                 SEQ ID 
                 EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGL 
               
               
                   
                 NO: 203 
                 EWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAED 
               
               
                   
                   
                 TAVYYCSRWGGDGFYAMDYWGQGTLVTVSSAKTTAPSVYPLAPV 
               
               
                   
                   
                 CGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSD 
               
               
                   
                   
                 LYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCP 
               
               
                   
                   
                 PCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPD 
               
               
                   
                   
                 VQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSG 
               
               
                   
                   
                 KEFKCKVNNKDLGAPIERTISKPKGSVRAPQVYVLPPPEKEMTKKQ 
               
               
                   
                   
                 VSLTCLVKDFMPEDIYVEWTNNGKTELNYKNTEPVLKSDGSYFMY 
               
               
                   
                   
                 SKLTVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGGGGGSG 
               
               
                   
                   
                 GGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKD 
               
               
                   
                   
                 TYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSK 
               
               
                   
                   
                 NTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVS 
               
               
                   
                   
                 SASGGGGSGGGGSGGGGSHASDIQMTQSPSSLSASVGDRVTITCRA 
               
               
                   
                   
                 SQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDF 
               
               
                   
                   
                 TLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGGGSGGGSHH 
               
               
                   
                   
                 HHHH 
               
               
                   
               
               
                 AF4393 
                 SEQ ID 
                 DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPK 
               
               
                   
                 NO: 64 
                 LLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYT 
               
               
                   
                   
                 TPPTFGQGTKVEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFY 
               
               
                   
                   
                 PKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDE 
               
               
                   
                   
                 YERHNSYTCEATHKTSTSPIVKSFNRNEC 
               
               
                   
                 SEQ ID 
                 EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGL 
               
               
                   
                 NO: 202 
                 EWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAED 
               
               
                   
                   
                 TAVYYCSRWGGDGFYAMDYWGQGTLVTVSSAKTTAPSVYPLAPV 
               
               
                   
                   
                 CGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSD 
               
               
                   
                   
                 LYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCP 
               
               
                   
                   
                 PCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPD 
               
               
                   
                   
                 VQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSG 
               
               
                   
                   
                 KEFKCKVNNKDLGAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQ 
               
               
                   
                   
                 VTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFM 
               
               
                   
                   
                 YSDLRVEKKNWVERNSYSCSVVHEGLHNHHTTESFSRTPGGGGGS 
               
               
                   
                   
                 GGGGSGGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKL 
               
               
                   
                   
                 TDMLTFEFYMPKKATELKHLQCLERELKPLEEVLNLAQSKNFHLRP 
               
               
                   
                   
                 RDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIIST 
               
               
                   
                   
                 LT 
               
               
                   
                 SEQ ID 
                 EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGL 
               
               
                   
                 NO: 204 
                 EWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAED 
               
               
                   
                   
                 TAVYYCSRWGGDGFYAMDYWGQGTLVTVSSAKTTAPSVYPLAPV 
               
               
                   
                   
                 CGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSD 
               
               
                   
                   
                 LYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCP 
               
               
                   
                   
                 PCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPD 
               
               
                   
                   
                 VQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSG 
               
               
                   
                   
                 KEFKCKVNNKDLGAPIERTISKPKGSVRAPQVYVLPPPEKEMTKKQ 
               
               
                   
                   
                 VSLTCLVKDFMPEDIYVEWTNNGKTELNYKNTEPVLKSDGSYFMY 
               
               
                   
                   
                 SKLTVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGGGGGSG 
               
               
                   
                   
                 GGGSGGGGSGGGGSEVQLVESGGGLVKPGGSLRLSCAASGFTFSS 
               
               
                   
                   
                 YTLAWVRQAPGKGLEWVAAIDSSSYTYSPDTVRGRFTISRDNAKN 
               
               
                   
                   
                 SLYLQMNSLRAEDTAVYYCARDSNWDALDYWGQGTLVTVSSASG 
               
               
                   
                   
                 GGGSGGGGSGGGGSHASDIQMTQSPSSLSASVGDRVSITCKASQNV 
               
               
                   
                   
                 GTNVGWYQQKPGKAPKALIYSASFRYSGVPSRFSGSGSGTDFTLTI 
               
               
                   
                   
                 SSLQPEDFATYFCQQYYTYPYTFGGGTKLEIKGGGSGGGSHHHHH 
               
               
                   
                   
                 H 
               
               
                   
               
            
           
         
       
     
     Example 27 
     Improved Regulation by Engineering DBA Affinity Using Standard Methods 
     This example describes the use of standard techniques to modify DBA affinity and improve the range of sensor-dependent activation of a DBA-cytokine construct. A series of variants of the DBA PDL1-IFN R01 A05 (EXAMPLE 1) were prepared as described in EXAMPLE 4, assembled into DBA-cytokine-complexes and assayed as described in EXAMPLE 9. Results with six exemplary variant DBA-cytokine complexes are shown in  FIG.  21 A-F . Each plot includes data for IFNAR2 binding in the presence (triangles) and absence (squares) of PD-L1. Binding to a control IFNα antibody (circles) is used to confirm the presence of intact DBA-complex and provide a relative estimate of the amount of DBA-complex captured in the wells. AF2719 and the five variant DBA-cytokine complexes show negligible IFNAR2 binding in the absence of PD-L1 and different levels of IFNAR2 binding in the presence of PD-L1. AF3099 (SEQ ID NO: 293) shows negligible IFNAR2 binding at all concentrations of PD-L1, while the IFNAR2 binding signal with AF3092 (SEQ ID NO: 292) is similar to the anti-IFNα antibody binding. AF2719 (SEQ ID NO: 41), AF3101 (SEQ ID NO: 289), AF3093 (SEQ ID NO: 290) and AF3094 (SEQ ID NO: 291) show intermediate levels of IFNAR binding relative to anti-IFNα binding. Together, these results demonstrate that standard methods of antibody engineering may be used to improve the regulation of the therapeutic activity of a DBA-cytokine complex. Protein complex sequences are provided in TABLE 25 below. 
     
       
         
           
               
             
               
                 TABLE 25 
               
             
            
               
                   
               
               
                 DBA-cytokine protein complexes 
               
            
           
           
               
               
               
            
               
                 Protein 
                 SEQ ID 
                   
               
               
                 Complex 
                 NO: 
                 Sequence 
               
               
                   
               
               
                 AF3099 
                 SEQ ID 
                 MSTSTCDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAE 
               
               
                   
                 NO: 293 
                 TIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTET 
               
               
                   
                   
                 PLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEG 
               
               
                   
                   
                 GGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFSGYYIH 
               
               
                   
                   
                 WVRQAPGQGLEWMGWMDSNSGGTGYAQKFQGRVTMTRDTSTSTVYMELSSL 
               
               
                   
                   
                 RSEDTAVYYCAKEVFSGWYDYWGQGTLVTVSSASGGGGSGGGGSGGGGSHASDI 
               
               
                   
                   
                 QMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVP 
               
               
                   
                   
                 SRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPYTFGQGTKVEIKGKPIPNPLLGL 
               
               
                   
                   
                 DST 
               
               
                   
               
               
                 AF3092 
                 SEQ ID 
                 MSTSTCDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAE 
               
               
                   
                 NO: 292 
                 TIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTET 
               
               
                   
                   
                 PLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEG 
               
               
                   
                   
                 GGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFSNYYIH 
               
               
                   
                   
                 WVRQAPGQGLEWMGWMDPNSGGTGYAQKFQGRVTMTRDTSTSTVYMELSSL 
               
               
                   
                   
                 RSEDTAVYYCAKEVFSGWYDYWGQGTLVTVSSASGGGGSGGGGSGGGGSHASDI 
               
               
                   
                   
                 QMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVP 
               
               
                   
                   
                 SRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPYTFGQGTKVEIKGKPIPNPLLGL 
               
               
                   
                   
                 DST 
               
               
                   
               
               
                 AF2719 
                 SEQ ID 
                 MSTSTCDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAE 
               
               
                   
                 NO: 41 
                 TIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTET 
               
               
                   
                   
                 PLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEG 
               
               
                   
                   
                 GGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFSNYYV 
               
               
                   
                   
                 HWVRQAPGQGLEWMGWMDPNSGGTGYAHQFQGRVTMTRDTSTSTVYMELS 
               
               
                   
                   
                 SLRSEDTAVYYCAKEVFSGWYDYWGQGTLVTVSSASGGGGSGGGGSGGGGSHAS 
               
               
                   
                   
                 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSG 
               
               
                   
                   
                 VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPYTFGQGTKVEIKGKPIPNPLL 
               
               
                   
                   
                 GLDST 
               
               
                   
               
               
                 AF3101 
                 SEQ ID 
                 MSTSTCDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAE 
               
               
                   
                 NO: 289 
                 TIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTET 
               
               
                   
                   
                 PLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEG 
               
               
                   
                   
                 GGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFSNYYIH 
               
               
                   
                   
                 WVRQAPGQGLEWMGWMDGNSGGTGYAQKFQGRVTMTRDTSTSTVYMELSSL 
               
               
                   
                   
                 RSEDTAVYYCAKEVFSGWYDYWGQGTLVTVSSASGGGGSGGGGSGGGGSHASDI 
               
               
                   
                   
                 QMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVP 
               
               
                   
                   
                 SRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPYTFGQGTKVEIKGKPIPNPLLGL 
               
               
                   
                   
                 DST 
               
               
                   
               
               
                 AF3093 
                 SEQ ID 
                 MSTSTCDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAE 
               
               
                   
                 NO: 290 
                 TIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTET 
               
               
                   
                   
                 PLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEG 
               
               
                   
                   
                 GGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFSNYYIH 
               
               
                   
                   
                 WVRQAPGQGLEWMGWMDSNSGYTGYAQKFQGRVTMTRDTSTSTVYMELSSL 
               
               
                   
                   
                 RSEDTAVYYCAKEVFSGWYDYWGQGTLVTVSSASGGGGSGGGGSGGGGSHASDI 
               
               
                   
                   
                 QMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVP 
               
               
                   
                   
                 SRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPYTFGQGTKVEIKGKPIPNPLLGL 
               
               
                   
                   
                 DST 
               
               
                   
               
               
                 AF3094 
                 SEQ ID 
                 MSTSTCDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAE 
               
               
                   
                 NO: 291 
                 TIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTET 
               
               
                   
                   
                 PLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEG 
               
               
                   
                   
                 GGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFSNYYIH 
               
               
                   
                   
                 WVRQAPGQGLEWMGWMDPNSGYTGYAHQFQGRVTMTRDTSTSTVYMELSSL 
               
               
                   
                   
                 RSEDTAVYYCAKEVFSGWYDYWGQGTLVTVSSASGGGGSGGGGSGGGGSHASDI 
               
               
                   
                   
                 QMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVP 
               
               
                   
                   
                 SRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPYTFGQGTKVEIKGKPIPNPLLGL 
               
               
                   
                   
                 DST 
               
               
                   
               
            
           
         
       
     
     Example 28 
     Regulated Interferon Receptor Binding by PD-L1/IFNα Dual Binding Antibodies 
     This example describes regulated interferon receptor binding by PD-L1/IFNα DBA-cytokine complexes. DBA-cytokine complexes AF2659 (SEQ ID NO: 276-277), AF2666 (SEQ ID NO: 96 and 277), AF2645 (SEQ ID NO: 95 and 112), AF2615 (SEQ ID NO: 279-280) and AF2616 (SEQ ID NO: 281-282) were chosen for analysis. The DBAs used to assemble these complexes were derived from three parental PD-L1/IFNα DBAs described in EXAMPLE 1: PDL1-IFN-R01-A05 (AF2659 and AF2666), PDL1-IFN-R01-C08 (AF2645) and PDL1-IFN-R01-B12 (AF2615 and AF2616) using standard antibody engineering techniques as described in EXAMPLE 4. Three control antibody-cytokine constructs were assembled from monospecific antibodies: AF2696 (anti-PDL1, SEQ ID NO: 283-284), AF2697 (anti-Her2, SEQ ID NO: 214 and 286) and AF2698 (anti-IFNα, SEQ ID NO: 287-288). Protein complex sequences are provided in TABLE 26 below. The eight antibody-cytokine complexes were produced as symmetric IgG molecules with IFNα appended to the N-terminus of the heavy chain through a flexible linker (AF2659, AF2666 and AF2645, shown schematically in  FIG.  9   e   ) or the N-terminus of the light chain through a flexible linker (AF2615, AF2616, AF2696, AF2697 and AF2698, shown schematically in  FIG.  9   d   ). The proteins were expressed in mammalian cells and purified using standard protocols. An ELISA assay was performed with a constant amount of the antibody-cytokine construct coated on each well probed with biotinylated interferon receptor (IFNAR2) in the presence of varying amounts of PD-L1-Fc or Her2-Fc. 384-well ELISA plates were coated with anti-Fc antibody at 10 micrograms/ml overnight at 4° C. and washed twice with SuperBlock. Twenty microliters of a 66.7 nM antibody-cytokine complex was added to each well, incubated for one hour and washed three times in PBS. Biotinylated IFNAR2 protein was added to each well at a final concentration of 5 nM with PD-L1-Fc protein or HER2-Fc protein at a final concentration of 0.64 nM, 3.2 nM, 16 nM, 80 nM, or 400 nM. The plates were incubated for an hour, washed and biotinylated IFNAR2 detection was performed using streptavidin-HRP and standard ELISA protocols. As shown in  FIG.  22 A-H , DBA-cytokine complexes AF2659, AF2666, AF2645, AF2615 and AF2616 show a dose-dependent induction of IFNAR2 binding in the presence of PD-L1 protein but were not affected by the same dose range of Her2 protein, demonstrating PD-L1 dependent INF activity in five DBA constructs derived from three different parental PD-L1/IFNA dual-binding antibodies. IFNAR2 binding to the three control antibody complexes AF2696, AF2697 and AF2698 was not significantly affected by PD-L1 concentration. Protein complexes of the present invention can be produced from multiple dual-binding antibodies using standard antibody engineering methods. 
     
       
         
           
               
             
               
                 TABLE 26 
               
             
            
               
                   
               
               
                 DBA-cytokine protein complexes 
               
            
           
           
               
               
               
            
               
                 Protein 
                 SEQ ID 
                   
               
               
                 Complex 
                 NO: 
                 Sequence 
               
               
                   
               
               
                 AF2659 
                 SEQ ID 
                 CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVL 
               
               
                   
                 NO: 276 
                 HEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMK 
               
               
                   
                   
                 EDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEVPGVG 
               
               
                   
                   
                 VPGAGVPGVGVPGGGVPGVGVPGGGVPGAGVPGGGVPGVGVPGAGVPGVGV 
               
               
                   
                   
                 PGGGQVQLVQSGAEVKKPGASVKVSCKASGYTFSNYYIHWVRQAPGQGLEWMG 
               
               
                   
                   
                 WMDSNSGYTGYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAKEVFSG 
               
               
                   
                   
                 WYDYWGQGTLVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLT 
               
               
                   
                   
                 WNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKI 
               
               
                   
                   
                 EPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPD 
               
               
                   
                   
                 VQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNN 
               
               
                   
                   
                 KDLGAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEW 
               
               
                   
                   
                 TNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHH 
               
               
                   
                   
                 TTKSFSRTPGK 
               
               
                   
                 SEQ ID 
                 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSG 
               
               
                   
                 NO: 277 
                 VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPYTFGQGTKVEIKRADAAPTV 
               
               
                   
                   
                 SIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDS 
               
               
                   
                   
                 TYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC 
               
               
                   
               
               
                 AF2666 
                 SEQ ID 
                 CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVL 
               
               
                   
                 NO: 96 
                 HEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMK 
               
               
                   
                   
                 EDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEVPGVG 
               
               
                   
                   
                 VPGAGVPGVGVPGGGVPGVGVPGGGVPGAGVPGGGVPGVGVPGAGVPGVGV 
               
               
                   
                   
                 PGGGQVQLVQSGAEVKKPGASVKVSCKASGYTFSNYYIHWVRQAPGQGLEWMG 
               
               
                   
                   
                 WMDSNSGYTGYAQQFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAKEVFSG 
               
               
                   
                   
                 WYDYWGQGTLVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLT 
               
               
                   
                   
                 WNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKI 
               
               
                   
                   
                 EPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPD 
               
               
                   
                   
                 VQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNN 
               
               
                   
                   
                 KDLGAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEW 
               
               
                   
                   
                 TNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHH 
               
               
                   
                   
                 TTKSFSRTPGK 
               
               
                   
                 SEQ ID 
                 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSG 
               
               
                   
                 NO: 277 
                 VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPYTFGQGTKVEIKRADAAPTV 
               
               
                   
                   
                 SIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDS 
               
               
                   
                   
                 TYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC 
               
               
                   
               
               
                 AF2645 
                 SEQ ID 
                 CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVL 
               
               
                   
                 NO: 95 
                 HEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMK 
               
               
                   
                   
                 EDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEVPGVG 
               
               
                   
                   
                 VPGAGVPGVGVPGGGVPGVGVPGGGVPGAGVPGGGVPGVGVPGAGVPGVGV 
               
               
                   
                   
                 PGGGQVQLVQSGAEVKKPGASVKVSCKASGGTFSSYAISWVRQAPGQGLEWMG 
               
               
                   
                   
                 IIDPSVTYTRYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSLFPTIFGV 
               
               
                   
                   
                 EVAFDIWGQGTLVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLT 
               
               
                   
                   
                 WNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKI 
               
               
                   
                   
                 EPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPD 
               
               
                   
                   
                 VQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNN 
               
               
                   
                   
                 KDLGAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEW 
               
               
                   
                   
                 TNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHH 
               
               
                   
                   
                 TTKSFSRTPGK 
               
               
                   
                 SEQ ID 
                 DIQMTQSPSSLSASVGDRVTITCRASQSISNRLAWYQQKPGKAPKLLIYKASSLESG 
               
               
                   
                 NO: 112 
                 VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSNSTPFTFGQGTKVEIKRADAAPTV 
               
               
                   
                   
                 SIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDS 
               
               
                   
                   
                 TYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC 
               
               
                   
               
               
                 AF2615 
                 SEQ ID 
                 QVQLVQSGAEVKKPGASVKVSCKASGNTFTDYYMHWVRQAPGQGLEWMGWM 
               
               
                   
                 NO: 279 
                 NPNSGNTGYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSLFPTIFGV 
               
               
                   
                   
                 EVAFDIWGQGTLVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLT 
               
               
                   
                   
                 WNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKI 
               
               
                   
                   
                 EPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPD 
               
               
                   
                   
                 VQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNN 
               
               
                   
                   
                 KDLGAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEW 
               
               
                   
                   
                 TNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHH 
               
               
                   
                   
                 TTKSFSRTPGK 
               
               
                   
                 SEQ ID 
                 CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVL 
               
               
                   
                 NO: 280 
                 HEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMK 
               
               
                   
                   
                 EDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEGGGGS 
               
               
                   
                   
                 GGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQK 
               
               
                   
                   
                 PGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTF 
               
               
                   
                   
                 GQGTRLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSER 
               
               
                   
                   
                 QNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNR 
               
               
                   
                   
                 NEC 
               
               
                   
               
               
                 AF2616 
                 SEQ ID 
                 QVQLVQSGAEVKKPGASVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGIIDPS 
               
               
                   
                 NO: 281 
                 VTYTRYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSLFPTIFGVEVAF 
               
               
                   
                   
                 DIWGQGTLVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNS 
               
               
                   
                   
                 GSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRG 
               
               
                   
                   
                 PTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQIS 
               
               
                   
                   
                 WFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLG 
               
               
                   
                   
                 APIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNN 
               
               
                   
                   
                 GKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTK 
               
               
                   
                   
                 SFSRTPGK 
               
               
                   
                 SEQ ID 
                 CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVL 
               
               
                   
                 NO: 282 
                 HEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMK 
               
               
                   
                   
                 EDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEGGGGS 
               
               
                   
                   
                 GGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCQASQSISNRLAWYQQK 
               
               
                   
                   
                 PGKAPKLLIYKASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYSTPITFG 
               
               
                   
                   
                 QGTKVEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQ 
               
               
                   
                   
                 NGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRN 
               
               
                   
                   
                 EC 
               
               
                   
               
               
                 AF2696 
                 SEQ ID 
                 QVQLVQSGAEVKKPGASVKVSCKASGYTFSGYYMHWVRQAPGQGLEWMGWM 
               
               
                   
                 NO: 283 
                 DPNSGYTGYAHQFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAKEVFSGWYD 
               
               
                   
                   
                 YWGQGTLVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSG 
               
               
                   
                   
                 SLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGP 
               
               
                   
                   
                 TIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISW 
               
               
                   
                   
                 FVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLGAP 
               
               
                   
                   
                 lERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDlYVEWTNNGKT 
               
               
                   
                   
                 ELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSR 
               
               
                   
                   
                 TPGK 
               
               
                   
                 SEQ ID 
                 CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVL 
               
               
                   
                 NO: 284 
                 HEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMK 
               
               
                   
                   
                 EDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEGGGGS 
               
               
                   
                   
                 GGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQTISSYLNWYQQKP 
               
               
                   
                   
                 GKAPKLLIYAASTLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGYSTPITFG 
               
               
                   
                   
                 PGTKVDIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQ 
               
               
                   
                   
                 NGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRN 
               
               
                   
                   
                 EC 
               
               
                   
               
               
                 AF2697 
                 SEQ ID 
                 EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTN 
               
               
                   
                 NO: 214 
                 GYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDY 
               
               
                   
                   
                 WGQGTLVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGS 
               
               
                   
                   
                 LSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPT 
               
               
                   
                   
                 IKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWF 
               
               
                   
                   
                 VNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLGAPI 
               
               
                   
                   
                 ERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKT 
               
               
                   
                   
                 ELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSR 
               
               
                   
                   
                 TPGK 
               
               
                   
                 SEQ ID 
                 CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVL 
               
               
                   
                 NO: 286 
                 HEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMK 
               
               
                   
                   
                 EDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEGGGGS 
               
               
                   
                   
                 GGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQ 
               
               
                   
                   
                 KPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPT 
               
               
                   
                   
                 FGQGTKVEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSE 
               
               
                   
                   
                 RQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFN 
               
               
                   
                   
                 RNEC 
               
               
                   
               
               
                 AF2698 
                 SEQ ID 
                 EVQLVESGGGLVQPGGSLRLSCATSGYTFTEYIIHWVRQAPGKGLEWVASINPDYD 
               
               
                   
                 NO: 287 
                 ITNYNQRFKGRFTISLDKSKRTAYLQMNSLRAEDTAVYYCASWISDFFDYWGQGTL 
               
               
                   
                   
                 VTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHT 
               
               
                   
                   
                 FPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPC 
               
               
                   
                   
                 KCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEV 
               
               
                   
                   
                 HTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLGAPIERTISKP 
               
               
                   
                   
                 KGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKN 
               
               
                   
                   
                 TEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK 
               
               
                   
                 SEQ ID 
                 CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVL 
               
               
                   
                 NO: 288 
                 HEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMK 
               
               
                   
                   
                 EDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEGGGGS 
               
               
                   
                   
                 GGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSVSTSSYSYMHW 
               
               
                   
                   
                 YQQKPGKAPKVLISYASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHSW 
               
               
                   
                   
                 GIPRTFGQGTKVEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKI 
               
               
                   
                   
                 DGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIV 
               
               
                   
                   
                 KSFNRNEC 
               
               
                   
               
            
           
         
       
     
     Example 29 
     Selection of IL-15 and PD-L1 Specific Dual Binding Antibodies 
     This example describes isolation of sensor domains of the present disclosure, specifically, selection of IL-15 and PD-L1 specific dual binding antibodies (DBAs). Anti-PD-L1 and anti-IL-15 DBAs were isolated from the IFNα Tumbler antibody phage display library described in EXAMPLE 1. The selection was similar to the protocol described in EXAMPLE 1, alternating between PD-L1 selection and IL-15 selection. 
     The final selection was plated as single colonies and 380 colonies were picked for Sanger sequencing. Thirty-eight unique clones were screened for PD-L1 and IL-15 binding. The scFv DNA sequence for each clone was synthesized as a gBlock (Integrated DNA Technologies, Inc.) with a T7 promoter, a translation initiation site, a Myc tag, the scFv sequence, a V5 tag and a T7 terminator. Proteins were expressed using the PUREfrex2.1 cell-free transcription/translation system as described in previous examples. The scFv samples were subjected to ELISA analysis to detect PDL1 and IL15 binding. In these experiments, wells of a 384-well plate were coated with an anti-V5 antibody (Sv5-Pk1, BioRad) at 1 ug/ml overnight at 4 degrees. After washing, wells were blocked with SuperBlock (ThermoFisher, 37515) followed by addition of saturating levels of scFvs in SuperBlock. After washing, antigens were added and plates incubated for one hour (PDL1-hFc-Avi, Acro Biosystems, PDL-H82F2); AF33 (SEQ ID NO: 298-299), biotinylated using standard methods; controls of PD1-hFc-Avi (Acro Biosystems, PD1-H82F1); AF35 (SEQ ID 63-64), biotinylated using standard methods). Biotinylated antigens were detected using streptavidin HRP using standard methods. Varying amounts of labeled test antigen were added to show binding and to estimate relative affinities of the different scFvs.  FIGS.  23 A-D  show the ELISA binding data for four exemplary dual-binding scFvs AF635 (SEQ ID NO: 216), AF636 (SEQ ID NO: 217), AF666 (SEQ ID NO: 218) and AF614 (SEQ ID NO: 219). All four antibodies show binding to both PD-L1 and IL-15, with binding to PD-L1 detectable at a lower concentration of the antigen. Protein complex sequences are summarized in TABLE 27 below. 
     
       
         
           
               
             
               
                 TABLE 27 
               
             
            
               
                   
               
               
                 DBA-cytokine protein complexes, and control protein complexes 
               
            
           
           
               
               
               
            
               
                 Protein 
                 SEQ ID 
                   
               
               
                 Complex 
                 NO: 
                 Sequence 
               
               
                   
               
               
                 AF33 
                 SEQ ID 
                 EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTN 
               
               
                   
                 NO: 298 
                 GYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDY 
               
               
                   
                   
                 WGQGTLVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGS 
               
               
                   
                   
                 LSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPT 
               
               
                   
                   
                 IKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWF 
               
               
                   
                   
                 VNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLGAPI 
               
               
                   
                   
                 ERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKT 
               
               
                   
                   
                 ELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSR 
               
               
                   
                   
                 TPGKGGGSGGGSHHHHHH 
               
               
                   
                 SEQ ID 
                 ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWT 
               
               
                   
                 NO: 299 
                 TPSLKCIRDPALVHQRPAPPSGGSGGGGSGGGSGGGGSLQNWVNVISDLKKIEDLI 
               
               
                   
                   
                 QSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSL 
               
               
                   
                   
                 SSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGGGGSGGGGSGGGGSG 
               
               
                   
                   
                 GGGSDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSA 
               
               
                   
                   
                 SFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRA 
               
               
                   
                   
                 DAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTD 
               
               
                   
                   
                 QDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC 
               
               
                   
               
               
                 AF35 
                 SEQ ID 
                 CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVL 
               
               
                   
                 NO: 63 
                 HEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMK 
               
               
                   
                   
                 EDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEGGGGS 
               
               
                   
                   
                 GGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQ 
               
               
                   
                   
                 APGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYY 
               
               
                   
                   
                 CSRWGGDGFYAMDYWGQGTLVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLV 
               
               
                   
                   
                 KGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAH 
               
               
                   
                   
                 PASSTKVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCV 
               
               
                   
                   
                 VVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMS 
               
               
                   
                   
                 GKEFKCKVNNKDLGAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTD 
               
               
                   
                   
                 FMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCS 
               
               
                   
                   
                 VVHEGLHNHHTTKSFSRTPGK 
               
               
                   
                 SEQ ID 
                 DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYS 
               
               
                   
                 NO: 64 
                 GVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRADAAP 
               
               
                   
                   
                 TVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSK 
               
               
                   
                   
                 DSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC 
               
               
                   
               
               
                 AF635 
                 SEQ ID 
                 MSTSTEQKLISEEDLQVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYIHWVRQAP 
               
               
                   
                 NO: 216 
                 GQGLEWMGIINPSGGSTRYAOKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYC 
               
               
                   
                   
                 ARSLFPTIFGVEVAFDIWGQGTLVTVSSASGGGGSGGGGSGGGGSHASDIQMTQ 
               
               
                   
                   
                 SPSSLSASVGDRVTITCRASQSIRTYLNWYQQKPGKAPKLLIYSASNLQSGVPSRFSG 
               
               
                   
                   
                 SGSGTDFTLTISSLQPEDFATYYCQQANSFPFTFGPGTKVDIKGKPIPNPLLGLDST 
               
               
                   
               
               
                 AF636 
                 SEQ ID 
                 MSTSTEQKLISEEDLQVQLVQSGAEVKKPGASVKVSCKASGYSFTSYYLHWVRQAP 
               
               
                   
                 NO: 217 
                 GQGLEWMGRISPRSGGTKNAQNFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYY 
               
               
                   
                   
                 CVRSLFPTIFGVEVAFDIWGQGTLVTVSSASGGGGSGGGGSGGGGSHASDIQMT 
               
               
                   
                   
                 QSPSSLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYYASSLQSGVPSRFS 
               
               
                   
                   
                 GSGSGTDFTLTISSLQPEDFATYYCQQGYQYPYTFGQGTKLEIKGKPIPNPLLGLDST 
               
               
                   
               
               
                 AF666 
                 SEQ ID 
                 MSTSTEQKLISEEDLQVQLVQSGAEVKKPGASVKVSCKASGYTFSTYYIHWVRQAP 
               
               
                   
                 NO: 218 
                 GQGLEWMGWMNPNSGNTGYAQTFQGRVTMTRDTSTSTVYMELSSLRSEDTAV 
               
               
                   
                   
                 YYCARSLFPTIFGVEVAFDIWGQGTLVTVSSASGGGGSGGGGSGGGGSHASDIQM 
               
               
                   
                   
                 TQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRF 
               
               
                   
                   
                 SGSGSGTDFTLTISSLQPEDFATYYCQQGYSTPRTFGQGTKVEIKGKPIPNPLLGLDS 
               
               
                   
                   
                 T 
               
               
                   
               
               
                 AF614 
                 SEQ ID 
                 MSTSTEQKLISEEDLQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYMHWVRQA 
               
               
                   
                 NO: 219 
                 PGQGLEWMGWMNPNSGNTGYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTA 
               
               
                   
                   
                 VYYCARSLFPTIFGVEVAFDIWGQGTLVTVSSASGGGGSGGGGSGGGGSHASDIQ 
               
               
                   
                   
                 MTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPS 
               
               
                   
                   
                 RFSGSGSGTDFTLTISSLOPEDFATYYCQQSYSTPRTFGQGTKLEIKGKPIPNPLLGLD 
               
               
                   
                   
                 ST 
               
               
                   
               
            
           
         
       
     
     Example 30 
     Regulated IL-2 Receptor Signaling by a PD-L1/IL-15 Dual Binding Antibody (DBA) Cytokine Complex 
     This example describes regulated IL-15 activity in a reporter cell line by PD-L1/IL-15 DBA-cytokine complexes. HEK-Blue™ IL-2 reporter cells (Invivogen Catalog #hkb-il2) were grown according to the vendors instructions. The cells express IL-2 receptor and respond to IL-2 or IL-15 signaling by induction of an enzyme that can be read with a colorimetric assay. The exemplary dual-binding scFv sequences AF635 (SEQ ID NO: 216), AF636 (SEQ ID NO: 217), AF666 (SEQ ID NO: 218) and AF614 (SEQ ID NO: 219) described in EXAMPLE 29 and TABLE 28, were used to assemble scFv DBA-cytokine complexes AF4455 (SEQ ID NO: 220), AF4456 (SEQ ID NO: 221), AF4457 (SEQ ID NO: 222), and AF4440 (SEQ ID NO: 223) respectively. Two monospecific anti-IL-15 scFv sequences were assembled into cytokine-scFv complexes AF4478 (SEQ ID NO: 224) and AF4479 (SEQ ID NO: 225) to serve as negative controls. Protein complex sequences are provided in TABLE 28 below. The proteins were expressed using a PUREfrex2.1 cell-free transcription/translation system. 384-well ELISA plates (Corning 3700) were coated with 25 microliters per well of 1 microgram per ml anti-V5 antibody (SV5-pk1) in 100 mM bicarbonate solution pH 9.0 overnight at 4° C. The plates were washed three times with 100 microliters PBS+ Tween and once with 50 ul/well of SuperBlock. The PUREfrex reactions for each sample were diluted 1:2, 160 in Superblock, added to the anti-V5-coated plates at 20 μl/well and incubated for 1 hour at room temperature to capture a uniform quantity of the scFv protein on the plate. Each plate was then washed three times with PBS with 0.05% Tween 20 (PBST). PDL-1 protein (Acro Biosystems Product #PD1-H5358) or Fc protein (Acro Biosystems Product #FCC-H5214) was diluted in growth media (DMEM complete) to 2-fold higher than the final concentration and 12.5 microliters added per well. After a 15 minute incubation 12.5 microliters of HEK-Blue™ IL-2 reporter cells (12,500 cells) were added to each well and incubated overnight. Five microliters from each well was transferred to a new plate containing 45 microliters of QuantiBlue solution (Invivogen Product #rep-qbs). After 30 to 60 minutes the absorbance at 630 nm was determined using a Perkin-Elmer Envision. 
     
       
         
           
               
             
               
                 TABLE 28 
               
             
            
               
                   
               
               
                 DBA-cytokine protein complexes, and control protein complexes 
               
            
           
           
               
               
            
               
                 Protein 
                 SEQ ID 
               
            
           
           
               
               
               
            
               
                 Complex 
                 NO: 
                 Sequence 
               
               
                   
               
               
                 AF4455 
                 SEQ ID 
                 MSTSTITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATN 
               
               
                   
                 NO: 220 
                 VAHWTTPSLKCIRDPALVHQRPAPPSGGSGGGGSGGGSGGGGSLQNWVNVISDL 
               
               
                   
                   
                 KKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIIL 
               
               
                   
                   
                 ANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGGSGSGSGGSG 
               
               
                   
                   
                 GSGSGGSGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYIHWVRQAPGQGL 
               
               
                   
                   
                 EWMGIINPSGGSTRYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSLF 
               
               
                   
                   
                 PTIFGVEVAFDIWGQGTLVTVSSASGGGGSGGGGSGGGGSHASDIQMTQSPSSLS 
               
               
                   
                   
                 ASVGDRVTITCRASQSIRTYLNWYQQKPGKAPKLLIYSASNLQSGVPSRFSGSGSGT 
               
               
                   
                   
                 DFTLTISSLQPEDFATYYCQQANSFPFTFGPGTKVDIKAAAGSGSEQKLISEEDLGKPI 
               
               
                   
                   
                 PNPLLGLDSTNA 
               
               
                   
               
               
                 AF4456 
                 SEQ ID 
                 MSTSTITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATN 
               
               
                   
                 NO: 221 
                 VAHWTTPSLKCIRDPALVHQRPAPPSGGSGGGGSGGGSGGGGSLQNWVNVISDL 
               
               
                   
                   
                 KKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIIL 
               
               
                   
                   
                 ANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGGSGSGSGGSG 
               
               
                   
                   
                 GSGSGGSGSQVQLVQSGAEVKKPGASVKVSCKASGYSFTSYYLHWVRQAPGQGL 
               
               
                   
                   
                 EWMGRISPRSGGTKNAQNFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCVRSL 
               
               
                   
                   
                 FPTIFGVEVAFDIWGQGTLVTVSSASGGGGSGGGGSGGGGSHASDIQMTQSPSSL 
               
               
                   
                   
                 SASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYYASSLQSGVPSRFSGSGSG 
               
               
                   
                   
                 TDFTLTISSLQPEDFATYYCQQGYQYPYTFGQGTKLEIKAAAGSGSEQKLISEEDLGK 
               
               
                   
                   
                 PIPNPLLGLDSTNA 
               
               
                   
               
               
                 AF4457 
                 SEQ ID 
                 MSTSTITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATN 
               
               
                   
                 NO: 222 
                 VAHWTTPSLKCIRDPALVHQRPAPPSGGSGGGGSGGGSGGGGSLQNWVNVISDL 
               
               
                   
                   
                 KKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIIL 
               
               
                   
                   
                 ANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGGSGSGSGGSG 
               
               
                   
                   
                 GSGSGGSGSQVQLVQSGAEVKKPGASVKVSCKASGYTFSTYYIHWVRQAPGQGLE 
               
               
                   
                   
                 WMGWMNPNSGNTGYAQTFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARS 
               
               
                   
                   
                 LFPTIFGVEVAFDIWGQGTLVTVSSASGGGGSGGGGSGGGGSHASDIQMTQSPSS 
               
               
                   
                   
                 LSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSG 
               
               
                   
                   
                 TDFTLTISSLQPEDFATYYCQQGYSTPRTFGQGTKVEIKAAAGSGSEQKLISEEDLGK 
               
               
                   
                   
                 PIPNPLLGLDSTNA 
               
               
                   
               
               
                 AF4440 
                 SEQ ID 
                 MSTSTITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATN 
               
               
                   
                 NO: 223 
                 VAHWTTPSLKCIRDPALVHQRPAPPSGGSGGGGSGGGSGGGGSLQNWVNVISDL 
               
               
                   
                   
                 KKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIIL 
               
               
                   
                   
                 ANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGGSGSGSGGSG 
               
               
                   
                   
                 GSGSGGSGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYMHWVRQAPGQG 
               
               
                   
                   
                 LEWMGWMNPNSGNTGYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCA 
               
               
                   
                   
                 RSLFPTIFGVEVAFDIWGQGTLVTVSSASGGGGSGGGGSGGGGSHASDIQMTQSP 
               
               
                   
                   
                 SSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSG 
               
               
                   
                   
                 SGTDFTLTISSLQPEDFATYYCQQSYSTPRTFGQGTKLEIKAAAGSGSEQKLISEEDLG 
               
               
                   
                   
                 KPIPNPLLGLDSTNA 
               
               
                   
               
               
                 AF4478 
                 SEQ ID 
                 MSTSTITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATN 
               
               
                   
                 NO: 224 
                 VAHWTTPSLKCIRDPALVHQRPAPPSGGSGGGGSGGGSGGGGSLQNWVNVISDL 
               
               
                   
                   
                 KKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIIL 
               
               
                   
                   
                 ANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGGSGSGSGGSG 
               
               
                   
                   
                 GSGSGGSGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYIHWVRQAPGQGL 
               
               
                   
                   
                 EWMGWMNPNSGNTGYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCA 
               
               
                   
                   
                 RSLFPTIFGVEVAFDIWGQGTLVTVSSASGGGGSGGGGSGGGGSHASDIQMTQSP 
               
               
                   
                   
                 SSLSASVGDRVTITCRASRSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSG 
               
               
                   
                   
                 SGTDFTLTISSLQPEDFATYYCQQSYSTPRTFGQGTKVEIKAAAGSGSEQKLISEEDL 
               
               
                   
                   
                 GKPIPNPLLGLDSTNA 
               
               
                   
               
               
                 AF4479 
                 SEQ ID 
                 MSTSTITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATN 
               
               
                   
                 NO: 225 
                 VAHWTTPSLKCIRDPALVHQRPAPPSGGSGGGGSGGGSGGGGSLQNWVNVISDL 
               
               
                   
                   
                 KKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIIL 
               
               
                   
                   
                 ANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGGSGSGSGGSG 
               
               
                   
                   
                 GSGSGGSGSQVQLVQSGAEVKKPGSSVKVSCKASGYSFTDYYVHWVRQAPGQGL 
               
               
                   
                   
                 EWVGGINPKRGDTVFAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGGL 
               
               
                   
                   
                 GVFGVVDVWGQGTTVTVSSASGGGGSGGGGSGGGGSHASDIVMTQSPLSLPVT 
               
               
                   
                   
                 PGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYAATTLQSGVPDRFSGSG 
               
               
                   
                   
                 SGTDFTLKISRVEAEDVGVYYCMQALQTPLTFGGGTKLEIKAAAGSGSEQKLISEEDL 
               
               
                   
                   
                 GKPIPNPLLGLDST 
               
               
                   
               
            
           
         
       
     
     The results are shown in  FIGS.  24 A-F . IL-15 activity increased in a dose dependent manner with the addition of PD-L1 (circles) but not with the addition of Fc protein (squares) for DBA-cytokine complexes AF4455 (SEQ ID NO: 220), AF4456 (SEQ TD NO: 221), AF4457 (SEQ TD NO: 222) and AF4440 (SEQ IDF: 223). IL-15 activity from the monospecific IL-15 scFv cytokine complexes AF4478 (SEQ TD NO: 224) and AF4479 (SEQ TD NO: 225) did not change with the addition of PD-L1 or Fc protein. 
     The exemplary dual-binding antibody sequences from AF614 (SEQ TD NO: 219) and AF666 (SEQ TD NO: 218) were assembled into asymmetric IgG molecules with IL-15 appended to the N-terminus of one heavy chain through a flexible linker (as shown schematically in  FIG.  9   b   ) to create AF4591 (SEQ TD NO: 226-228) and AF4592 (SEQ TD NO: 229-231) respectively. Two controls were assembled in the same format from an anti-G-15 antibody (AF4659, SEQ ID NO: 232-234) and a PDL1-IFN dual-binding antibody (AF4660, SEQ TD NO: 235-237). Protein complex sequences are summarized in TABLE 29. The proteins were expressed in mammalian cells and purified using standard protocols. The four antibody-cytokine complexes were assayed for IL-15 activity using HEK-Blue™ IL-2 reporter cells in an assay similar to that described above, with the exception that all of the proteins were in solution in the growth media. The purified antibody-cytokine complexes were diluted to a final concentration of 100 pM and assayed in varying concentrations of PD-L1 or a control IgG1 antibody. 
     
       
         
           
               
             
               
                 TABLE 29 
               
             
            
               
                   
               
               
                 DBA-cytokine protein complexes, and control protein complexes 
               
            
           
           
               
               
               
            
               
                 Protein 
                 SEQ ID 
                   
               
               
                 Complex 
                 NO: 
                 Sequence 
               
               
                   
               
               
                 AF4591 
                 SEQ ID 
                 ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATN 
               
               
                   
                 NO: 226 
                 VAHWTTPSLKCIRDPALVHQRPAPPSGGSGGGGSGGGSGGGGSLQNW 
               
               
                   
                   
                 VNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESG 
               
               
                   
                   
                 DASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQ 
               
               
                   
                   
                 MFINTSGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVS 
               
               
                   
                   
                 CKASGYTFTNYYMHWVRQAPGQGLEWMGWMNPNSGNTGYAQKFQ 
               
               
                   
                   
                 GRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSLFPTIFGVEVAFDIWGQ 
               
               
                   
                   
                 GTLVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNS 
               
               
                   
                   
                 GSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVD 
               
               
                   
                   
                 KKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVV 
               
               
                   
                   
                 DVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQD 
               
               
                   
                   
                 WMSGKEFKCKVNNKDLGAPIERTISKPKGSVRAPQVYVLPPPEEEMTKK 
               
               
                   
                   
                 QVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSDL 
               
               
                   
                   
                 RVEKKNWVERNSYSCSVVHEGLHNHHTTESFSRTPGK 
               
               
                   
                 SEQ ID 
                 QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYMHWVRQAPGQGLEW 
               
               
                   
                 NO: 227 
                 MGWMNPNSGNTGYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVY 
               
               
                   
                   
                 YCARSLFPTIFGVEVAFDIWGQGTLVTVSSAKTTAPSVYPLAPVCGDTTGS 
               
               
                   
                   
                 SVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSS 
               
               
                   
                   
                 TWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVF 
               
               
                   
                   
                 IFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTH 
               
               
                   
                   
                 REDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLGAPIERTISKPKGS 
               
               
                   
                   
                 VRAPQVYVLPPPEKEMTKKQVSLTCLVKDFMPEDIYVEWTNNGKTELNY 
               
               
                   
                   
                 KNTEPVLKSDGSYFMYSKLTVEKKNWVERNSYSCSVVHEGLHNHHTTKSF 
               
               
                   
                   
                 SRTPGK 
               
               
                   
                 SEQ ID 
                 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAA 
               
               
                   
                 NO: 228 
                 SSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPRTFGQGTK 
               
               
                   
                   
                 LEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSER 
               
               
                   
                   
                 QNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPI 
               
               
                   
                   
                 VKSFNRNEC 
               
               
                   
               
               
                 AF4592 
                 SEQ ID 
                 ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATN 
               
               
                   
                 NO: 229 
                 VAHWTTPSLKCIRDPALVHQRPAPPSGGSGGGGSGGGSGGGGSLQNW 
               
               
                   
                   
                 VNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESG 
               
               
                   
                   
                 DASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQ 
               
               
                   
                   
                 MFINTSGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVS 
               
               
                   
                   
                 CKASGYTFSTYYIHWVRQAPGQGLEWMGWMNPNSGNTGYAQTFQGR 
               
               
                   
                   
                 VTMTRDTSTSTVYMELSSLRSEDTAVYYCARSLFPTIFGVEVAFDIWGQGT 
               
               
                   
                   
                 LVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGS 
               
               
                   
                   
                 LSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKI 
               
               
                   
                   
                 EPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDV 
               
               
                   
                   
                 SEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDW 
               
               
                   
                   
                 MSGKEFKCKVNNKDLGAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQV 
               
               
                   
                   
                 TLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSDLRV 
               
               
                   
                   
                 EKKNWVERNSYSCSVVHEGLHNHHTTESFSRTPGK 
               
               
                   
                 SEQ ID 
                 QVQLVQSGAEVKKPGASVKVSCKASGYTFSTYYIHWVRQAPGQGLEWM 
               
               
                   
                 NO: 230 
                 GWMNPNSGNTGYAQTFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYC 
               
               
                   
                   
                 ARSLFPTIFGVEVAFDIWGQGTLVTVSSAKTTAPSVYPLAPVCGDTTGSSV 
               
               
                   
                   
                 TLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSST 
               
               
                   
                   
                 WPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFI 
               
               
                   
                   
                 FPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTH 
               
               
                   
                   
                 REDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLGAPIERTISKPKGS 
               
               
                   
                   
                 VRAPQVYVLPPPEKEMTKKQVSLTCLVKDFMPEDIYVEWTNNGKTELNY 
               
               
                   
                   
                 KNTEPVLKSDGSYFMYSKLTVEKKNWVERNSYSCSVVHEGLHNHHTTKSF 
               
               
                   
                   
                 SRTPGK 
               
               
                   
                 SEQ ID 
                 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAA 
               
               
                   
                 NO: 231 
                 SSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGYSTPRTFGQGT 
               
               
                   
                   
                 KVEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSE 
               
               
                   
                   
                 RQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSP 
               
               
                   
                   
                 IVKSFNRNEC 
               
               
                   
               
               
                 AF4659 
                 SEQ ID 
                 ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATN 
               
               
                   
                 NO: 232 
                 VAHWTTPSLKCIRDPALVHQRPAPPSGGSGGGGSGGGSGGGGSLQNW 
               
               
                   
                   
                 VNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESG 
               
               
                   
                   
                 DASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQ 
               
               
                   
                   
                 MFINTSGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVS 
               
               
                   
                   
                 CKASGDTFSSYAISWVRQAPGQGLEWMGWMNPNSGNTGYAQKFQGR 
               
               
                   
                   
                 VTMTRDTSTSTVYMELSSLRSEDTAVYYCATGITMIGYWGQGTLVTVSSA 
               
               
                   
                   
                 KTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHT 
               
               
                   
                   
                 FPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTI 
               
               
                   
                   
                 KPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPD 
               
               
                   
                   
                 VQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFK 
               
               
                   
                   
                 CKVNNKDLGAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVT 
               
               
                   
                   
                 DFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSDLRVEKKNWV 
               
               
                   
                   
                 ERNSYSCSVVHEGLHNHHTTESFSRTPGK 
               
               
                   
                 SEQ ID 
                 QVQLVQSGAEVKKPGASVKVSCKASGDTFSSYAISWVRQAPGQGLEWM 
               
               
                   
                 NO: 233 
                 GWMNPNSGNTGYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYC 
               
               
                   
                   
                 ATGITMIGYWGQGTLVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVK 
               
               
                   
                   
                 GYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITC 
               
               
                   
                   
                 NVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVL 
               
               
                   
                   
                 MISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTL 
               
               
                   
                   
                 RVVSALPIQHQDWMSGKEFKCKVNNKDLGAPIERTISKPKGSVRAPQVY 
               
               
                   
                   
                 VLPPPEKEMTKKQVSLTCLVKDFMPEDIYVEWTNNGKTELNYKNTEPVLK 
               
               
                   
                   
                 SDGSYFMYSKLTVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK 
               
               
                   
                 SEQ ID 
                 DIQMTQSPSSLSASVGDRVTITCQASQDISSYLNWYQQKPGKAPKLLIYAA 
               
               
                   
                 NO: 234 
                 STLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPYTFGQGTK 
               
               
                   
                   
                 VEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSER 
               
               
                   
                   
                 QNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPI 
               
               
                   
                   
                 VKSFNRNEC 
               
               
                   
               
               
                 AF4660 
                 SEQ ID 
                 ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATN 
               
               
                   
                 NO: 235 
                 VAHWTTPSLKCIRDPALVHQRPAPPSGGSGGGGSGGGSGGGGSLQNW 
               
               
                   
                   
                 VNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESG 
               
               
                   
                   
                 DASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQ 
               
               
                   
                   
                 MFINTSGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVS 
               
               
                   
                   
                 CKASGGTFSSYAISWVRQAPGQGLEWMGIIDPSMTYTRYAQKFQGRVT 
               
               
                   
                   
                 MTRDTSTSTVYMELSSLRSEDTAVYYCARSLFPTIFGLEVAFDIWGQGTLV 
               
               
                   
                   
                 TVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLS 
               
               
                   
                   
                 SGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEP 
               
               
                   
                   
                 RGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSE 
               
               
                   
                   
                 DDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMS 
               
               
                   
                   
                 GKEFKCKVNNKDLGAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLT 
               
               
                   
                   
                 CMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSDLRVEKK 
               
               
                   
                   
                 NWVERNSYSCSVVHEGLHNHHTTESFSRTPGK 
               
               
                   
                 SEQ ID 
                 QVQLVQSGAEVKKPGASVKVSCKASGGTFSSYAISWVRQAPGQGLEWM 
               
               
                   
                 NO: 236 
                 G11DPSMTYTRYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR 
               
               
                   
                   
                 SLFPTIFGLEVAFDIWGQGTLVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLG 
               
               
                   
                   
                 CLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPS 
               
               
                   
                   
                 QSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPK 
               
               
                   
                   
                 IKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDY 
               
               
                   
                   
                 NSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLGAPIERTISKPKGSVRAP 
               
               
                   
                   
                 QVYVLPPPEKEMTKKQVSLTCLVKDFMPEDIYVEWTNNGKTELNYKNTE 
               
               
                   
                   
                 PVLKSDGSYFMYSKLTVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTP 
               
               
                   
                   
                 GK 
               
               
                   
                 SEQ ID 
                 DIQMTQSPSSLSASVGDRVTITCQASQSISNRLAWYQQKPGKAPKLLIYKA 
               
               
                   
                 NO: 237 
                 SSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYSTPITFGQGTK 
               
               
                   
                   
                 VEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSER 
               
               
                   
                   
                 QNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPI 
               
               
                   
                   
                 VKSFNRNEC 
               
               
                   
               
            
           
         
       
     
     The results are shown in  FIGS.  25 A-D . IL-15 activity increased in a dose dependent manner with the addition of PD-L1 but not with the addition of Fc protein for DBA-cytokine complexes AF4591 (SEQ ID NO: 226-228) and AF4592 (SEQ ID NO: 229-231). IL-15 activity from the monospecific IL-15 scFv cytokine complexes AF4659 (SEQ ID NO: 232-234) and AF4660 (SEQ ID NO: 235-237) did not change with the addition of PD-L1 or Fc protein. 
     Protein complexes of the present invention based on four different PD-L1/IL-15 dual-binding antibodies produced in two different formats showed PD-L1-dependent IL-15 activity. 
     Example 31 
     Selection and Binding of IFN and CEA Specific Dual Binding Antibodies 
     This example describes isolation of sensor domains of the present disclosure, specifically, selection of IFNα and CEA specific dual binding antibodies (DBAs). Anti-CEA and anti-IFNα DBAs were isolated from a Tumbler antibody phage display library similar to the library described in EXAMPLE 1. The antibody phage display library was constructed to incorporate the heavy chain CDR1, heavy chain CDR2, and light chain diversity of the Superhuman 2.0 antibody library combined with various heavy chain (“HC”) CDR3 sequences from anti-IFNα antibodies (TABLE 18). The selection was similar to the protocol described in EXAMPLE 1, using one round of IFNα selection (IFNα2b, GenScript, Z03002, biotinylated using standard protocols) and one round of CEA selection (CEA-hFc, Sino Biologicals, 11077-H02H). 
     
       
         
           
               
             
               
                 TABLE 18 
               
             
            
               
                   
               
               
                 HC-CDR3 of IFNα binders 
               
            
           
           
               
               
               
            
               
                 SEQ ID NO 
                 Sequence 
                 Description 
               
               
                   
               
               
                 SEQ ID NO: 238 
                 CASGGSYSPWYFDLW 
                 HC-CDR3 of IFNα binder 
               
               
                   
               
               
                 SEQ ID NO: 239 
                 CASLAAAGPYYYYGMDVW 
                 HC-CDR3 of IFNα binder 
               
               
                   
               
               
                 SEQ ID NO: 240 
                 CVSSVGAGAYYYQGLDVW 
                 HC-CDR3 of IFNα binder 
               
               
                   
               
               
                 SEQ ID NO: 241 
                 CARDHDYLTSFGYW 
                 HC-CDR3 of IFNα binder 
               
               
                   
               
               
                 SEQ ID NO: 242 
                 CAFSSPTYYYYYGMDVW 
                 HC-CDR3 of IFNα binder 
               
               
                   
               
               
                 SEQ ID NO: 243 
                 CARVNYDFWSGQSLRFDPW 
                 HC-CDR3 of IFNα binder 
               
               
                   
               
               
                 SEQ ID NO: 244 
                 CATIKGLGAYYYYGMDVW 
                 HC-CDR3 of IFNα binder 
               
               
                   
               
               
                 SEQ ID NO: 245 
                 CASDHGWLDAFDIW 
                 HC-CDR3 of IFNα binder 
               
               
                   
               
               
                 SEQ ID NO: 246 
                 CARDWYGDYFDYW 
                 HC-CDR3 of IFNα binder 
               
               
                   
               
               
                 SEQ ID NO: 247 
                 CARGILSDYGDHAFDYW 
                 HC-CDR3 of IFNα binder 
               
               
                   
               
               
                 SEQ ID NO: 248 
                 CARVDSSSSLHFDYW 
                 HC-CDR3 of IFNα binder 
               
               
                   
               
               
                 SEQ ID NO: 249 
                 CARTSGYDLLFDYW 
                 HC-CDR3 of IFNα binder 
               
               
                   
               
               
                 SEQ ID NO: 250 
                 CARVGGWGIYYYYGMDVW 
                 HC-CDR3 of IFNα binder 
               
               
                   
               
               
                 SEQ ID NO: 251 
                 CARDPSYSTGYYDYW 
                 HC-CDR3 of IFNα binder 
               
               
                   
               
               
                 SEQ ID NO: 252 
                 CARGSRADYW 
                 HC-CDR3 of IFNα binder 
               
               
                   
               
            
           
         
       
     
     Following two rounds of selection in phage, the resulting library of DBAs was subcloned into a yeast surface display vector and transformed into yeast for further screening using standard protocols. The yeast library was sorted four times for binding to CEA and IFNα. In each round of sorting, the library was labeled with either CEA-Fc-biotin or IFNα-biotin, then with Streptavidin-PE (Abcam #ab239759), and sorted based on PE fluorescence on a Sony MA900 cell sorter. The four sorts were carried out with labelling at 100 nM CEA-Fc-biotin, 1000 nM IFNα-biotin, 10 nM CEA-Fc-biotin, and 20 nM CEA-Fc-biotin. Plasmids were rescued from the yeast after the final sort using a Zymoprep Yeast Plasmid Miniprep II kit (Zymo research D2004) and transformed into DH5a  E. coli  for cloning. Ninety-six colonies were picked for Sanger sequencing, from which thirty-four unique clones were identified and screened for IFNα and CEA binding. The scFv DNA sequence for each clone, including c-myc and V5 tags, was amplified by PCR using a forward primer containing a T7 promoter and a translation initiation site, and a reverse primer containing a T7 terminator. Proteins were expressed using the PUREfrex2.1 cell-free transcription/translation system as described in previous examples. The scFv samples were subjected to ELISA analysis to detect CEA and IFNα binding. In these experiments, wells of a 384-well plate are coated with an anti-V5 antibody (Sv5-Pk1, BioRad) at 1 ug/ml overnight at 4 degrees. After washing, wells are blocked with SuperBlock (ThermoFisher, 37515) followed by addition of saturating levels of scFvs in SuperBlock. After washing, antigens are added and plates incubated for one hour. Biotinylated IFNα is detected using streptavidin HRP and CEA-Fc is detected using anti-hFc-HRP, and developed using standard methods. Varying amounts of labelled test antigen were added to show binding and to estimate relative affinities of the different scFvs.  FIG.  26    shows the ELISA binding data for five exemplary dual-binding scFvs binding to CEA. Because the binding affinity for IFNα was too low to detect by ELISA, the binding to IFNα was measured by Biolayer Interferometry (OctetRED96e) as described in Example 5. Results are tabulated in TABLE 19. All five show binding to both CEA and IFN, with binding to CEA detectable at a lower concentration of the antigen. 
     
       
         
           
               
             
               
                 TABLE 19 
               
             
            
               
                   
               
               
                 Anti-IFNα Binding Results 
               
            
           
           
               
               
               
               
               
            
               
                   
                 IFNα 
                   
                   
                   
               
               
                   
                 binding 
                   
                   
                   
               
               
                 CloneID 
                 (@5 uM) 
                 ELISA 
                 SEQUENCES 
                 HC-CDR3 
               
               
                   
               
               
                 8b2_A09 
                 + 
                 - 
                 (SEQ ID NO: 253) 
                 SEQ ID 
               
               
                   
                   
                   
                 MSTSTEVQLLESGGGLVQPGGSLRLSCAAS 
                 NO: 240 
               
               
                   
                   
                   
                 GFTFSSYAMHWVRQAPGKGLEWVSAIGA 
                   
               
               
                   
                   
                   
                 GGGTYYADSVKGRFTISRDDSKNTLYLQM 
                   
               
               
                   
                   
                   
                 NSLKTEDTAVYYCVSSVGAGAYYYQGLDV 
                   
               
               
                   
                   
                   
                 WGQGTLVTVSSASGGGGSGGGGSGGGG 
                   
               
               
                   
                   
                   
                 SHASDIQMTQSPSSLSASVGDRVTITCRAS 
                   
               
               
                   
                   
                   
                 QDIFTYLNWYQQRPGKAPKLLIYDASRLQT 
                   
               
               
                   
                   
                   
                 GVPSRFSGSGSGTDFTLTISSLQPEDFATYY 
                   
               
               
                   
                   
                   
                 CQQSYSIPYTFGQGTKLEIKRAAAGSGSEQ 
                   
               
               
                   
                   
                   
                 KLISEEDLGKPIPNPLLGLDST 
                   
               
               
                   
               
               
                 8b2_B10 
                 + 
                 - 
                 (SEQ ID NO: 254) 
                 SEQ ID 
               
               
                   
                   
                   
                 MSTSTEVQLLESGAEVKKPGGSLR 
                 NO: 240 
               
               
                   
                   
                   
                 LSCAASGFTVSSNYMSWVRQAPG 
                   
               
               
                   
                   
                   
                 KGLEWVSAISGSGGSTYYADFVKG 
                   
               
               
                   
                   
                   
                 RFTISRDNSKNTLYLQMNSLRAED 
                   
               
               
                   
                   
                   
                 TAVYYCVSSVGAGAYYYQGLDV 
                   
               
               
                   
                   
                   
                 WGQGTLVTVSSASGGGGSGGGGS 
                   
               
               
                   
                   
                   
                 GGGGSHASDIQMTQSPSSLSASVG 
                   
               
               
                   
                   
                   
                 DRVTITCRASQGVGNFLAWYQQKP 
                   
               
               
                   
                   
                   
                 GKAPKLLIYGASTLQSGVPSRFSGS 
                   
               
               
                   
                   
                   
                 GSGTDFTLTISSLQPEDFATYYCQQ 
                   
               
               
                   
                   
                   
                 SYSTPFTFGGGTKLEIKRAAAGSGS 
                   
               
               
                   
                   
                   
                 EQKLISEEDLGKPIPNPLLGLDST 
                   
               
               
                   
               
               
                 862_C02 
                 + 
                 - 
                 — 
                   
               
               
                   
               
               
                 8b2_C03 
                 + 
                 - 
                 — 
                   
               
               
                   
               
               
                 8b2_C08 
                 + 
                 - 
                 (SEQ ID NO: 256) 
                 SEQ ID 
               
               
                   
                   
                   
                 MSTSTEVQLLESGAEVKKPGGSLR 
                 NO: 248 
               
               
                   
                   
                   
                 LSCAASGFTFSSYAMSWVRQAPGK 
                   
               
               
                   
                   
                   
                 GLEWVSAISGSGGSTYYADSVKGR 
                   
               
               
                   
                   
                   
                 FTISRDNSKNTLYLQMNSLRAEDT 
                   
               
               
                   
                   
                   
                 AVYYCARVDSSSSLHFDYWGQGT 
                   
               
               
                   
                   
                   
                 LVTVSSASGGGGSGGGGSGGGGSH 
                   
               
               
                   
                   
                   
                 ASDIQMTQSPSSLSASVGDRVTITC 
                   
               
               
                   
                   
                   
                 RASQRIGTYLNWYQQKPGKAPKLL 
                   
               
               
                   
                   
                   
                 IYAASNLQSGVPSRFSGSGSGTDFT 
                   
               
               
                   
                   
                   
                 LTISSLQPEDFATYYCLQTFNTPFTF 
                   
               
               
                   
                   
                   
                 GPGTKVDIKRAAAGSGSEQKLISEE 
                   
               
               
                   
                   
                   
                 DLGKPIPNPLLGLDST 
                   
               
               
                   
               
               
                 AF317 
                 (KD &lt; 10nM) 
                 +++ 
                 (SEQ ID NO: 257) 
                 SEQ ID 
               
               
                   
                   
                   
                 MSTSTEQKLISEEDLQVQLVQSGAE 
                 NO: 244 
               
               
                   
                   
                   
                 VKKPGASVKVSCKASGYSFTSYDI 
                   
               
               
                   
                   
                   
                 NWVRQAPGQGLEWIGMINPSSGFT 
                   
               
               
                   
                   
                   
                 SAAQTFQGRVTMTRDTSTSTVYME 
                   
               
               
                   
                   
                   
                 LSSLRSEDTAVYYCATIKGLGAYY 
                   
               
               
                   
                   
                   
                 YYGMDVWGQGTTVTVSSASGGGG 
                   
               
               
                   
                   
                   
                 SGGGGSGGGGSHASDIQMTQSPSS 
                   
               
               
                   
                   
                   
                 LSASVGDRVTITCRASQSIDRYLNW 
                   
               
               
                   
                   
                   
                 YQQKPGKAPKLLIYAASSLQSGVPS 
                   
               
               
                   
                   
                   
                 RFSGSGSGTDFTLTISSLQPEDFATY 
                   
               
               
                   
                   
                   
                 YCQQSYSPPLTFGGGTKVEIKGSGL 
                   
               
               
                   
                   
                   
                 NDIFEAQKIEWHEGKPIPNPLLGLD 
                   
               
               
                   
                   
                   
                 ST 
                   
               
               
                   
               
               
                 AF372 
                 - 
                 - 
                 (SEQ ID NO: 258) 
                 — 
               
               
                   
                   
                   
                 MSTSTEQKLISEEDLEVQLVESGGG 
                   
               
               
                   
                   
                   
                 LVQPGGSLRLSCAASGFNIKDTYIH 
                   
               
               
                   
                   
                   
                 WVRQAPGKGLEWVARIYPTNGYT 
                   
               
               
                   
                   
                   
                 RYADSVKGRFTISADTSKNTAYLQ 
                   
               
               
                   
                   
                   
                 MNSLRAEDTAVYYCSRWGGDGFY 
                   
               
               
                   
                   
                   
                 AMDYWGQGTLVTVSSASGGGGSG 
                   
               
               
                   
                   
                   
                 GGGSGGGGSHASDIQMTQSPSSLS 
                   
               
               
                   
                   
                   
                 ASVGDRVTITCRASQDVNTAVAW 
                   
               
               
                   
                   
                   
                 YQQKPGKAPKLLIYSASFLYSGVPS 
                   
               
               
                   
                   
                   
                 RFSGSRSGTDFTLTISSLQPEDFATY 
                   
               
               
                   
                   
                   
                 YCQQHYTTPPTFGQGTKVEIKGSG 
                   
               
               
                   
                   
                   
                 LNDIFEAQKIEWHEGKPIPNPLLGL 
                   
               
               
                   
                   
                   
                 DST 
               
               
                   
               
            
           
         
       
     
     Example 32 
     Regulated IFNAR2 (IFNα Receptor 2) Binding by a CEA-IFNα Dual Binding Antibody (DBA) Cytokine Complex 
     This example describes regulated binding of IFN to the receptor IFNAR2 by CEA/IFN DBA-cytokine complexes in the form shown in  FIG.  8   . DBA-cytokine complexes of SEQ ID NO: 259, SEQ ID NO: 260 and SEQ ID NO: 261 were chosen for analysis with a negative control with a similar structure based on an IFNα monospecific binding scFv (SEQ ID NO: 300) and two non-IFNα binding scFvs (one with SEQ ID NO: 301 and one with SEQ ID NO: 302). DBA-cytokine complex and control protein complex sequences are summarized in TABLE 30. To test the ability of Carcino Embryonic Antigen (CEA) to regulate the binding of IFNAR2, V5-tagged constructs were generated consisting of a CEA-IFN-α DBA scFv linked to IFN-α. scFvs were synthesized using the PUREfrex 2.1 in vitro translation system and added to a 384 well plate at a single dilution. Titrating concentrations of either CEA-Fc (SINO Biological) or PDL1-Fc (ACROBiosystems) were added, and the plate was incubated for 30 minutes at room temperature. Eu-labeled IFNAR2 (ACROBiosystems) and Alexa Fluor 647-labeled anti-V5 antibody (Perkin Elmer) were added to all wells and incubated for 24 hours at room temperature. The HTRF signal was then read on an Envision 2105 microplate reader (Perkin Elmer). IFNAR2 was able to bind IFN-α on the DBA-scFv complex in the presence of increasing concentrations of CEA-Fc. No increase in IFNAR2 binding was seen in the presence of the PD-L1-Fc control protein, which does not bind the DBA-scFv protein complex. Results shown in  FIGS.  27 A-F  demonstrate IFNAR2 binding increased in a dose dependent manner with the addition of CEA but not with the addition of control protein PDL1 for DBA-cytokine complexes of SEQ ID NO: 259 ( FIG.  27 A ), SEQ ID NO: 260 ( FIG.  27 B ) and SEQ ID NO: 261 ( FIG.  27 C ). IFNAR2 binding from the monospecific IFN scFv cytokine complexes did not change with the addition of CEA or PD-L1. 
     
       
         
           
               
             
               
                 TABLE 30 
               
             
            
               
                   
               
               
                 DBA-cytokine protein complexes, and control protein complexes 
               
            
           
           
               
               
               
            
               
                 Protein 
                 SEQ ID 
                   
               
               
                 Complex 
                 NO: 
                 Sequence 
               
               
                   
               
               
                 DBA- 
                 SEQ ID 
                 MSTSTCDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQE 
               
               
                 cytokine 
                 NO: 259 
                 EFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTEL 
               
               
                 complex 
                   
                 YQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKK 
               
               
                   
                   
                 YSPCAWEVVRAEIMRSFSLSTNLQESLRSKEGGSGSGSGGSGGSGS 
               
               
                   
                   
                 GGSGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMHWVRQA 
               
               
                   
                   
                 PGKGLEWVSAIGAGGGTYYADSVKGRFTISRDDSKNTLYLQMNSL 
               
               
                   
                   
                 KTEDTAVYYCVSSVGAGAYYYQGLDVWGQGTLVTVSSASGGGGS 
               
               
                   
                   
                 GGGGSGGGGSHASDIQMTQSPSSLSASVGDRVTITCRASQDIFTYL 
               
               
                   
                   
                 NWYQQRPGKAPKLLIYDASRLQTGVPSRFSGSGSGTDFTLTISSLQP 
               
               
                   
                   
                 EDFATYYCQQSYSIPYTFGQGTKLEIKRAAAGSGSEQKLISEEDLGK 
               
               
                   
                   
                 PIPNPLLGLDST 
               
               
                   
               
               
                 DBA- 
                 SEQ ID 
                 MSTSTCDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQE 
               
               
                 cytokine 
                 NO: 260 
                 EFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTEL 
               
               
                 complex 
                   
                 YQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKK 
               
               
                   
                   
                 YSPCAWEVVRAEIMRSFSLSTNLQESLRSKEGGSGSGSGGSGGSGS 
               
               
                   
                   
                 GGSGSEVQLLESGAEVKKPGGSLRLSCAASGFTVSSNYMSWVRQA 
               
               
                   
                   
                 PGKGLEWVSAISGSGGSTYYADFVKGRFTISRDNSKNTLYLQMNSL 
               
               
                   
                   
                 RAEDTAVYYCVSSVGAGAYYYQGLDVWGQGTLVTVSSASGGGGS 
               
               
                   
                   
                 GGGGSGGGGSHASDIQMTQSPSSLSASVGDRVTITCRASQGVGNFL 
               
               
                   
                   
                 AWYQQKPGKAPKLLIYGASTLQSGVPSRFSGSGSGTDFTLTISSLQP 
               
               
                   
                   
                 EDFATYYCQQSYSTPFTFGGGTKLEIKRAAAGSGSEQKLISEEDLGK 
               
               
                   
                   
                 PIPNPLLGLDST 
               
               
                   
               
               
                 DBA- 
                 SEQ ID 
                 MSTSTCDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQE 
               
               
                 cytokine 
                 NO: 261 
                 EFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTEL 
               
               
                 complex 
                   
                 YQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKK 
               
               
                   
                   
                 YSPCAWEVVRAEIMRSFSLSTNLQESLRSKEGGSGSGSGGSGGSGS 
               
               
                   
                   
                 GGSGSEVQLLESGAEVKKPGGSLRLSCAASGFTFSSYAMSWVRQA 
               
               
                   
                   
                 PGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSL 
               
               
                   
                   
                 RAEDTAVYYCARVDSSSSLHFDYWGQGTLVTVSSASGGGGSGGG 
               
               
                   
                   
                 GSGGGGSHASDIQMTQSPSSLSASVGDRVTITCRASQRIGTYLNWY 
               
               
                   
                   
                 QQKPGKAPKLLIYAASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDF 
               
               
                   
                   
                 ATYYCLQTFNTPFTFGPGTKVDIKRAAAGSGSEQKLISEEDLGKPIP 
               
               
                   
                   
                 NPLLGLDST 
               
               
                   
               
               
                 AF2589 
                 SEQ ID 
                 MSTSTCDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAE 
               
               
                   
                 NO: 300 
                 TIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTET 
               
               
                   
                   
                 PLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEG 
               
               
                   
                   
                 GGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYSFTSYDIN 
               
               
                   
                   
                 WVRQAPGQGLEWIGMINPSSGFTSAAQTFQGRVTMTRDTSTSTVYMELSSLRSE 
               
               
                   
                   
                 DTAVYYCATIKGLGAYYYYGMDVWGQGTTVTVSSASGGGGSGGGGSGGGGSHA 
               
               
                   
                   
                 SDIQMTQSPSSLSASVGDRVTITCRASQSIDRYLNWYQQKPGKAPKLLIYAASSLQS 
               
               
                   
                   
                 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSPPLTFGGGTKVEIKGKPIPNPL 
               
               
                   
                   
                 LGLDST 
               
               
                   
               
               
                 AF2592 
                 SEQ ID 
                 MSTSTCDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAE 
               
               
                   
                 NO: 301 
                 TIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTET 
               
               
                   
                   
                 PLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEG 
               
               
                   
                   
                 GGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGGTFSSYAIS 
               
               
                   
                   
                 WVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSTSTVYMELSSLR 
               
               
                   
                   
                 SEDTAVYYCARSLFPTIFGVEVAFDIWGQGTTVTVSSASGGGGSGGGGSGGGGSH 
               
               
                   
                   
                 ASDIQMTQSPSSLSASVGDRVTITCRASQSIIDRLAWYQQKPGKAPKLLIYKASSLES 
               
               
                   
                   
                 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPFTFGPGTKVDIKGKPIPNPL 
               
               
                   
                   
                 LGLDST 
               
               
                   
               
               
                 AF2594 
                 SEQ ID 
                 MSTSTCDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAE 
               
               
                   
                 NO: 302 
                 TIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTET 
               
               
                   
                   
                 PLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEG 
               
               
                   
                   
                 GGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIH 
               
               
                   
                   
                 WVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAED 
               
               
                   
                   
                 TAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASGGGGSGGGGSGGGGSHASDI 
               
               
                   
                   
                 QMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGV 
               
               
                   
                   
                 PSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGKPIPNPLL 
               
               
                   
                   
                 GLDST 
               
               
                   
               
            
           
         
       
     
     Example 33 
     Selection and Binding of LRRC15 and IFNα Specific Dual Binding Antibodies 
     This example describes isolation of sensor domains of the present disclosure, specifically, selection of LRRC15 and IFNα specific dual binding antibodies (DBAs). Anti-LRRC15 and anti-IFNα DBAs were isolated from the IFNα Tumbler antibody phage display library described in EXAMPLE 31. The selection was similar to the protocol described in EXAMPLE 1, using one round of IFNα selection (IFNα2b, GenScript, Z03002, biotinylated using standard protocols) and one round of LRRC15 selection (LRRC15-hFc, Sino Biologicals, 15786-H02H). 
     After a first round of selection in phage on 100 nM IFNα and a second round on 100 nM LRRC15, the resulting library of DBAs was subcloned into a yeast surface display vector and transformed into yeast for further screening using standard protocols. The yeast library was sorted four times for binding to LRRC15 and IFNα. In each round of sorting, the library was labeled with either LRRC15-Fc-biotin or IFNα-biotin, then with Streptavidin-PE (Abcam #ab239759) and sorted based on PE fluorescence on a Sony MA900 cell sorter. The four sorts were carried out with labelling at 100 nM LRRC15-Fc-biotin, 1000 nM IFNα-biotin, 10 nM LRRC15-Fc-biotin, and 10 nM LRRC15-Fc-biotin. Plasmids were rescued from the yeast after the final sort using a Zymoprep Yeast Plasmid Miniprep II kit (Zymo research D2004) and transformed into DH5a  E. coli  for cloning. Ninety-six colonies were picked for Sanger sequencing, from which twenty-four unique clones were identified and screened for IFNα and LRRC15 binding. The scFv DNA sequence for each clone, including c-myc and V5 tags, was amplified by PCR using a forward primer containing a T7 promoter and a translation initiation site, and a reverse primer containing a T7 terminator. Proteins were expressed using the PUREfrex2.1 cell-free transcription/translation system as described in previous examples. The scFv samples were subjected to ELISA analysis to detect LRRC15 and IFNα binding.  FIG.  28 A  shows the ELISA binding data for six exemplary scFvs to LRRC15. Because the IFNα binding affinity was too low to detect by ELISA for some scFvs, the binding to IFNα was also measured by Biolayer Interferometry (OctetRED96e) as described in Example 5. Results and antibody sequences are tabulated in  FIG.  28 B . All six show binding to both LRRC15 and IFNα, with binding to LRRC15 detectable at a lower concentration of the antigen. Protein complex sequences are provided in TABLE 31. 
     
       
         
           
               
             
               
                 TABLE 31 
               
             
            
               
                   
               
               
                 DBA-cytokine protein complexes, and control protein complexes 
               
            
           
           
               
               
               
            
               
                 Protein 
                 SEQ ID 
                   
               
               
                 Complex 
                 NO: 
                 Sequence 
               
               
                   
               
               
                 A03 
                 SEQ ID 
                 MSTSTQVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEW 
               
               
                   
                 NO: 262 
                 MGWMDPNNDDADYAQRFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCASLA 
               
               
                   
                   
                 AAGPYYYYGMDVWGQGTLVTVSSASGGGGSGGGGSGGGGSHASDIQMTQSPS 
               
               
                   
                   
                 SLSASVGDRVTITCRASQSINNYLNWYQQKPGKAPKLLIYGASNLETGVPSRFSGSG 
               
               
                   
                   
                 SGTDFTLTISSLQPEDFATYYCQQSYGTPLTFGGGTKVEIKRAAAGSGSEQKLISEED 
               
               
                   
                   
                 LGKPIPNPLLGLDST 
               
               
                   
               
               
                 i47_A11 
                 SEQ ID 
                 MSTSTQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYDINWVRQA 
               
               
                   
                 NO: 314 
                 PGQGLEWMGTINPSDGDTTYAQKFQGRVTITADESTSTAYMELSSL 
               
               
                   
                   
                 RSEDTAVYYCARVGGWGIYYYYGMDVWGQGTLVTVSSASGGGG 
               
               
                   
                   
                 SGGGGSGGGGSHASDIQMTQSPSSLSASVGDRVTITCRASQSINSW 
               
               
                   
                   
                 LAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQ 
               
               
                   
                   
                 PEDFATYYCQQTYTVPFSFGQGTKLEIKRAAAGSGSEQKLISEEDLG 
               
               
                   
                   
                 KPIPNPLLGLDST 
               
               
                   
               
               
                 i47_A12 
                 SEQ ID 
                 MSTSTQVQLVQSGAEVKKPGSSVKVSCKASGYTFINNDINWVRQA 
               
               
                   
                 NO: 315 
                 PGQGLEWMGGTIPIFGVHIYAQKFQGRVTITADESTSTAYMELSSL 
               
               
                   
                   
                 RSEDTAVYYCVSSVGAGAYYYYGMDVWGQGTLVTVSSASGGGG 
               
               
                   
                   
                 SGGGGSGGGGSHASDIQMTQSPSSLSASVGDRVTITCQASQDISNY 
               
               
                   
                   
                 LNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQ 
               
               
                   
                   
                 PEDFATYYCQQSYSLPYTFGQGTRLEIKRAAAGSGSEQKLISEEDLG 
               
               
                   
                   
                 KPIPNPLLGLDST 
               
               
                   
               
               
                 B03 
                 SEQ ID 
                 MSTSTQVQLVQSGAEVKKPGASVEVSCKASGGTFSSYAINWVRQAPGQGLEWM 
               
               
                   
                 NO: 263 
                 GWIDPKSGDTTYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCASGGSYSP 
               
               
                   
                   
                 WYFDLWGQGTLVTVSSASGGGGSGGGGSGGGGSHASDIQMTQSPSSLSASVGD 
               
               
                   
                   
                 RVTITCRASQSISSWLAWYQQKSGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTI 
               
               
                   
                   
                 SSLQPEDFATYYCQQAYSFPFTFGPGTKVDIKRAAAGSGSEQKLISEEDLGKPIPNPL 
               
               
                   
                   
                 LGLDST 
               
               
                   
               
               
                 B07 
                 SEQ ID 
                 MSTSTQVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEW 
               
               
                   
                 NO: 264 
                 MGWMDPNNDDADYAQRFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCASLA 
               
               
                   
                   
                 AAGPYYYYGMDVWGQGTLVTVSSASGGGGSGGGGSGGGGSHASDIQMTQSPS 
               
               
                   
                   
                 SLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYGASILEAGVPSRFSGSG 
               
               
                   
                   
                 SGTDFTLTISSLQPEDFATYYCQQSYSAPITFGQGTRLEIKRAAAGSGSEQKLISEEDL 
               
               
                   
                   
                 GKPIPNPLLGLDST 
               
               
                   
               
               
                 B11 
                 SEQ ID 
                 MSTSTQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYDINWVRQAPGQGLEWLG 
               
               
                   
                 NO: 265 
                 GTVPLFGISHYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCVSSVGAGAYY 
               
               
                   
                   
                 YQGLDVWGQGTLVTVSSASGGGGSGGGGSGGGGSHASDIQMTQSPSSLSASVG 
               
               
                   
                   
                 DRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLT 
               
               
                   
                   
                 ISSLLPEDFATYYCQQSYLPPYSFGQGTKLEIKRAAAGSGSEQKLISEEDLGKPIPNPLL 
               
               
                   
                   
                 GLDST 
               
               
                   
               
               
                 AF372 
                 SEQ ID 
                 MSTSTEQKLISEEDLEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAP 
               
               
                   
                 NO: 258 
                 GKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCS 
               
               
                   
                   
                 RWGGDGFYAMDYWGQGTLVTVSSASGGGGSGGGGSGGGGSHASDIQMTQSP 
               
               
                   
                   
                 SSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGS 
               
               
                   
                   
                 RSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGSGLNDIFEAQKIEW 
               
               
                   
                   
                 HEGKPIPNPLLGLDST 
               
               
                   
               
            
           
         
       
     
     Example 34 
     Regulated IFNAR2 (IFNα Receptor 2) Binding by a LRRC15-IFNα Dual Binding Antibody (DBA) Cytokine Complex 
     This example describes LRRC15 dependent binding of IFN to the receptor IFNAR2 by LRRC15/IFN DBA-cytokine complexes. The cytokine complexes of this example are composed of a LRRC15-IFN-αL DBA IgG with IFN-α linked to the N-terminus of one heavy chain (a schematic of the structure is shown in  FIG.  9 B ). DBA-cytokine complexes AF4581 (SEQ ID NO: 266-268), AF4586 (SEQ TD NO: 269-271), AF4587 (SEQ TD NO: 266-267, 272) and AF4588 (SEQ TD NO: 273-275) were chosen for analysis. Two cytokine complexes with the same structure were included as controls: AF4305 (SEQ TD NO: 294-296), which is based on a neutralizing anti-IFNα antibody, and AF4306 (SEQ TD NO: 64, 192, 297), which is based on an anti-Her2 antibody. Protein complex sequences are provided in TABLE 32. The six IgG-cytokines were expressed in mammalian cells using standard protocols and added to a 384 well plate at a single dilution. Titrating concentrations of either LRRC15-Fc (SINO Biological), PDL1-Fc (ACROBiosystems) or CEA-Fc (SINO Biological) were added, and the plate was incubated for 30 minutes at room temperature. Eu-labeled IFNAR2 (ACROBiosystems) and Alexa Fluor 647-labeled anti-V5 antibody (Perkin Elmer) were added to all wells and incubated for 24 hours at room temperature. The HTRF signal was then read on an Envision 2105 microplate reader (Perkin Elmer). Results shown in  FIGS.  29 A-F  demonstrate IFNAR2 binding increased in a dose dependent manner with the addition of LRRC15 but not with the addition of control protein PDL1 for DBA-cytokine complexes of AF4581, AF4586 and AF4587, demonstrating LRRC15-dependent regulation of IFNAR-binding activity. IFNAR2 binding from AF4588 and the monospecific IFN scFv cytokine complexes AF4305 and AF4306 did not change with the addition of LRRC15 or PD-L1. 
     While preferred embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments of the disclosure described herein may be employed in practicing the disclosure. It is intended that the following claims define the scope of the disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby. 
     
       
         
           
               
             
               
                 TABLE 32 
               
             
            
               
                   
               
               
                 DBA-cytokine protein complexes, and control protein complexes 
               
            
           
           
               
               
               
            
               
                 Protein 
                 SEQ ID 
                   
               
               
                 Complex 
                 NO: 
                 Sequence 
               
               
                   
               
               
                 AF4581 
                 SEQ ID 
                 CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVL 
               
               
                   
                 NO: 266 
                 HEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMK 
               
               
                   
                   
                 EDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEGGGGS 
               
               
                   
                   
                 GGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWV 
               
               
                   
                   
                 RQAPGQGLEWMGWMDPNNDDADYAQRFQGRVTMTRDTSTSTVYMELSSLRS 
               
               
                   
                   
                 EDTAVYYCASLAAAGPYYYYGMDVWGQGTLVTVSSAKTTAPSVYPLAPVCGDTTG 
               
               
                   
                   
                 SSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPS 
               
               
                   
                   
                 QSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLM 
               
               
                   
                   
                 ISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPI 
               
               
                   
                   
                 QHQDWMSGKEFKCKVNNKDLGAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQ 
               
               
                   
                   
                 VTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSDLRVEKKN 
               
               
                   
                   
                 WVERNSYSCSVVHEGLHNHHTTESFSRTPGK 
               
               
                   
                 SEQ ID 
                 QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWIVIGWIVI 
               
               
                   
                 NO: 267 
                 DPNNDDADYAQRFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCASLAAAGPYY 
               
               
                   
                   
                 YYGMDVWGQGTLVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTL 
               
               
                   
                   
                 TWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKK 
               
               
                   
                   
                 lEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPD 
               
               
                   
                   
                 VQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNN 
               
               
                   
                   
                 KDLGAPIERTISKPKGSVRAPQVYVLPPPEKEMTKKQVSLTCLVKDFIVIPEDIYVEWT 
               
               
                   
                   
                 NNGKTELNYKNTEPVLKSDGSYFMYSKLTVEKKNWVERNSYSCSVVHEGLHNHHT 
               
               
                   
                   
                 TKSFSRTPGK 
               
               
                   
                 SEQ ID 
                 DIQMTQSPSSLSASVGDRVTITCRASQSINNYLNWYQQKPGKAPKLLIYGASNLET 
               
               
                   
                 NO: 268 
                 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYGTPLTFGGGTKVEIKRADAAPT 
               
               
                   
                   
                 VSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSK 
               
               
                   
                   
                 DSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC 
               
               
                   
               
               
                 AF4586 
                 SEQ ID 
                 CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVL 
               
               
                   
                 NO: 269 
                 HEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMK 
               
               
                   
                   
                 EDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEGGGGS 
               
               
                   
                   
                 GGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVEVSCKASGGTFSSYAINWVR 
               
               
                   
                   
                 QAPGQGLEWMGWIDPKSGDTTYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDT 
               
               
                   
                   
                 AVYYCASGGSYSPWYFDLWGQGTLVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLG 
               
               
                   
                   
                 CLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCN 
               
               
                   
                   
                 VAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIV 
               
               
                   
                   
                 TCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDW 
               
               
                   
                   
                 MSGKEFKCKVNNKDLGAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMV 
               
               
                   
                   
                 TDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSDLRVEKKNWVERNSYS 
               
               
                   
                   
                 CSVVHEGLHNHHTTESFSRTPGK 
               
               
                   
                 SEQ ID 
                 QVQLVQSGAEVKKPGASVEVSCKASGGTFSSYAINWVRQAPGQGLEWMGWIDP 
               
               
                   
                 NO: 270 
                 KSGDTTYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCASGGSYSPWYFDL 
               
               
                   
                   
                 WGQGTLVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGS 
               
               
                   
                   
                 LSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPT 
               
               
                   
                   
                 IKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWF 
               
               
                   
                   
                 VNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLGAPI 
               
               
                   
                   
                 ERTISKPKGSVRAPQVYVLPPPEKEMTKKQVSLTCLVKDFMPEDIYVEWTNNGKTE 
               
               
                   
                   
                 LNYKNTEPVLKSDGSYFMYSKLTVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRT 
               
               
                   
                   
                 PGK 
               
               
                   
                 SEQ ID 
                 DIQMTQSPSSLSASVGDRVTITCRASQSISSWLAWYQQKSGKAPKLLIYAASSLQSG 
               
               
                   
                 NO: 271 
                 VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYSFPFTFGPGTKVDIKRADAAPTV 
               
               
                   
                   
                 SIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDS 
               
               
                   
                   
                 TYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC 
               
               
                   
               
               
                 AF4587 
                 SEQ ID 
                 CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVL 
               
               
                   
                 NO: 266 
                 HEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMK 
               
               
                   
                   
                 EDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEGGGGS 
               
               
                   
                   
                 GGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWV 
               
               
                   
                   
                 RQAPGQGLEWMGWMDPNNDDADYAQRFQGRVTMTRDTSTSTVYMELSSLRS 
               
               
                   
                   
                 EDTAVYYCASLAAAGPYYYYGMDVWGQGTLVTVSSAKTTAPSVYPLAPVCGDTTG 
               
               
                   
                   
                 SSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPS 
               
               
                   
                   
                 QSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLM 
               
               
                   
                   
                 ISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPI 
               
               
                   
                   
                 QHQDWMSGKEFKCKVNNKDLGAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQ 
               
               
                   
                   
                 VTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSDLRVEKKN 
               
               
                   
                   
                 WVERNSYSCSVVHEGLHNHHTTESFSRTPGK 
               
               
                   
                 SEQ ID 
                 QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWM 
               
               
                   
                 NO: 267 
                 DPNNDDADYAQRFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCASLAAAGPYY 
               
               
                   
                   
                 YYGMDVWGQGTLVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTL 
               
               
                   
                   
                 TWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKK 
               
               
                   
                   
                 IEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPD 
               
               
                   
                   
                 VQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNN 
               
               
                   
                   
                 KDLGAPIERTISKPKGSVRAPQVYVLPPPEKEMTKKQVSLTCLVKDFMPEDIYVEWT 
               
               
                   
                   
                 NNGKTELNYKNTEPVLKSDGSYFMYSKLTVEKKNWVERNSYSCSVVHEGLHNHHT 
               
               
                   
                   
                 TKSFSRTPGK 
               
               
                   
                 SEQ ID 
                 DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYGASILEAG 
               
               
                   
                 NO: 272 
                 VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSAPITFGQGTRLEIKRADAAPTV 
               
               
                   
                   
                 SIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDS 
               
               
                   
                   
                 TYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC 
               
               
                   
               
               
                 AF4588 
                 SEQ ID 
                 CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVL 
               
               
                   
                 NO: 273 
                 HEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMK 
               
               
                   
                   
                 EDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEGGGGS 
               
               
                   
                   
                 GGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYDINWVRQ 
               
               
                   
                   
                 APGQGLEWLGGTVPLFGISHYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYY 
               
               
                   
                   
                 CVSSVGAGAYYYQGLDVWGQGTLVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGC 
               
               
                   
                   
                 LVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNV 
               
               
                   
                   
                 AHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVT 
               
               
                   
                   
                 CVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDW 
               
               
                   
                   
                 MSGKEFKCKVNNKDLGAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMV 
               
               
                   
                   
                 TDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSDLRVEKKNWVERNSYS 
               
               
                   
                   
                 CSVVHEGLHNHHTTESFSRTPGK 
               
               
                   
                 SEQ ID 
                 QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYDINWVRQAPGQGLEWLGGTVPL 
               
               
                   
                 NO: 274 
                 FGISHYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCVSSVGAGAYYYQGLD 
               
               
                   
                   
                 VWGQGTLVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNS 
               
               
                   
                   
                 GSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRG 
               
               
                   
                   
                 PTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQIS 
               
               
                   
                   
                 WFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLG 
               
               
                   
                   
                 APIERTISKPKGSVRAPQVYVLPPPEKEMTKKQVSLTCLVKDFMPEDIYVEWTNNG 
               
               
                   
                   
                 KTELNYKNTEPVLKSDGSYFMYSKLTVEKKNWVERNSYSCSVVHEGLHNHHTTKSF 
               
               
                   
                   
                 SRTPGK 
               
               
                   
                 SEQ ID 
                 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSG 
               
               
                   
                 NO: 275 
                 VPSRFSGSGSGTDFTLTISSLLPEDFATYYCQQSYLPPYSFGQGTKLEIKRADAAPTVS 
               
               
                   
                   
                 IFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDST 
               
               
                   
                   
                 YSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC 
               
               
                   
               
               
                 AF4305 
                 SEQ ID 
                 CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVL 
               
               
                   
                 NO: 294 
                 HEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMK 
               
               
                   
                   
                 EDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEGGGGS 
               
               
                   
                   
                 GGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTAYDINWVR 
               
               
                   
                   
                 QAPGQGLEWVGIINPGSGSPMYAQKFQGRVTITADESTSTAYMELSSLRSEDTAV 
               
               
                   
                   
                 YYCVSSVGAGAYYYQGLDVWGQGTLVTVSSAKTTAPSVYPLAPVCGDTTGSSVTL 
               
               
                   
                   
                 GCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITC 
               
               
                   
                   
                 NVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPI 
               
               
                   
                   
                 VTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQD 
               
               
                   
                   
                 WMSGKEFKCKVNNKDLGAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTC 
               
               
                   
                   
                 MVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSDLRVEKKNWVERN 
               
               
                   
                   
                 SYSCSVVHEGLHNHHTTESFSRTPGK 
               
               
                   
                 SEQ ID 
                 QVQLVQSGAEVKKPGSSVKVSCKASGYTFTAYDINWVRQAPGQGLEWVGIINPGS 
               
               
                   
                 NO: 295 
                 GSPMYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCVSSVGAGAYYYQGLD 
               
               
                   
                   
                 VWGQGTLVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNS 
               
               
                   
                   
                 GSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRG 
               
               
                   
                   
                 PTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQIS 
               
               
                   
                   
                 WFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLG 
               
               
                   
                   
                 APIERTISKPKGSVRAPQVYVLPPPEKEMTKKQVSLTCLVKDFMPEDIYVEWTNNG 
               
               
                   
                   
                 KTELNYKNTEPVLKSDGSYFMYSKLTVEKKNWVERNSYSCSVVHEGLHNHHTTKSF 
               
               
                   
                   
                 SRTPGGGGSGGGSHHHHHH 
               
               
                   
                 SEQ ID 
                 DIQMTQSPSSLSASVGDRVTITCQASQDIANYLNWYQQKPGKAPKLLIYSASNLQS 
               
               
                   
                 NO: 296 
                 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTQWTFGQGTKVEIKRADAA 
               
               
                   
                   
                 PTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDS 
               
               
                   
                   
                 KDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC 
               
               
                   
               
               
                 AF4306 
                 SEQ ID 
                 DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYS 
               
               
                   
                 NO: 64 
                 GVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRADAAP 
               
               
                   
                   
                 TVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSK 
               
               
                   
                   
                 DSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC 
               
               
                   
                 SEQ ID 
                 EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRY 
               
               
                   
                 NO: 192 
                 ADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVS 
               
               
                   
                   
                 SAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDL 
               
               
                   
                   
                 YTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVF 
               
               
                   
                   
                 IFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVV 
               
               
                   
                   
                 SALPIQHQDWMSGKEFKCKVNNKDLGAPIERTISKPKGSVRAPQVYVLPPPEKEMTKKQV 
               
               
                   
                   
                 SLTCLVKDFMPEDIYVEWTNNGKTELNYKNTEPVLKSDGSYFMYSKLTVEKKNWVERNSYS 
               
               
                   
                   
                 CSVVHEGLHNHHTTKSFSRTPGGGGSGGGSHHHHHH 
               
               
                   
                 SEQ ID 
                 CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVL 
               
               
                   
                 NO: 297 
                 HEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMK 
               
               
                   
                   
                 EDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEGGGGS 
               
               
                   
                   
                 GGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQ 
               
               
                   
                   
                 APGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYY 
               
               
                   
                   
                 CSRWGGDGFYAMDYWGQGTLVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLV 
               
               
                   
                   
                 KGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAH 
               
               
                   
                   
                 PASSTKVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCV 
               
               
                   
                   
                 VVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMS 
               
               
                   
                   
                 GKEFKCKVNNKDLGAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTD 
               
               
                   
                   
                 FMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSDLRVEKKNWVERNSYSCS 
               
               
                   
                   
                 VVHEGLHNHHTTESFSRTPGK