Patent Publication Number: US-11028174-B1

Title: Bifunctional molecules targeting PD-L1 and TGF-β

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
     This application claims priority to PCT/CN2020/105286, filed Jul. 28, 2020, which is hereby incorporated by reference in its entirety. 
     SEQUENCE LISTING 
     The instant application contains a Sequence Listing which has been filed electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Aug. 28, 2020, is named 320293US_ST25.txt and is 112,479 bytes in size. 
     BACKGROUND 
     The exciting advances in cancer immunotherapy in recent years have led to paradigm shifts in oncology. The most noticeable results have been with T-cell-based therapies including immune checkpoint inhibitors (ICI), genetically engineered T-cells and bispecific antibodies (BsAb). T-cells represent a major class of immunosurveillance and tumor eradication with exquisite specificity and long-term memory. However, in the tumor microenvironment, T-cells can become exhausted or tolerized to tumor cells. T-cell exhaustion is commonly associated with overexpression of inhibitory receptors, including programmed death receptor-1 (PD-1), cytotoxic T lymphocyte antigen-4 (CTLA-4), lymphocyte-activation gene-3 (LAG-3), T-cell immunoglobulin domain and mucin domain-3 (TIM-3), IL-10 receptor, and killer immunoglobulin receptors. 
     Monoclonal antibody (mAb) based therapies to counteract these checkpoint molecules can remove the brake that restrains tumor-infiltrating T-cells, thereby achieving significant clinical benefits in different malignancies. For instance, blocking PD-1/PD-L1 interactions can enhance immune normalization and reinforce anticancer responses. However, the noticeable deficiency of PD-1/PD-L1 blockades is inconsistency across a homogeneous study population with similar tumor characteristics. Also, PD-1/PD-L1 blockade treatments may also cause certain inflammatory side effects in some patients. The limitations of monotherapy with PD-1/PD-L1 blockades and the lack of promising alternatives has made it necessary to seek combination treatment methods which can activate antitumor immunity and enhance treatment efficacy. 
     M7824 (bintrafusp alfa) is a bifunctional protein composed of a monoclonal antibody against programmed death ligand 1 (PD-L1) fused to the extracellular domain of human transforming growth factor-β (TGF-β) receptor II, which functions as a “trap” for all three TGF-θ isoforms. The PD-L1 portion is the based on avelumab, which has been approved for the treatment of Merkel cell carcinoma and urothelial cancer. Current clinical data show, however, that the use of M7824 is associated with undesired skin growth and the overall response rate was only about 35% to 40% in a phase II trial for patients with HPV-positive malignancies. Improved therapies are needed, therefore. 
     SUMMARY 
     The present disclosure provides, in some embodiments, bifunctional molecules that target both the PD-L1 protein and TGF-β. The disclosed PD-L1 targeting unit, comprised of an anti-PD-L1 antibody, is fused to an extracellular domain of human transforming growth factor-β (TGF-β) receptor II which functions as a trap for TGF-β. Experimental data show that these new bifunctional molecules are more effective than M7824, the current lead candidate in clinical development. 
     In accordance with one embodiment of the present disclosure, therefore, provided is a multifunctional molecule, comprising an anti-PD-L1 (programmed death-ligand 1) antibody or fragment thereof and an extracellular domain of human TGF-β RII (TGF-beta receptor type-2), wherein the anti-PD-L1 antibody or fragment thereof has specificity to the human PD-L1 protein and comprises a heavy chain variable region (VH) comprising a VH CDR1, a VH CDR2 and a VH CDR3, and a light chain variable region (VL) comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3, respectively, comprise the amino acid sequences of SEQ ID NO:7-12, 13-18, 19; 20; 21-24, or 19; 91; 21-24, or 19; 92; 21-24, wherein the human TGF-β RII extracellular domain comprises the amino acid sequence of SEQ ID NO:72 and is fused to the anti-PD-L1 antibody or fragment thereof. 
     In one embodiment, provided is an anti-PD-L1 (programmed death-ligand 1) antibody or fragment thereof, which has specificity to the human PD-L1 protein and comprises a heavy chain variable region (VH) comprising a VH CDR1, a VH CDR2 and a VH CDR3, and a light chain variable region (VL) comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3, respectively, comprise the amino acid sequences of SEQ ID NO:7-12, 13-18, 19; 20; 21-24, or 19; 91; 21-24, or 19; 92; 21-24. 
     Also provided is a multifunctional molecule, comprising an antibody or antigen-binding fragment thereof fused, through a peptide linker, to the N-terminus of the amino acid sequence of SEQ ID NO:72, wherein the peptide linker (a) is at least 30 amino acid residues in length, or (b) is at least 25 amino acid residues in length and comprises an alpha helix motif. 
     Also provided are uses and methods for treating cancer with any of the molecules of the present disclosure. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  shows that 47C6A3, 67F3G7 and 89C10H8 can bind to human PD-L1 with high affinity. 
         FIG. 2  shows the 47C6A3, 67F3G7 and 89C10H8 antibodies can highly binding to PD-L1 expressed on mammalian cells. 
         FIG. 3  shows that 47C6A3, 67F3G7 and 89C10H8 antibodies can bind to cynomolgus PD-L1 with high affinity and cannot bind to rat or mouse PD-L1. 
         FIG. 4  shows that 47C6A3, 67F3G7 and 89C10H8 can efficiently inhibit the binding of human PD-L1 to human PD1. 
         FIG. 5  shows the binding kinetics of 47C6A3, 67F3G7 and 89C10H8 to recombinant PD-L1. 
         FIG. 6A-C  show that all tested humanized antibodies had comparable binding efficacy to human PD-L1 in contact to chimeric antibody. 
         FIG. 7  shows that tested humanized antibodies can high efficiently bind to PD-L1 expressed on mammalian cells, comparable with chimeric antibody. 
         FIG. 8A-C  show that some humanized antibodies can efficiently inhibit the binding of human PD-L1 to human PD1. 
         FIG. 9A-C  show that some humanized antibodies can efficiently inhibit the binding of human PD-L1 to human CD80. 
         FIG. 10  shows the binding kinetics of LP008-06, LP008-06a, LP008-06a-DA and LP008-06a-ES to recombinant PD-L1. 
         FIG. 11  shows the binding kinetics of LP008-02 to PD-L1 and TGF-0. 
         FIG. 12  shows that LP008-02 and LP008-06a-ES can blockade PD1 and PD-L1 interact with higher affinity. 
         FIG. 13  shows the M7824, LP008-02 and LP008-06a-ES can effectively blocked TGF-β canonical signaling. 
         FIG. 14  shows that LP008-02 and LP008-06a-ES bind to human PD-L1 with high affinity. 
         FIG. 15  shows that LP008-02 and LP008-06a-ES can bind to cynomolgus PD-L1 with higher affinity but cannot bind to rat PD-L1 or mouse PD-L1. 
         FIG. 16  shows that LP008-02 and LP008-06a-ES had comparable binding efficacy to human TGF-β in contrast to M7824. 
         FIG. 17  shows that LP008-02 and LP008-06a-ES had comparable binding efficacy to cynomolgus TGF-β, mouse TGF-β, and rat TGF-β in contrast to M7824. 
         FIG. 18A-B  show the drug effects of LP008-02 and LP008-06a-ES in animal models. 
         FIG. 19  shows that all tested modified bifunctional molecules had comparable binding efficacy to human TGF-β in contact to LP008-02-1. 
         FIG. 20  shows that all tested modified bifunctional molecules can effectively block TGF-β canonical signaling. 
     
    
    
     DETAILED DESCRIPTION 
     Definitions 
     It is to be noted that the term “a” or “an” entity refers to one or more of that entity; for example, “an antibody,” is understood to represent one or more antibodies. As such, the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein. 
     As used herein, an “antibody” or “antigen-binding polypeptide” refers to a polypeptide or a polypeptide complex that specifically recognizes and binds to an antigen. An antibody can be a whole antibody and any antigen binding fragment or a single chain thereof. Thus the term “antibody” includes any protein or peptide containing molecule that comprises at least a portion of an immunoglobulin molecule having biological activity of binding to the antigen. Examples of such include, but are not limited to a complementarity determining region (CDR) of a heavy or light chain or a ligand binding portion thereof, a heavy chain or light chain variable region, a heavy chain or light chain constant region, a framework (FR) region, or any portion thereof, or at least one portion of a binding protein. 
     The terms “antibody fragment” or “antigen-binding fragment”, as used herein, is a portion of an antibody such as F(ab′) 2 , F(ab) 2 , Fab′, Fab, Fv, scFv and the like. Regardless of structure, an antibody fragment binds with the same antigen that is recognized by the intact antibody. The term “antibody fragment” includes aptamers, spiegelmers, and diabodies. The term “antibody fragment” also includes any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex. 
     The term antibody encompasses various broad classes of polypeptides that can be distinguished biochemically. Those skilled in the art will appreciate that heavy chains are classified as gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ϵ) with some subclasses among them (e.g., γ1-γ4). It is the nature of this chain that determines the “class” of the antibody as IgG, IgM, IgA IgG, or IgE, respectively. The immunoglobulin subclasses (isotypes) e.g., IgG 1 , IgG 2 , IgG 3 , IgG 4 , IgG 5 , etc. are well characterized and are known to confer functional specialization. Modified versions of each of these classes and isotypes are readily discernable to the skilled artisan in view of the instant disclosure and, accordingly, are within the scope of the instant disclosure. All immunoglobulin classes are clearly within the scope of the present disclosure, the following discussion will generally be directed to the IgG class of immunoglobulin molecules. With regard to IgG, a standard immunoglobulin molecule comprises two identical light chain polypeptides of molecular weight approximately 23,000 Daltons, and two identical heavy chain polypeptides of molecular weight 53,000-70,000. The four chains are typically joined by disulfide bonds in a “Y” configuration wherein the light chains bracket the heavy chains starting at the mouth of the “Y” and continuing through the variable region. 
     By “specifically binds” or “has specificity to,” it is generally meant that an antibody binds to an epitope via its antigen-binding domain, and that the binding entails some complementarity between the antigen-binding domain and the epitope. According to this definition, an antibody is said to “specifically bind” to an epitope when it binds to that epitope, via its antigen-binding domain more readily than it would bind to a random, unrelated epitope. The term “specificity” is used herein to qualify the relative affinity by which a certain antibody binds to a certain epitope. For example, antibody “A” may be deemed to have a higher specificity for a given epitope than antibody “B,” or antibody “A” may be said to bind to epitope “C” with a higher specificity than it has for related epitope “D.” 
     As used herein, the terms “treat” or “treatment” refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder, such as the progression of cancer. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented. 
     By “subject” or “individual” or “animal” or “patient” or “mammal,” is meant any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired. Mammalian subjects include humans, domestic animals, farm animals, and zoo, sport, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, and so on. 
     As used herein, phrases such as “to a patient in need of treatment” or “a subject in need of treatment” includes subjects, such as mammalian subjects, that would benefit from administration of an antibody or composition of the present disclosure used, e.g., for detection, for a diagnostic procedure and/or for treatment. 
     Multifunctional Molecules 
     As demonstrated in the appended experimental examples, the instant inventors were able to identify a number of bifunctional fusion proteins that include an anti-PD-L1 unit and a TGF-β-targeting unit. As shown in Example 14, for instance, both of the tested bifunctional proteins, LP008-02 and LP008-06a-ES, exhibited greater efficacy than M7824 in a MC38 mouse model. M7824 is a PD-L1/TGF-β dual targeting fusion protein currently in phase H clinical trial for patients with HPV-positive malignancies. M7824&#39;s anti-PD-L1 unit is based on avelumab, which is a leading PD-L1 antibody and has been approved for the treatment of Merkel cell carcinoma and urothelial cancer. The superior performance of the newly disclosed bifunctional proteins, as compared to M7824, is therefore surprising. 
     Further, as shown in Example 12, the instantly disclosed bifunctional proteins have better species specificity. Unlike M7824 which reacts with mouse and rat PD-L1 as well, the new bifunctional proteins bind to only human and cynomolgus PD-L1, in addition to its superior PD-L1 binding activity. 
     In one embodiment, therefore, the present disclosure provides a multifunctional molecules having at least an anti-PD-L1 unit and a TGF-β-targeting unit. The anti-PD-L1 unite can include an anti-PD-L1 antibody or fragment of the present disclosure. The TGF-β-targeting unit is preferably an extracellular domain of human transforming growth factor-β (TGF-β) receptor II (TGF-β RII or TGFBR2). 
     TGF-β RII has two isoforms. Isoform A (NP_001020018.1; SEQ ID NO:70) has a longer extracellular fragment than Isoform B (NP_003233.4; SEQ ID NO:71), but they share the same core ectodomain (SEQ ID NO:72). Their sequences are provided in Table A below. 
     
       
         
           
               
             
               
                 TABLE A 
               
             
            
               
                   
               
               
                 Sequences related to TGF-β RII (underlined and 
               
               
                 bold: core ectodomain; underlined and italic: different 
               
               
                  residues between isoforms; underlined only: mutations) 
               
            
           
           
               
               
               
            
               
                   
                   
                 SEQ ID 
               
               
                 Name 
                 Sequence 
                 NO: 
               
               
                   
               
               
                 TGEBR2 isoform A 
                 MGRGLLRGLWPLHIVLWTRIASTIPPHVQKS   DVEMEAQKDETICPSCNRT     
                 70 
               
               
                   
                     AHPLRHI   NNDMIVTDNNGAVKFP   QLCKFCDVRFSTCDNQKSCMSNCSITS     
                   
               
               
                   
                 
                   
                     ICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKE 
                   
                 
                   
               
               
                   
                     KKKPGETFFMCSCSSDECNDNIIFS   EEYNTSNPDLLLVIFQVTGISLLPP 
                   
               
               
                   
                 LGVAISVIIIFYCYRVNRQQKLSSTWETGKTRKLMEFSEHCAIILEDDRS 
                   
               
               
                   
                 DISSTCANNINHNTELLPIELDTLVGKGRFAEVYKAKLKQNTSEQFETVA 
                   
               
               
                   
                 VKIFPYEEYASWKTEKDIFSDINLKHENILQFLTAEERKTELGKQYWLIT 
                   
               
               
                   
                 AFHAKGNLQEYLTRHVISWEDLRKLGSSLARGIAHLHSDHTPCGRPKMPI 
                   
               
               
                   
                 VHRDLKSSNILVKNDLTCCLCDFGLSLRLDPTLSVDDLANSGQVGTARYM 
                   
               
               
                   
                 APEVLESRMNLENVESFKQTDVYSMALVLWEMTSRCNAVGEVKDYEPPFG 
                   
               
               
                   
                 SKVREHPCVESMKDNVLRDRGRPEIPSFWLNHQGIQMVCETLTECWDHDP 
                   
               
               
                   
                 EARLTAQCVAERFSELEHLDRLSGRSCSEEKIPEDGSLNTTK 
                   
               
               
                   
               
               
                 TGEBR2 isoform B 
                 MGRGLLRGLWPLHIVLWTRIASTIPPH   V   QKSVNNDMIVTDNNGAVKFP   QL     
                 71 
               
               
                   
                 
                   
                     CKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETV 
                   
                 
                   
               
               
                   
                 
                   
                     CHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFS 
                   
                 
                   
               
               
                   
                 EEYNTSNPDLLLVIFQVTGISLLPPLGVAISVIIIFYCYRVNRQQKLSST 
                   
               
               
                   
                 WETGKTRKLMEFSEHCAIILEDDRSDISSTCANNINHNTELLPIELDTLV 
                   
               
               
                   
                 GKGRFAEVYKAKLKQNTSEQFETVAVKIFPYEEYASWKTEKDIFSDINLK 
                   
               
               
                   
                 HENILQFLTAEERKTELGKQYWLITAFHAKGNLQEYLTRHVISWEDLRKL 
                   
               
               
                   
                 GSSLARGIAHLHSDHTPCGRPKMPIVHRDLKSSNILVKNDLTCCLCDFGL 
                   
               
               
                   
                 SLRLDPTLSVDDLANSGQVGTARYMAPEVLESRMNLENVESFKQTDVYSM 
                   
               
               
                   
                 ALVLWEMTSRCNAVGEVKDYEPPFGSKVREHPCVESMKDNVLRDRGRPEI 
                   
               
               
                   
                 PSFWLNHQGIQMVCETLTECWDHDPEARLTAQCVAERFSELEHLDRLSGR 
                   
               
               
                   
                 SCSEEKIPEDGSLNTTK 
                   
               
               
                   
               
               
                 Ectodomain 
                 QLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLE 
                 72 
               
               
                   
                 TVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNII 
                   
               
               
                   
                 FS 
                   
               
               
                   
               
               
                 Variant 1 (Longer 
                 IPPHVQKSVNNDMIVTDNNGAVKFP QLCKFCDVRFSTCDNQKSCMSNCSI   
                 61 
               
               
                 extracellular fragment) 
                 
                   TSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIM 
                 
                   
               
               
                   
                   KEKKKPGETFFMCSCSSDECNDNIIFS EEYNTSNPD 
                   
               
               
                   
               
               
                 Variant 2 (N-terminal 25 
                 
                   QLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLE 
                 
                 73 
               
               
                 amino acid truncation 
                 
                   TVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNII 
                 
                   
               
               
                 from variant 1) 
                   FS EEYNTSNPD 
                   
               
               
                   
               
               
                 Variant 3 (removal of 
                 IPPHVQ XX VNNDMIVTDN X GAVKFP QLCKFCDVRFSTCDNQKSCMSNCSI   
                 74 
               
               
                 potential glycosylation 
                 
                   TSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIM 
                 
                   
               
               
                 sites from variant 1) 
                   KEKKKPGETFFMCSCSSDECNDNIIFS EEYNTSNPD 
                   
               
               
                   
               
               
                 Variant 4 (Longer 
                 TAGHTQTSTGGGAITTGTSGAGHGP QLCKFCDVRFSTCDNQKSCMSNCSI   
                 75 
               
               
                 peptides substitute) 
                 
                   TSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIM 
                 
                   
               
               
                   
                 
                   KEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD 
                 
                   
               
               
                   
               
               
                 Variant 5 (Longer 
                 TAGHTQTSTGGGAITTGTSGAGHGP QLCKFCDVRFSTCDNQKSCMSNCSI   
                 76 
               
               
                 peptides substitute and 
                 
                   TSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIM 
                 
                   
               
               
                 C terminal deletion) 
                 
                   KEKKKPGETFFMCSCSSDECNDNIIFS 
                 
                   
               
               
                   
               
               
                 Variant 6 (Shorter 
                 HYP QLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENI   
                 77 
               
               
                 peptides substitute and 
                 
                   TLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECND 
                 
                   
               
               
                 C terminal deletion) 
                 
                   NIIFS 
                 
                   
               
               
                   
               
               
                 Variant 7 (shorter 
                 HYP QLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENI   
                 78 
               
               
                 peptides substitute) 
                 
                   TLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECND 
                 
                   
               
               
                   
                   NIIFS EEYNTSNPD 
                   
               
               
                   
               
               
                 Substitute N-terminal 
                 IPPHVQ XX VNNDMIVTDN X GAVKF 
                 88 
               
               
                 fragment of Variant 3 
                   
                   
               
               
                   
               
            
           
         
       
     
     In some embodiments, the TGF-β RII extracellular domain includes the core ectodomain (SEQ ID NO:72) as well as some flanking residues. For instance, Variant 1 (SEQ ID NO:61) which was tested in Examples 8-15 includes additional 25 residues at the N-terminal side and nine on the C-terminal side. Another variant, Variant 2 (SEQ ID NO:73) only includes the nine C-terminal flanking residues. Other variants such as Variants 4-7 (SEQ ID NO:75-78) include alternative linkers replacing part of the N-terminal sequences of SEQ ID NO:61. 
     In some embodiments, the TGF-β RII extracellular domain does not include the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acids of SEQ ID NO:61. In some embodiments, the TGF-β RII extracellular domain does not include the last 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acids of SEQ ID NO:61. 
     Yet another variant, Variant 3, is based on Variant 1 but includes at least an amino acid substitution at the X positions within the N-terminal portion (SEQ ID NO:88). These X positions are potential glycosylation sites. The substitution, therefore, is with an amino acid other than K, S, and N. Examples of substitutions are R, A, G, Q, I, L, D, or E, without limitation. 
     The ani-PD-L1 unit, in some embodiments, is comprised of an anti-PD-L1 antibody of fragment thereof as further described below. The antibody or fragment may take any antibody format, such as the conventional full IgG format, a Fab fragment, a single chain fragment, or a single domain antibody, without limitation. When the antibody or fragment thereof has a light chain and a separate heavy chain, the TGF-β RII extracellular domain can be fused to either the light chain or the heavy chain. When the antibody or fragment thereof has a light chain and a heavy chain on a single protein chain (e.g., scFv), the TGF-β RII extracellular domain can be fused to be closer to either the light chain or the heavy chain. 
     In some embodiments, the TGF-β RII extracellular domain is fused to the N-terminus of a chain of the anti-PD-L1 unit. In some embodiments, the TGF-β RII extracellular domain is fused to the C-terminus of a chain of the anti-PD-L1 unit. In a preferred embodiment, the TGF-β RII extracellular domain is fused to the C-terminus of the heavy chain of the anti-PD-L1 unit, optionally though a peptide linker (e.g., SEQ ID NO:60, or one, two, or three GGGGS (SEQ ID NO:86) repeats). 
     In some embodiments, the anti-PD-L1 unit includes a VH (heavy chain variable region) and a VL (light chain variable region). The VH and VL regions include VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3, such as those illustrated in Tables 1A-1C. 
     In one embodiment, the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 comprise the sequences of SDYAWN (SEQ ID NO:7), YIIYSGSTSYNPSLKS (SEQ ID NO:8), STMIATNWFAY (SEQ ID NO:9), KASQDVSLAVA (SEQ ID NO:10), WASTRHT (SEQ ID NO:11), and QQHYITPWT (SEQ ID NO:12), respectively. Examples of such VH sequences are provided in SEQ ID NO:25 (mouse) and 26-28 (humanized). Examples of such VL sequences are provided in SEQ ID NO:29 (mouse) and 30 (humanized). Example humanized antibodies include those that have a VH of SEQ ID NO:26, or 27, or 28 and a VL of SEQ ID NO:30. 
     In one embodiment, the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 comprise the sequences of DFWVS (SEQ ID NO:13), EIYPNSGVSRYNEKFKG (SEQ ID NO:14), YFGYTYWFGY (SEQ ID NO:15), RASKSVSTYMH (SEQ ID NO:16), SASHLES (SEQ ID NO:17) and QQSNELPVT (SEQ ID NO:18), respectively. Examples of such VH sequences are provided in SEQ ID NO:31 (mouse) and 32-37 (humanized). Examples of such VL sequences are provided in SEQ ID NO:38 (mouse) and 39-43 (humanized). Example humanized antibodies include those that have a VH of SEQ ID NO:34 and a VL of SEQ ID NO:39, 40, or 43, have a VH of SEQ ID NO:35 and a VL of SEQ ID NO:39, or have a VH of SEQ ID NO:37 and VL of SEQ ID NO:39. In one embodiment, the humanized antibody includes a VH of SEQ ID NO:34 and a VL of SEQ ID NO:43. 
     In one embodiment, the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 comprise the sequences of NYWMT (SEQ ID NO:19), SITNTGSSTFYPDSVKG (SEQ ID NO:20), DTTIAPFDY (SEQ ID NO:21), KASQNLNEYLN (SEQ ID NO:22), KTNTLQA (SEQ ID NO:23) and SQYNSGNT (SEQ ID NO:24), respectively. Alternatively, VH CDR2 can includes SITNTGSSTFYPDAVKG (SEQ ID NO:91) or SITNTGSSTFYPESVKG (SEQ ID NO:92). Examples of such VH sequences are provided in SEQ ID NO:44 (mouse) and 45-49 (humanized) and 57-58 (humanized). Examples of such VL sequences are provided in SEQ ID NO:50 (mouse) and 51-55 (humanized) and 56 (humanized). 
     Example humanized antibodies include those that have a VH of SEQ ID NO:49 and a VL of SEQ ID NO:52 or 54, or have a VH of SEQ ID NO:48 and a VL of SEQ ID NO:53 or 54. In one embodiment, the humanized antibody includes a VH of SEQ ID NO:48 and a VL of SEQ ID NO:53. In one embodiment, the humanized antibody includes a VH of SEQ ID NO:48 and a VL of SEQ ID NO:56. In one embodiment, the humanized antibody includes a VH of SEQ ID NO:57 and a VL of SEQ ID NO:56. In one embodiment, the humanized antibody includes a VH of SEQ ID NO:58 and a VL of SEQ ID NO:56. 
     In some embodiments, the antibody or fragment thereof further a heavy chain constant region (e.g., CH1, CH2 and/or CH3) and/or a light chain constant region (e.g., CL). An example heavy chain constant region is provided in SEQ ID NO:59, and an example light chain constant region is provided in SEQ ID NO:67 (residues 108-214). 
     TGF-β RII x Antibody Fusions 
     Testing with different fusion protein designs (e.g., Table 15) demonstrated that only the core ectodomain (SEQ ID NO:72) of TGF-β RII is required for activity. Further, the extracellular domain of TGF-β RII should not be directly fused to the antibody. There should be a sufficient distance, provided by a peptide linker. 
     With reference to the ectodomain, the peptide linker (which may be entirely an artificial linker, or include part of extracellular fragment N-terminal to the ectodomain, SEQ ID NO:89) should have a minimum length. If the distance is too short, the fusion protein has reduced stability or activity. The minimum length, in some embodiment, is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acid residents. In some embodiments, the linker is not longer than 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 170 or 200 amino acid residues. 
     Inclusion of a flexible linker, e.g., one or more G4S (SEQ ID NO:86) units and, in some embodiment, can be useful for the stability and/or activity of the multifunctional molecule. In some embodiments, the flexible linker includes at least 40%, 50%, 60%, 70% or 80% glycine. In some embodiments, the flexible linker includes one or more serine. In some embodiments, the flexible linker includes 1, 2, 3, 4, 5 or 6 G4S (SEQ ID NO:86) repeats. 
     It is shown that, in some embodiments, the natural N-terminal fragment (IPPHVQKSVNNDMIVTDNNGAVKFP; SEQ ID NO:89) can be relaced with a substitute peptide to increase stability, without sacrifice or even with improvement of activity. In some embodients, the substitute peptide is different from SEQ ID NO:89 but has at least 30%, 40%, 50%, 60%, 70%, 80% or 90% sequence identity to SEQ ID NO:89. 
     An example substitute peptide is IPPHVQXXVNNDMIVTDNXGAVKFP (SEQ ID NO:88), wherein X is any amino acid except K, S, or N. In some embodiments, substitutions can be made to remove the rigid di-peptide PP, removal of potential cleavage sites QK, N and/or K, include multiple glycine residues to increase flexibility, and/or reduce hydrophobic residues. One such example is TAGHTQTSTGGGAITTGTSGAGHGP (SEQ ID NO:87) or a variant having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to SEQ ID NO:87. In some embodiments, the variant includes at least 4 G, no PP dipeptide, no more than 3 hydrophobic amino acid residues selected from the group consisting of I, L, M, F, V, W, Y and P. In some embodiments, the variant includes at least 5 G and no more than 1 hydrophobic amino acid residue selected from the group consisting of I, L, M, F, V, W, Y and P. 
     In some embodiments, the peptide linker between the antibody or fragment thereof and the ectodomain (SEQ ID NO:72) of TGF-β RII include a flexible linker. In some embodiments, the peptide linker includes a substitute peptide of SEQ ID NO:89. In some embodiments, the peptide linker includes both a flexible linker and a substitute peptide. In some embodments, the flexible linker is N-terminal to the substitute peptide. In some embodments, the flexible linker is C-terminal to the substitute peptide. 
     In some embodiments, the multifunctional molecule at least does not include the entire sequence of EEYNTSNPD (SEQ ID NO:90). The multifunctional molecule may have the entire SEQ ID NO:90 removed from the extracellular domain of TGF-β RII. In some embodiments, the multifunctional molecule does not include more than 1, 2, 3, 4, 5, 6, 7 or 8 amino acid residues of EEYNTSNPD (SEQ ID NO:90). 
     The antibody or antigen-binding fragment thereof of the multifunctional molecule may target any antigen. Non-limiting examples are PD-1, PD-L1, CTLA-4, LAG-3, CD28, CD122, 4-1BB, TIM3, OX-40, OX40L, CD40, CD40L, LIGHT, ICOS, ICOSL, GITR, GITRL, TIGIT, CD27, VISTA, B7H3, B7H4, BTLA, CD47 and CD73. They can also be any antibodies or fragments as disclosed herein. 
     The ectodomain of TGF-β RII may be fused to any part of the antibody or fragment. In some embodiments, the ectodomain is fused to the C-terminus of a heavy chain or a light chain of the antibody or fragment. In some embodiments, ectodomain is fused to the C-terminus of a Fc fragment of the antibody or fragment. 
     Anti-PD-L1 Antibodies and Fragments 
     Anti-PD-L1 antibodies and fragments are also provided, which can be used as an anti-PD-L1 unit in a multifunctional molecule, a bi- or multi-specific antibody, or alone in a monospecific antibody. 
     In some embodiments, the anti-PD-L1 antibody or fragment includes a VH (heavy chain variable region) and a VL (light chain variable region). The VH and VL regions include VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3, such as those illustrated in Tables 1A-1C. 
     In one embodiment, the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 comprise the sequences of SDYAWN (SEQ ID NO:7), YIIYSGSTSYNPSLKS (SEQ ID NO:8), STMIATNWFAY (SEQ ID NO:9), KASQDVSLAVA (SEQ ID NO:10), WASTRHT (SEQ ID NO:11), and QQHYITPWT (SEQ ID NO:12), respectively. Examples of such VH sequences are provided in SEQ ID NO:25 (mouse) and 26-28 (humanized). Examples of such VL sequences are provided in SEQ ID NO:29 (mouse) and 30 (humanized). Example humanized antibodies include those that have a VH of SEQ ID NO:26, or 27, or 28 and a VL of SEQ ID NO:30. 
     In one embodiment, the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 comprise the sequences of DFWVS (SEQ ID NO:13), EIYPNSGVSRYNEKFKG (SEQ ID NO:14), YFGYTYWFGY (SEQ ID NO:15), RASKSVSTYMH (SEQ ID NO:16), SASHLES (SEQ ID NO:17) and QQSNELPVT (SEQ ID NO:18), respectively. Examples of such VH sequences are provided in SEQ ID NO:31 (mouse) and 32-37 (humanized). Examples of such VL sequences are provided in SEQ ID NO:38 (mouse) and 39-43 (humanized). Example humanized antibodies include those that have a VH of SEQ ID NO:34 and a VL of SEQ ID NO:39, 40, or 43, have a VH of SEQ ID NO:35 and a VL of SEQ ID NO:39, or have a VH of SEQ ID NO:37 and VL of SEQ ID NO:39. In one embodiment, the humanized antibody includes a VH of SEQ ID NO:34 and a VL of SEQ ID NO:43. 
     In one embodiment, the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 comprise the sequences of NYWMT (SEQ ID NO:19), SITNTGSSTFYPDSVKG (SEQ ID NO:20), DTTIAPFDY (SEQ ID NO:21), KASQNLNEYLN (SEQ ID NO:22), KTNTLQA (SEQ ID NO:23) and SQYNSGNT (SEQ ID NO:24), respectively. Alternatively, VH CDR2 can includes SITNTGSSTFYPDAVKG (SEQ ID NO:91) or SITNTGSSTFYPESVKG (SEQ ID NO:92). Examples of such VH sequences are provided in SEQ ID NO:44 (mouse) and 45-49 (humanized) and 57-58 (humanized). Examples of such VL sequences are provided in SEQ ID NO:50 (mouse) and 51-55 (humanized) and 56 (humanized). 
     Example humanized antibodies include those that have a VH of SEQ ID NO:49 and a VL of SEQ ID NO:52 or 54, or have a VH of SEQ ID NO:48 and a VL of SEQ ID NO:53 or 54. In one embodiment, the humanized antibody includes a VH of SEQ ID NO:48 and a VL of SEQ ID NO:53. In one embodiment, the humanized antibody includes a VH of SEQ ID NO:48 and a VL of SEQ ID NO:56. In one embodiment, the humanized antibody includes a VH of SEQ ID NO:57 and a VL of SEQ ID NO:56. In one embodiment, the humanized antibody includes a VH of SEQ ID NO:58 and a VL of SEQ ID NO:56. 
     In some embodiments, the antibody or fragment thereof further a heavy chain constant region (e.g., CH1, CH2 and/or CH3) and/or a light chain constant region (e.g., CL). An example heavy chain constant region is provided in SEQ ID NO:59, and an example light chain constant region is provided in SEQ ID NO:67 (residues 108-214). 
     It is contemplated that small changes (e.g., one amino acid addition, deletion or substitution) can be designed among these CDR sequences that can retain the antibodies&#39; activities or even improve them. Such modified CDR sequences are referred to as CDR variants. It will also be understood by one of ordinary skill in the art that antibodies as disclosed herein may be modified such that they vary in amino acid sequence from the naturally occurring binding polypeptide from which they were derived. For example, a polypeptide or amino acid sequence derived from a designated protein may be similar, e.g., have a certain percent identity to the starting sequence, e.g., it may be 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical to the starting sequence. In some embodiments, the modified antibody or fragment retains the designate CDR sequences. 
     In certain embodiments, the antibody comprises an amino acid sequence or one or more moieties not normally associated with an antibody. Exemplary modifications are described in more detail below. For example, an antibody of the disclosure may comprise a flexible linker sequence, or may be modified to add a functional moiety (e.g., PEG, a drug, a toxin, or a label). 
     Polynucleotides Encoding the Proteins and Methods of Preparing the Proteins 
     The present disclosure also provides isolated polynucleotides or nucleic acid molecules encoding the multifunctional proteins, antibodies, variants or derivatives thereof of the disclosure. The polynucleotides of the present disclosure may encode the entire heavy and light chain variable regions of the antigen-binding polypeptides, variants or derivatives thereof on the same polynucleotide molecule or on separate polynucleotide molecules. Additionally, the polynucleotides of the present disclosure may encode portions of the heavy and light chain variable regions of the antigen-binding polypeptides, variants or derivatives thereof on the same polynucleotide molecule or on separate polynucleotide molecules. 
     Methods of making antibodies are well known in the art and described herein. In certain embodiments, both the variable and constant regions of the antigen-binding polypeptides of the present disclosure are fully human Fully human antibodies can be made using techniques described in the art and as described herein. For example, fully human antibodies against a specific antigen can be prepared by administering the antigen to a transgenic animal which has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled. Exemplary techniques that can be used to make such antibodies are described in U.S. Pat. Nos. 6,150,584; 6,458,592; 6,420,140 which are incorporated by reference in their entireties. 
     Cancer Treatment 
     As described herein, the antibodies, variants or derivatives of the present disclosure may be used in certain treatment and diagnostic methods. 
     The present disclosure is further directed to multifunctional molecule- or antibody-based therapies which involve administering the multifunctional molecules or the antibodies of the disclosure to a patient such as an animal, a mammal, and a human for treating one or more of the disorders or conditions described herein. Therapeutic compounds of the disclosure include, but are not limited to, antibodies of the disclosure (including variants and derivatives thereof as described herein) and nucleic acids or polynucleotides encoding antibodies of the disclosure (including variants and derivatives thereof as described herein). 
     The antibodies of the disclosure can also be used to treat or inhibit cancer. PD-L1 can be overexpressed in tumor cells. Tumor-derived PD-L1 can bind to PD-1 on immune cells thereby limiting antitumor T-cell immunity. Results with small molecule inhibitors, or monoclonal antibodies targeting PD-L1 in murine tumor models, indicate that targeted PD-L1 therapy is an important alternative and realistic approach to effective control of tumor growth. As demonstrated in the experimental examples, the anti-PD-L1 antibodies activated the adaptive immune response machinery, which can lead to improved survival in cancer patients. 
     Accordingly, in some embodiments, provided are methods for treating a cancer in a patient in need thereof. The method, in one embodiment, entails administering to the patient an effective amount of a multifunctional molecule or an antibody of the present disclosure. In some embodiments, at least one of the cancer cells (e.g., stromal cells) in the patient expresses, over-express, or is induced to express PD-L1. Induction of PD-L1 expression, for instance, can be done by administration of a tumor vaccine or radiotherapy. 
     Tumors that express the PD-L1 protein include those of bladder cancer, non-small cell lung cancer, renal cancer, breast cancer, urethral cancer, colorectal cancer, head and neck cancer, squamous cell cancer, Merkel cell carcinoma, gastrointestinal cancer, stomach cancer, oesophageal cancer, ovarian cancer, renal cancer, and small cell lung cancer. Accordingly, the presently disclosed antibodies can be used for treating any one or more such cancers. 
     Cellular therapies, such as chimeric antigen receptor (CAR) T-cell therapies, are also provided in the present disclosure. A suitable cell can be used, that is put in contact with an anti-PD-L1 antibody of the present disclosure (or alternatively engineered to express an anti-PD-L1 antibody of the present disclosure). Upon such contact or engineering, the cell can then be introduced to a cancer patient in need of a treatment. The cancer patient may have a cancer of any of the types as disclosed herein. The cell (e.g., T cell) can be, for instance, a tumor-infiltrating T lymphocyte, a CD4+ T cell, a CD8+ T cell, or the combination thereof, without limitation. 
     In some embodiments, the cell was isolated from the cancer patient him- or her-self. In some embodiments, the cell was provided by a donor or from a cell bank. When the cell is isolated from the cancer patient, undesired immune reactions can be minimized. 
     Additional diseases or conditions associated with increased cell survival, that may be treated, prevented, diagnosed and/or prognosed with the antibodies or variants, or derivatives thereof of the disclosure include, but are not limited to, progression, and/or metastases of malignancies and related disorders such as leukemia (including acute leukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemias (e.g., chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin&#39;s disease and non-Hodgkin&#39;s disease), multiple myeloma, Waldenstrom&#39;s macroglobulinemia, heavy chain disease, and solid tumors including, but not limited to, sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing&#39;s tumor, leiomyosarcoma, rhabdomyo sarcoma, colon carcinoma, pancreatic cancer, breast cancer, thyroid cancer, endometrial cancer, melanoma, prostate cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm&#39;s tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma and retinoblastoma. 
     Compositions 
     The present disclosure also provides pharmaceutical compositions. Such compositions comprise an effective amount of an antibody, and an acceptable carrier. In some embodiments, the composition further includes a second anticancer agent (e.g., an immune checkpoint inhibitor). 
     In a specific embodiment, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. Further, a “pharmaceutically acceptable carrier” will generally be a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. 
     The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents such as acetates, citrates or phosphates. Antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; and agents for the adjustment of tonicity such as sodium chloride or dextrose are also envisioned. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in Remington&#39;s Pharmaceutical Sciences by E. W. Martin, incorporated herein by reference. Such compositions will contain a therapeutically effective amount of the antigen-binding polypeptide, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration. The parental preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. 
     In an embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration. 
     EXAMPLES 
     Example 1: Generation of Mouse Monoclonal Antibodies Against Human PD-L1 
     This example describes anti-human-PD-L1 mouse monoclonal antibodies generated using the hybridoma technology. 
     Antigen: human PDL1-Fc protein and human PD-L1 highly expressed on the CHOK1 cell line (PDL1-CHOK1 cell line). 
     Immunization: To generate mouse monoclonal antibodies to human PD-L1, Balb/c mice and Wistar Rat were firstly immunized with PD-L1-Fc protein. The immunized mice and Rat were subsequently boosted with the fusion PD-L1-Fc protein, CHO-K1/PD-L1 stable cells and PD-L1-Fc protein. To select mice or rat producing antibodies that bound PD-L1 protein, the serum of immunized mice was subjected to the antibody titer evaluation by ELISA. Briefly, microtiter plates were coated with human PD-L1 protein at 0.5 μg/ml in ELISA coating buffer, 100 μl/well at 4° C. overnight, then blocked with 150 μl/well of 1% BSA. Dilutions of serum from immunized mice were added to each well and incubated for 1-2 hours at 37° C. The plates were washed with PBS/Tween and then incubate with anti-mouse IgG antibody conjugated with Horse Radish Peroxidase (HRP) or anti-rat IgG antibody conjugated with HRP for 1 hour at 37° C. After washing, the plates were developed with TMB substrate and analyzed by spectrophotometer at OD 450 nm. Post 3 rounds of immunization, Immune response will also be tested by serum ELISA against rhPD-L1 protein and FACS against CHO-K1/PDL-1 stable cell line while CHO-K1 parental cell line served as negative control. Mice with sufficient titers of anti-PDL1 IgG were boosted with 25 μg human PDL1-Fc protein after 4 rounds of immunization. The resulting mice were used for fusions. The hybridoma supernatants were tested for anti-PD-L1 IgGs by ELISA. 
     Cell fusion: Fusion was performed by electro fusion. Fused cells were plated into 50 96-well plates for each fusion. 
     Screening: The supernatants were screened by ELISA against recombinant human (rh) PD-L1 Fc protein and counter screening antigen. Then, positive supernatants underwent confirmative screening from primary screening with receptor blocking FACS against CHO-K1/PD-L1 stable cell line and rhPD1-Fc protein. 
     Subcloning and screening: positive primary clones from each fusion were subcloned by limiting dilution to ensure that the subclones were derived from a single parental cell. Subcloning were screened the same as primary clones and culture supernatant of positive clones underwent additional confirmative screening by affinity ranking. 
     Hybridoma clones 47C6A3, 67F3G7 and 89C10H8 were selected for further analysis. The amino acid sequences of the variable regions of 47C6A3, 67F3G7 and 89C10H8 are provided in Table 1 below. 
     
       
         
           
               
             
               
                 TABLE 1 
               
               
                   
               
               
                 Sequences of the variable regions of 47C6A3, 67F3G7 and 89C10H8 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                   
                   
                 SEQ ID 
               
               
                 Name 
                 Sequence 
                 NO: 
               
               
                   
               
               
                 47C6A3 
                 DVQLQESGPGLVKPSQSLSLTCTVTGYSIT   SDYAWN   WIRQFPGNKLEWMG   YIIYSGSTS     
                 1 
               
               
                 VH 
                     YNPSLKS   RISITRDTSKNQFFLQLNSVTTEDTATYYCAR   STMIATNWFAY   WGQGTLVTV 
                   
               
               
                   
                 SA 
                   
               
               
                   
               
               
                 47C6A3 
                 DIVMTQSHKFMSTSVVDRVSITC   KASQDVSLAVA   WYQQKPGQSPKLLIY   WASTRHT   GVP 
                 2 
               
               
                 VL 
                 DRFTGSGSGTDFALTISSVQAEDLALYYC   QQHYITPWT   FGGGTKLEIK 
                   
               
               
                   
               
               
                 67F3G7 
                 QVKLLQSGAALVKPGASVKMSCKASGYIFT   DFWVS   WVKQSHEKSLEWIG   EIYPNSGVSR     
                 3 
               
               
                 VH 
                     YNEKFKG   RATMTVDKSTSTAYLELSRLTSEDSAIYYCTK   YFGYTYWFGY   WGQGTLVTVS 
                   
               
               
                   
                 S 
                   
               
               
                   
               
               
                 67F3G7 
                 DTVLTQSPALAVSLGQRITISC   RASKSVSTYMH   WYQQRSGLQPKLLIY   SASHLES   GVPS 
                 4 
               
               
                 VL 
                 RFSGSGSGTDFTLTIDPVEADDIANYYC   QQSNELPVT   FGSGTKLEIK 
                   
               
               
                   
               
               
                 89C10H8 
                 EVQLVESGGGLVQPGRSLTLSCVASGFTFS   NYWMT   WIRQAPGKGLEWVA   SITNTGSSTF     
                 5 
               
               
                 VH 
                     YPDSVKG   RFTISRDNTRSTLFLQINSLRSEDTATYYCTR   DTTIAPFDY   WGQGVMVTVSS 
                   
               
               
                   
               
               
                 89C10H8 
                 DIQMTQSPSFLSASVGDRVTITC   KASQNLNEYLN   WYQQKLGEAPKRLIY   KTNTLQA   GIP 
                 6 
               
               
                 VL 
                 SRFSGSGSGIDYTLTISSLQPEDVATYFC   SQYNSGNT   FGAGTKLELK 
               
               
                   
               
            
           
           
               
            
               
                 Table 1A. CDR sequences of 47C6A3 
               
            
           
           
               
               
               
            
               
                   
                   
                 SEQ ID 
               
               
                 47C6A3 
                 Sequence 
                 NO: 
               
               
                   
               
               
                 CDRH1 
                 SDYAWN 
                 7 
               
               
                   
               
               
                 CDRH2 
                 YIIYSGSTSYNPSLKS 
                 8 
               
               
                   
               
               
                 CDRH3 
                 STMIATNWFAY 
                 9 
               
               
                   
               
               
                 CDRL1 
                 KASQDVSLAVA 
                 10 
               
               
                   
               
               
                 CDRL2 
                 WASTRHT 
                 11 
               
               
                   
               
               
                 CDRL3 
                 QQHYITPWT 
                 12 
               
               
                   
               
            
           
           
               
            
               
                 Table IB. CDR sequences of 67F3G7 
               
            
           
           
               
               
               
            
               
                   
                   
                 SEQ ID 
               
               
                 67F3G7 
                 Sequence 
                 NO: 
               
               
                   
               
               
                 CDRH1 
                 DFWVS 
                 13 
               
               
                   
               
               
                 CDRH2 
                 EIYPNSGVSRYNEKFKG 
                 14 
               
               
                   
               
               
                 CDRH3 
                 YFGYTYWFGY 
                 15 
               
               
                   
               
               
                 CDRL1 
                 RASKSVSTYMH 
                 16 
               
               
                   
               
               
                 CDRL2 
                 SASHLES 
                 17 
               
               
                   
               
               
                 CDRL3 
                 QQSNELPVT 
                 18 
               
               
                   
               
            
           
           
               
            
               
                 Table IC. CDR sequences of 89C10H8 
               
            
           
           
               
               
               
            
               
                   
                   
                 SEQ ID 
               
               
                 89C10H8 
                 Sequence 
                 NO: 
               
               
                   
               
               
                 CDRH1 
                 NYWMT 
                 19 
               
               
                   
               
               
                 CDRH2 
                 SITNTGSSTFYPDSVKG 
                 20 
               
               
                   
               
               
                 ver DA 
                 SITNTGSSTFYPD A VKG 
                 91 
               
               
                   
               
               
                 ver ES 
                 SITNTGSSTFYP E SVKG 
                 92 
               
               
                   
               
               
                 CDRH3 
                 DTTIAPFDY 
                 21 
               
               
                   
               
               
                 CDRL1 
                 KASQNLNEYLN 
                 22 
               
               
                   
               
               
                 CDRL2 
                 KTNTLQA 
                 23 
               
               
                   
               
               
                 CDRL3 
                 SQYNSGNT 
                 24 
               
               
                   
               
            
           
         
       
     
     Example 2: The Binding Activity to PD-L1 Antigen 
     ELISA Testing 
     To evaluate the binding activity of hybridoma clones 47C6A3, 67F3G7 and 89C10H8, the chimeric mAb from these clones were subjected to ELISA test. 
     Briefly, microtiter plates were coated with human PD-L1-Fc protein at 0.5 μg/ml in PBS, 100 μl/well at 4° C. overnight, then blocked with 150 μl/well of 1% BSA. Three-fold dilutions of 47C6A3, 67F3G7 and 89C10H8 antibodies starting from 10 μg/ml were added to each well and incubated for 1 hour at 37° C. The plates were washed with PBS/Tween and then incubated with Mouse-anti-human IgG Fab antibody conjugated with Horse Radish Peroxidase (HRP) for 30 mins at 37° C. After washing, the plates were developed with TMB substrate and analyzed by spectrophotometer at OD 450 nm. As shown in  FIG. 1 , 47C6A3, 67F3G7 and 89C10H8 bound to human PD-L1 with high affinity (EC 50 =10.24 ng/ml for 47C6A3, EC 50 =10.76 ng/ml for 67F3G7, EC 50 =8.112 ng/ml for 89C10H8). 
     Cell-based binding: FACS was used to evaluate the binding activity of 47C6A3, 67F3G7 and 89C10H8 chimeric mAbs on human PD-L1 over-expressed CHOK1 cells. 
     Briefly, PDL1-CHOK1 cells were firstly incubated with 3-fold serially diluted 47C6A3, 67F3G7 and 89C10H8 chimeric mAbs starting at 100 nM at 4° C. for 40 mins. After washing by PBS, Alexa Fluor® 647 AffiniPure Goat Anti-Human IgG (H+L) were added to each well and incubated at 4° C. for 30 mins. Samples were twice washed with FACS buffer. The mean florescence intensity (MFI) of Alexa Fluor® 647 were evaluated by FACSCanto. As shown in  FIG. 2 , 47C6A3, 67F3G7 and 89C10H8 bound to PDL1-CHOK1 cells with high affinity (EC 50 =0.1476 nM for 47C6A3, EC 50 =0.1035 nM for 67F3G7, EC 50 =0.1696 nM for 89C10H8). 
     Cross Species Activity 
     ELISA testing was carried out to evaluate the binding of chimeric antibodies to human PD-L1, mouse PD-L1, rat PD-L1, and cynomolgus PD-L1, respectively. 
     Briefly, microtiter plates were coated with human, mouse, rat and cynomolgus PD-L1 proteins at 0.5 μg/ml in PBS, 100 μl/well at 4° C. overnight, then blocked with 150 μl/well of 1% BSA. Three-fold dilutions of chimeric antibodies starting from 10 μg/ml were added to each well and incubated for 1 hour at 37° C. The plates were washed with PBS/Tween and then incubate with mouse-anti-human IgG Fab antibody conjugated with Horse Radish Peroxidase (HRP) for 30 mins at 37° C. After washing, the plates were developed with TMB substrate and analyzed by spectrophotometer at OD 450 nm. 47C6A3, 67F3G7 and 89C10H8 antibodies bound to cynomolgus PD-L1 and did not bind to rat and mouse PD-L1 ( FIG. 3  and Table 2). 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Cross species activity of 47C6A3, 67F3G7 and 89C10H8 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Human 
                 Cynomolgus 
                 Rat 
                 Mouse 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 EC50 of 47C6A3 
                 10.24  
                 ng/ml 
                 6.336  
                 ng/ml 
                 No binding 
                 No binding 
               
               
                 EC50 of 67F3G7 
                 10.76  
                 ng/ml 
                 6.797  
                 ng/ml 
                 No binding 
                 No binding 
               
               
                 EC50 of 89C10H8 
                 8.112  
                 ng/ml 
                 6.774 
                 ng/ml 
                 No binding 
                 No binding 
               
               
                   
               
            
           
         
       
     
     Example 3. Blockade of PD-L1 Binding to PD-1 by Antibodies 
     To evaluate the blocking effects of 47C6A3, 67F3G7 and 89C10H8 chimeric mAbs on recombinant human PD-L1 to bind to its receptor PD-1, the ELISA based receptor blocking assay was employed. 
     Briefly, microtiter plates were coated with human PD-L1-Fc protein at 0.5 μg/ml in PBS, 100 μl/well at 4° C. overnight, then blocked with 150 μl/well of 1% BSA. 50 μl biotin-labeled human PD-1-Fc protein and 3-fold dilutions of 47C6A3, 67F3G7 and 89C10H8 antibodies starting from 10 μg/ml at 50 μl were added to each well and incubated for 1 hour at 37° C. The plates were washed with PBS/Tween and then incubated with Streptavidin-HRP for 10 mins at 37° C. After washing, the plates were developed with TMB substrate and analyzed by spectrophotometer at OD 450 nm. As shown in  FIG. 4 , 47C6A3, 67F3G7 and 89C10H8 efficiently inhibited the binding of human PD-L1 to human PD1 at IC 50 =91.18 ng/ml, 139.8 ng/ml, 129.8 ng/ml, respectively. 
     Example 4: Binding Affinity of mAbs 
     The binding of the 47C6A3, 67F3G7 and 89C10H8 antibodies to recombinant PD-L1 protein (human PD-L1-his tag) was tested with Biacore® using a capture method. The 47C6A3, 67F3G7 and 89C10H8 mAbs was captured using Protein A chip. A serial dilution of human PD-L1-his tag protein was injected over captured antibody for 2 mins at a flow rate of 30 μl/min. The antigen was allowed to dissociate for 480-1500 s. All the experiment were carried out on a Biacore T200. Data analysis was carried out using Biacore T200 evaluation software. The results are shown in  FIG. 5  and Table 3 below. 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Affinity measured by biacore 
               
            
           
           
               
               
               
            
               
                   
                   
                 PDL1-His 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Abs 
                 ka (1/Ms) 
                 kd (1/s) 
                 KD (M) 
               
               
                   
                   
               
               
                   
                 47C6A3 
                 1.851E+6 
                 6.785E−4 
                 3.665E−10 
               
               
                   
                 67F3G7 
                 2.538E+6 
                 6.193E−5 
                 2.441E−11 
               
               
                   
                 89C10H8 
                 1.597E+6 
                 6.636E−5 
                 4.154E−11 
               
               
                   
                   
               
            
           
         
       
     
     Example 5. Humanization of the Mouse Antibodies 
     The 47C6A3, 67F3G7, and 89C10H8 variable region genes were employed to create a humanized mAb. In the first step of this process, the amino acid sequences of the VH and VL or VK of 47C6A3, 67F3G7, and 89C10H8 were compared against the available database of human Ig gene sequences to find the overall best-matching human germline Ig gene sequences. For the light chain of 47C6A3, human Vk1-4 was the best fit germline, and for the heavy chain, human VH1-2 was chosen as the backbone. For the light chain of 67F3G7, the closest human match was the Vk1-39/JK4 gene, and for the heavy chain the closest human match was the VH1-2/JH4-FW4 gene. For the light chain of 89C10H8, the closest human match was the Vk1-17/JK2 gene, and for the heavy chain the closest human match was the VH3-21/JH3 gene. 
     For the VL of 47C6A3, human Vk1-4 was the best fit germline, and for VH of 47C6A3, human VH1-2 was chosen as the backbone. Humanized variable domain sequences of 47C6A3 were then designed where the CDRL1, L2, and L3 were grafted onto framework sequences of the Vk1-4 gene, and the CDRH1, H2, and H3 onto framework sequences of the VH1-2 gene. A 3D model was then generated to determine if there were any framework positions where replacing the mouse amino acid to the human amino acid could affect binding and/or CDR conformation. In the case of the heavy chain, R, M, and I in the framework was involved in back-mutations. 
     Humanized variable domain sequences of 67F3G7 were then designed where the CDRL1, L2 and L3 were grafted onto framework sequences of the Vk1-39/JK4 gene, and the CDRH1, H2, and H3 onto framework sequences of the VH1-2/JH4-FW4 gene. A 3D model was then generated to determine if there were any framework positions where replacing the mouse amino acid to the human amino acid could affect binding and/or CDR conformation. In the case of the heavy chain, V, K, T, and I in the framework was involved in back-mutations. In the case of the light chain, T, V, L, and Q in the framework was involved in back-mutations. 
     Humanized variable domain sequences of 89C10H8 were then designed where the CDRL1, L2 and L3 were grafted onto framework sequences of the Vk1-17/JK2 gene, and the CDRH1, H2, and H3 onto framework sequences of the VH3-21/JH3 gene. A 3D model was then generated to determine if there were any framework positions where replacing the mouse amino acid to the human amino acid could affect binding and/or CDR conformation. In the case of the heavy chain, A, T, I, and S in the framework was involved in back-mutations. In the case of the light chain, Y, I, E, and F in the framework was involved in back-mutations. 
     The amino acid and nucleotide sequences of some of the humanized antibody are listed in Table 4 below. 
     
       
         
           
               
             
               
                 TABLE 4 
               
               
                   
               
               
                 Humanized antibody sequences (underlining indicates CDR; bold/italic 
               
               
                 indicates back mutations) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                   
                   
                 SEQ ID 
               
               
                 47C6A3 
                 Sequence 
                 NO: 
               
               
                   
               
               
                 VH 
                 DVQLQESGPGLVKPSQSLSLTCTVTGYSIT SDYAWN WIRQFPGNKLEWMG YIIYSGST   
                 25 
               
               
                 mouse 
                   SYNPSLKS RISITRDTSKNQFFLQLNSVTTEDTATYYCAR STMIATNWFAY WGQGTLV 
                   
               
               
                   
                 TVSA 
                   
               
               
                   
               
               
                 VH-V0 
                 QVQLQESGPGLVKPSETLSLTCTVSGYSIT SDYAWN WIRQPPGKGLEWIG YIIYSGST   
                 26 
               
               
                   
                   SYNPSLKS RVTISVDTSKNQFSLKLSSVTAADTAVYYCAR STMIATNWFAY WGQGTLV 
                   
               
               
                   
                 TVSS 
                   
               
               
                   
               
               
                 VH-V1 
                 QVQLQESGPGLVKPSETLSLTCTVSGYSIT SDYAWN WIRQPPGKGLEWIG YIIYSGST   
                 27 
               
               
                   
                   SYNPSLKSR VTISRDTSKNQFSLKLSSVTAADTAVYYCAR STMIATNWFAY WGQGTLV 
                   
               
               
                   
                 TVSS 
                   
               
               
                   
               
               
                 VH-V2 
                 QVQLQESGPGLVKPSETLSLTCTVSGYSIT SDYAWN WIRQPPGKGLEW M G YIIYSGST   
                 28 
               
               
                   
                   SYNPSLKSR   I TISRDTSKNQFSLKLSSVTAADTAVYYCAR STMIATNWFAY WGQGTLV 
                   
               
               
                   
                 TVSS 
                   
               
               
                   
               
               
                 VL mouse 
                 DIVMTQSHKFMSTSVVDRVSITC KASQDVSLAVA WYQQKPGQSPKLLIY WASTRHT GV 
                 29 
               
               
                   
                 PDRFTGSGSGTDFALTISSVQAEDLALYYC QQHYITPWT FGGGTKLEIK 
                   
               
               
                   
               
               
                 VL V0 
                 DIQMTQSPSSLSASVGDRVTITC KASQDVSLAVA WYQQKPGKAPKLLIY WASTRHT GV 
                 30 
               
               
                   
                 PSRFSGSGSGTDFTLTISSLQPEDFATYYC QQHYITPWT FGGGTKVEIK 
               
               
                   
               
               
                   
                   
                 SEQ ID  
               
               
                 67F3G7 
                 Sequence 
                 NO: 
               
               
                   
               
               
                 VH 
                 QVKLLQSGAALVKPGASVKMSCKASGYIFT DFWVS WVKQSHEKSLEWIG EIYPNSGVS   
                 31 
               
               
                 mouse 
                   RYNEKFKG RATMTVDKSTSTAYLELSRLTSEDSAIYYCTK YFGYTYWFGY WGQGTLVT 
                   
               
               
                   
                 VSS 
                   
               
               
                   
               
               
                 VH V0 
                 EVQLVQSGAEVKKPGASVKVSCKASGYTFT DFWVS WVRQAPGQGLEWMG EIYPNSGVS   
                 32 
               
               
                   
                   RYNEKFKG RVTMTRDTSISTAYMELSRLRSDDTAVYYCAR YFGYTYWFGY WGQGTLVT 
                   
               
               
                   
                 VSS 
                   
               
               
                   
               
               
                 VH V1 
                 EVQLVQSGAEVKKPGASVKVSCKASGYTFT DFWVS WVRQAPGQGLEWMG EIYPNSGVS   
                 33 
               
               
                   
                   RYNEKFKG RVTMTVDKSISTAYMELSRLRSDDTAVYYCAR YFGYTYWFGY WGQGTLVT 
                   
               
               
                   
                 VSS 
                   
               
               
                   
               
               
                 VH V2 
                 EVQLVQSGAEVKKPGASVKVSCKASGYTFT DFWVS WVRQAPGQGLEWMG EIYPNSGVS   
                 34 
               
               
                   
                   RYNEKFKG RVTMTVDKSISTAYMELSRLRSDDTAVYYC TK   YFGYTYWFGY WGQGTLVT 
                   
               
               
                   
                 VSS 
                   
               
               
                   
               
               
                 VH V3 
                 EVQLVQSGAEVKKPGASVKVSCKASGY I FT DFWVS WVRQAPGQGLEWIG EIYPNSGVS   
                 35 
               
               
                   
                   RYNEKFKG RVTMTVDKSISTAYMELSRLRSDDTAVYYC TK   YFGYTYWFGY WGQGTLVT 
                   
               
               
                   
                 VSS 
                   
               
               
                   
               
               
                 VH V4 
                 EVQLVQSGAEVKKPGASVKVSCKASGY I FT DFWVS WVRQAPGQGLEWIG EIYPNSGVS   
                 36 
               
               
                   
                   RYNEKFKG RVTMTVDKSISTAYMELSRLRSDDTAVYYCAR YFGYTYWFGY WGQGTLVT 
                   
               
               
                   
                 VSS 
                   
               
               
                   
               
               
                 VH V5 
                 EVQLVQSGAEVKKPGASVKVSCKASGY I FT DFWVS WVRQAPGKGLEWIG EIYPNSGVS   
                 37 
               
               
                   
                   RYNEKFKG RVTMTVDKSISTAYMELSRLRSDDTAVYYCTK YFGYTYWFGY WGQGTLVT 
                   
               
               
                   
                 VSS 
                   
               
               
                   
               
               
                 VL mouse 
                 DTVLTQSPALAVSLGQRITISC RASKSVSTYMH WYQQRSGLQPKLLIY SASHLES GV 
                 38 
               
               
                   
                 PSRFSGSGSGTDFTLTIDPVEADDIANYYC QQSNELPVT FGSGTKLEIK 
                   
               
               
                   
               
               
                 VL V0 
                 DIQMTQSPSSLSASVGDRVTITC RASKSVSTYMH WYQQKPGKAPKLLIY SASHLES GV 
                 39 
               
               
                   
                 PSRFSGSGSGTDFTLTISSLQPEDFATYYC QQSNELPVT FGGGTKVEIK 
                   
               
               
                   
               
               
                 VL V1 
                 DTVLTQSPSSLSASVGDRVTITC RASKSVSTYMH WYQQKPGKAPKLLIY SASHLES GV 
                 40 
               
               
                   
                 PSRFSGSGSGTDFTLTISSLQPEDFATYYC QQSNELPVT FGGGTKVEIK 
                   
               
               
                   
               
               
                 VL V2 
                 DTVLTQSPSSLSASVGDRVTITC RASKSVSTYMH WYQQKPGKQPKLLIY SASHLES GV 
                 41 
               
               
                   
                 PSRFSGSGSGTDFTLTISSLQPEDFATYYC QQSNELPVT FGGGTKVEIK 
                   
               
               
                   
               
               
                 VL V3 
                 DTVLTQSPSLSASVGDRVTITC RASKSVSTYMH WYQQKPGKQPKLLIY SASHLES GV 
                 42 
               
               
                   
                 PSRFSGSGSGTDFTLTISSLQPEDFATYYC QQSNELPVT FGGGTKVEIK 
                   
               
               
                   
               
               
                 VL V4 
                 DIQMTQSPSSLSASVGDRVTITC RASKSVSTYMH WYQQKPGKQPKLLIY SASHLES GV 
                 43 
               
               
                   
                 PSRFSGSGSGTDFTLTISSLQPEDFATYYC QQSNELPVT FGGGTKVEIK 
               
               
                   
               
               
                   
                   
                 SEQ ID  
               
               
                 89C10H8 
                 Sequence 
                 NO: 
               
               
                   
               
               
                 VH 
                 EVQLVESGGGLVQPGRSLTLSCVASGFTFS NYWMT WIRQAPGKGLEWVA SITNTGSST   
                 44 
               
               
                 mouse 
                   FYPDSVKG RFTISRDNTRSTLFLQINSLRSEDTATYYCTR DTTIAPFDY WGQGVMVTV 
                   
               
               
                   
                 SS 
                   
               
               
                   
               
               
                 VH V0 
                 EVQLVESGGGLVKPGGSLRLSCAASGFTFS NYWMT WVRQAPGKGLEWVS SITNTGSST   
                 45 
               
               
                   
                   FYPDSVKG RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR DTTIAPFDY WGQGTMVTV 
                   
               
               
                   
                 SS 
                   
               
               
                   
               
               
                 VH V1 
                 EVQLVESGGGLVKPGGSLRLSCAASGFTFS NYWMT WVRQAPGKGLEWVA SITNTGSST   
                 46 
               
               
                   
                   FYPDSVKG RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR DTTIAPFDY WGQGTMVTV 
                   
               
               
                   
                 SS 
                   
               
               
                   
               
               
                 VH V2 
                 EVQLVESGGGLVKPGGSLRLSCAASGFTFS NYWMT WVRQAPGKGLEWVA SITNTGSST   
                 47 
               
               
                   
                   FYPDSVKG RFTISRDNAKNSLYLQMNSLRAEDTAVYYC T R DTTIAPFDY WGQGTMVTV 
                   
               
               
                   
                 SS 
                   
               
               
                   
               
               
                 VH V3 
                 EVQLVESGGGLVKPGGSLRLSCAASGFTFS NYWMT WIRQAPGKGLEWVA SITNTGSST   
                 48 
               
               
                   
                   FYPDSVKG RFTISRDNAKNSLYLQMNSLRAEDTAVYYC T R DTTIAPFDY WGQGTMVTV 
                   
               
               
                   
                 SS 
                   
               
               
                   
               
               
                 VH V4 
                 EVQLVESGGGLVKPGGSLRLSCAASGFTFS NYWMT WIRQAPGKGLEWVA SITNTGSST   
                 49 
               
               
                   
                   FYPDSVKG RFTISRDNAKSSLYLQMNSLRAEDTAVYYC T R DTTIAPFDY WGQGTMVTV 
                   
               
               
                   
                 SS 
                   
               
               
                   
               
               
                 VL mouse 
                 DIQMTQSPSFLSASVGDRVTITC KASQNLNEYLN WYQQKLGEAPKRLIY KTNTLQA GI 
                 50 
               
               
                   
                 PSRFSGSGSGIDYTLTISSLQPEDVATYFC SQYNSGNT FGAGTKLELK 
                   
               
               
                   
               
               
                 VL V0 
                 DIQMTQSPSSLSASVGDRVTITC KASQNLNEYLN WYQQKPGKAPKRLIY KTNTLQA GV 
                 51 
               
               
                   
                 PSRFSGSGSGTDFTLTISSLQPEDFATYYC SQYNSGNT FGQGTKLEIK 
                   
               
               
                   
               
               
                 VL V1 
                 DIQMTQSPSSLSASVGDRVTITC KASQNLNEYLN WYQQKPGKAPKRLIY KTNTLQA GV 
                 52 
               
               
                   
                 PSRFSGSGSGTDYTLTISSLQPEDFATYYC SQYNSGNT FGQGTKLEIK 
                   
               
               
                   
               
               
                 VL V2 
                 DIQMTQSPSSLSASVGDRVTITC KASONLNEYLN WYQQKPGKAPKRLIY KTNTLQA G I   
                 53 
               
               
                   
                 PSRFSGSGSGTDYTLTISSLQPEDFATYYC SQYNSGNT FGQGTKLEIK 
                   
               
               
                   
               
               
                 VL V3 
                 DIQMTQSPSSLSASVGDRVTITC KASQNLNEYLN WYQQKPGKAPKRLIY KTNTLQA G I   
                 54 
               
               
                   
                 PSRFSGSGSGIDYTLTISSLQPEDFATYYC SQYNSGNT FGQGTKLEIK 
                   
               
               
                   
               
               
                 VL V4 
                 DIQMTQSPSSLSASVGDRVTITC KASQNLNEYLN WYQQKLGEAPKRLIY KTNTLQA G I   
                 55 
               
               
                   
                 PSRFSGSGSGIDYTLTISSLQPEDFATYFC SQYNSGNT FGQGTKLEIK 
               
               
                   
               
            
           
         
       
     
     The genes were cloned in pcDNA 3.4 vector and transfected into 293F cells. The antibodies were produced according to the following table. 
     The humanized VH and VL genes were produced synthetically and then respectively cloned into vectors containing the human gamma 1 and human kappa constant domains. The pairing of the human VH and the human VL created the 41 humanized antibodies (see Table 5). 
     
       
         
           
               
             
               
                 TABLE 5 
               
               
                   
               
               
                 Humanized antibodies with their VH an VL regions 
               
               
                   
               
             
            
               
                 A. 47C6A3 
               
            
           
           
               
               
               
               
               
            
               
                 VH VL 
                 47C6A3 VL 
                 47C6A3 VL-V0 
                   
                   
               
               
                   
               
               
                 47C6A3 VH 
                 47C6A3-chimera 
                   
                   
                   
               
               
                 47C6A3 VH.V0 
                   
                 Hu47C6A3-1 
                   
                   
               
               
                 47C6A3 VH.V1 
                   
                 Hu47C6A3-2 
                   
                   
               
               
                 47C6A3 VH.V2 
                   
                 Hu47C6A3-3 
               
               
                   
               
            
           
           
               
            
               
                 B. 67F3G7 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 VH VL 
                 67F3G7 VL.V0 
                 67F3G7 VL.V1 
                 67F3G7 VL.V2 
                 67F3G7 VL.V3 
                 67F3G7 VL.V4 
                 67F3G7 VL 
               
               
                   
               
               
                 67F3G7 VH.V1 
                 Hu 67F3G7-1 
                 Hu 67F3G7-6 
                 Hu 67F3G7-11 
                 Hu 67F3G7-16 
                 Hu 67F3G7-21 
                   
               
               
                 67F3G7 VH.V2 
                 Hu 67F3G7-2 
                 Hu 67F3G7-7 
                 Hu 67F3G7-12 
                 Hu 67F3G7-17 
                 Hu 67F3G7-22 
                   
               
               
                 67F3G7 VH.V3 
                 Hu 67F3G7-3 
                 Hu 67F3G7-8 
                 Hu 67F3G7-13 
                 Hu 67F3G7-18 
                   
                   
               
               
                 67F3G7 VH.V4 
                 Hu 67F3G7-4 
                 Hu 67F3G7-9 
                 Hu 67F3G7-14 
                 Hu 67F3G7-19 
                   
                   
               
               
                 67F3G7 VH.V5 
                 Hu 67F3G7-5 
                 Hu 67F3G7-10 
                 Hu 67F3G7-15 
                 Hu 67F3G7-20 
                   
                   
               
               
                 67F3G7 VH 
                   
                   
                   
                   
                   
                 67F3G7- 
               
               
                   
                   
                   
                   
                   
                   
                 chimera 
               
               
                   
               
            
           
           
               
            
               
                 C. 89C10H8 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 VH VL 
                 89C10H8 VL.V1 
                 89C10H8 VL.V2 
                 89C10H8 VL.V3 
                 89C10H8 VL.V4 
                 89C10H8 VL 
                   
               
               
                   
               
               
                 89C10H8 VH.V1 
                 Hu 89C10H8-1 
                 Hu 89C10H8-5 
                 Hu 89C10H8-9 
                 Hu 89C10H8-13 
                   
                   
               
               
                 89C10H8 VH.V2 
                 Hu 89C10H8-2 
                 Hu 89C10H8-6 
                 Hu 89C10H8-10 
                 Hu 89C10H8-14 
                   
                   
               
               
                 89C10H8 VH.V3 
                 Hu 89C10H8-3 
                 Hu 89C10H8-7 
                 Hu 89C10H8-11 
                 Hu 89C10H8-15 
                   
                   
               
               
                 89C10H8 VH.V4 
                 Hu 89C10H8-4 
                 Hu 89C10H8-8 
                 Hu 89C10H8-12 
                 Hu 89C10H8-16 
                   
                   
               
               
                 89C10H8 VH 
                   
                   
                   
                   
                 89C10H8- 
                   
               
               
                   
                   
                   
                   
                   
                 chimera 
               
               
                   
               
            
           
         
       
     
     Example 6: Antigen Binding Properties of the Humanized Antibodies 
     Binding to Recombinant Human PD-L1 
     To evaluate antigen binding activity, the humanized antibodies were subjected to ELISA test. Briefly, microtiter plates were coated with human PD-L1-Fc protein at 0.5 μg/ml in PBS, 100 μl/well at 4° C. overnight, then blocked with 200 μl/well of 1% BSA. Three-fold dilutions of humanized antibodies starting from 10 μg/ml were added to each well and incubated for 1 hour at 37° C. The plates were washed with PBS/Tween and then incubate with mouse-anti-human IgG Fab antibody conjugated with Horse Radish Peroxidase (HRP) for 1 hour at 37° C. After washing, the plates were developed with TMB substrate and analyzed by spectrophotometer at OD 450 nm. As shown in  FIG. 6 , all the humanized antibodies showed comparable binding efficacy to human PD-L1 to the chimeric antibody. 
     To explore the binding kinetics of the humanized antibody, this example performed the affinity ranking by using Biacore®. As shown in Table 6, Hu67F3G7-2, Hu67F3G7-3, Hu67F3G7-5, Hu67F3G7-7, Hu67F3G7-22, Hu89C10H8-4, Hu89C10H8-7, Hu89C10H8-11, and Hu89C10H8-12 showed high affinity, which is comparable to chimeric antibody. 
                     TABLE 6                  Affinity ranking of humanized antibodies                         PDL1-His                             Abs   ka (1/Ms)   kd (1/s)   KD (M)               Hu67F3G7-1   1.855E+6   1.214E−4   6.548E−11       Hu67F3G7-2   2.569E+6   9.748E−5   3.795E−11       Hu67F3G7-3   2.803E+6   9.716E−5   3.466E−11       Hu67F3G7-4   1.883E+6   1.031E−4   5.477E−11       Hu67F3G7-5   2.621E+6   9.711E−5   3.704E−11       Hu67F3G7-6   2.006E+6   1.177E−4   5.867E−11       Hu67F3G7-7   2.687E+6   9.493E−5   3.533E−11       Hu67F3G7-8   2.765E+6   9.844E−5   3.560E−11       Hu67F3G7-9   2.214E+6   1.004E−4   4.535E−11       Hu67F3G7-10   2.383E+6   9.335E−5   3.918E−11       Hu67F3G7-11   5.114E+6   1.643E−4   3.213E−11       Hu67F3G7-12   2.565E+6   9.013E−5   3.513E−11       Hu67F3G7-13   2.798E+6   9.535E−5   3.408E−11       Hu67F3G7-14   2.092E+6   1.086E−4   5.191E−11       Hu67F3G7-15   2.610E+6   9.688E−5   3.712E−11       Hu67F3G7-16   1.478E+6   1.114E−4   7.537E−11       Hu67F3G7-17   2.741E+6   1.014E−4   3.698E−11       Hu67F3G7-18   2.876E+6   9.511E−5   3.307E−11       Hu67F3G7-19   1.970E+6   1.074E−4   5.451E−11       Hu67F3G7-20   2.698E+6   9.363E−5   3.471E−11       Hu67F3G7-21   5.399E+5   8.425E−5   1.560E−10       Hu67F3G7-22   1.669E+6   7.261E−5   4.351E−11       Hu89C10H8-1   7.541E+5   4.654E−4   6.172E−10       Hu89C10H8-2   6.411E+5   8.506E−5   1.327E−10       Hu89C10H8-3   7.375E+5   5.793E−5   7.855E−11       Hu89C10H8-4   6.230E+5   5.944E−5   9.542E−11       Hu89C10H8-5   6.707E+5   4.939E−4   7.364E−10       Hu89C10H8-6   7.010E+5   8.560E−5   1.221E−10       Hu89C10H8-7   6.150E+5   6.613E−5   1.075E−10       Hu89C10H8-8   6.550E+5   6.663E−5   1.017E−10       Hu89C10H8-9   6.712E+5   4.784E−4   7.127E−10       Hu89C10H8-10   5.950E+5   8.988E−5   1.511E−10       Hu89C10H8-11   7.001E+5   6.732E−5   9.615E−11       Hu89C10H8-12   7.269E+5   7.036E−5   9.680E−11       Hu89C10H8-13   7.644E+5   4.327E−4   5.611E−10       Hu89C10H8-14   7.125E+5   9.752E−5   1.369E−10       Hu89C10H8-15   7.814E+5   7.170E−5   9.176E−11       Hu89C10H8-16   6.671E+5   6.537E−5   9.799E−11                    
Binding to Human PD-L1 Overexpressed on Mammalian Cells
 
     To evaluate the antigen binding property, the humanized antibodies were analyzed for their binding to PD-L1 overexpressed on mammalian cells by FACS. Briefly, PDL1-CHOK1 cells were firstly incubated with 3-fold serious diluted humanized antibodies starting at 15 μg/ml at 4° C. for 40 mins. After wash by PBS, the Alexa Fluor® 647 AffiniPure Goat Anti-Human IgG (H+L) antibody was added to each well and incubated at 4° C. for 30 mins. The MFI of Alexa Fluor® 647 were evaluated by FACSCanto. As shown in  FIG. 7 , all the humanized antibodies can high efficiently bind to PD-L1 expressed on mammalian cells. 
     Full Kinetic Affinity of Humanized Antibodies by Biacore® 
     The binding of the humanized antibodies to recombinant PD-L1 protein (human PD-L1-his tag) was tested by Biacore® using a capture method. Hu47C6A3-1, Hu47C6A3-2, Hu47C6A3-3, Hu67F3G7-2, Hu67F3G7-3, Hu67F3G7-5, Hu67F3G7-7, Hu67F3G7-22, Hu89C10H8-4, Hu89C10H8-7, Hu89C10H8-11, and Hu89C10H8-12 mAbs were captured using Protein A chip. A serial dilution of human PD-L1-his tag protein was injected over captured antibody for 2 mins at a flow rate of 30 μl/min. The antigen was allowed to dissociate for 1500 s. All the experiment were carried out on a Biacore T200. Data analysis was carried out using Biacore T200 evaluation software and is shown in Table 7 below. 
     
       
         
           
               
             
               
                 TABLE 7 
               
             
            
               
                   
               
               
                 Affinity by Biacore 
               
            
           
           
               
               
            
               
                   
                 PDL1-His 
               
            
           
           
               
               
               
               
            
               
                 Abs 
                 ka (1/Ms) 
                 kd (1/s) 
                 KD (M) 
               
               
                   
               
               
                 HSP209-chi 
                 1.851E+6 
                 6.785E−4 
                 3.665E−10 
               
               
                 Hu47C6A3-1 
                 1.235E+6 
                 5.933E−4 
                 4.805E−10 
               
               
                 Hu47C6A3-2 
                 2.451E+6 
                 9.000E−4 
                 3.672E−10 
               
               
                 Hu47C6A3-3 
                 2.176E+6 
                 9.661E−4 
                 4.440E−10 
               
               
                 Hu67F3G7-2 
                 1.915E+6 
                 7.032E−5 
                 3.672E−11 
               
               
                 Hu67F3G7-3 
                 2.088E+6 
                 7.737E−5 
                 3.706E−11 
               
               
                 Hu67F3G7-5 
                 1.838E+6 
                 7.920E−5 
                 4.310E−11 
               
               
                 Hu67F3G7-7 
                 2.211E+6 
                 7.252E−5 
                 3.280E−11 
               
               
                 Hu67F3G7-22 
                 1.779E+6 
                 5.791E−5 
                 3.256E−11 
               
               
                 Hu89C10H8-4 
                 5.623E+5 
                 5.408E−5 
                 9.618E−11 
               
               
                 Hu89C10H8-7 
                 5.685E+5 
                 5.313E−5 
                 9.346E−11 
               
               
                 Hu89C10H8-11 
                 5.640E+5 
                 5.542E−5 
                 9.826E−11 
               
               
                 Hu89C10H8-12 
                 5.867E+5 
                 5.587E−5 
                 9.522E−11 
               
               
                   
               
            
           
         
       
     
     Example 7: Blockade the Binding of PDL1 to PD1 by Humanized Antibodies 
     Receptor Blocking Assay by Using Recombinant Human PD-L1 
     There are two receptors, PD-1 and CD80, for human PD-L1. To explore the blocking property of humanized PD-L1 antibody to these two proteins, protein based receptor blocking assay was employed here. 
     Briefly, microtiter plates were coated with human PD-L1-Fc protein at 0.5 μg/ml in PBS, 100 μl/well at 4° C. overnight, then blocked with 150 μl/well of 1% BSA at 37° C. for 2 hours. 50 μl biotin-labeled human PD-1-Fc or CD80-Fc protein and 3-fold dilutions of PD-L1 antibodies starting from 10 μg/ml at 50 μl were added to each well and incubated for 1 hour at 37° C. The plates were washed with PBS/Tween and then incubated with Streptavidin-HRP for 10 mins at 37° C. After washing, the plates were developed with TMB substrate and analyzed by spectrophotometer at OD 450 nm. As shown in  FIG. 8 , Hu47C6A3-1, Hu47C6A3-2, Hu47C6A3-3, Hu67F3G7-2, Hu67F3G7-3, Hu67F3G7-5, Hu67F3G7-7, Hu67F3G7-22, Hu89C10H8-4, Hu89C10H8-7, Hu89C10H8-11, and Hu89C10H8-12 efficiently inhibited the binding of human PD-L1 to human PD1. Moreover, Hu47C6A3-1, Hu47C6A3-2, Hu47C6A3-3, Hu67F3G7-2, Hu67F3G7-3, Hu67F3G7-5, Hu67F3G7-7, Hu67F3G7-22, Hu89C10H8-4, Hu89C10H8-7, Hu89C10H8-11, and Hu89C10H8-12 efficiently inhibited the binding of human PD-L1 to human B7-1 dose dependently ( FIG. 9 ). 
     Example 8. Bifunctional Protein Targeting for Both PD-L1 and TGF-β Pathways 
     Bifunctional recombinant anti-PD-L1 antibody and TGF-β RII fusion proteins were prepared and tested in this example. 
     The light chain of the molecule is the light chain of an anti-PDL1 mAb. The heavy chain is a fusion of the heavy chain of the anti-PDL1 mAb, via a flexible (Gly 4 Ser) 4 Gly linker, to the N-terminus of the soluble extracellular domain of TGF-β RII. At the fusion junction, the C-terminal lysine residue of the antibody heavy chain was mutated to alanine to reduce potential proteolytic cleavage. 
     In some examples, potential modification sites in the CDRs were mutated to similar amino acids. The sequences of the anti-PD-L1 portion are shown in Table 8 below. 
     
       
         
           
               
             
               
                 TABLE 8 
               
             
            
               
                   
               
               
                 Sequences of the variable regions of antibody portion in  
               
               
                 the bifunctional molecules 
               
            
           
           
               
               
               
            
               
                   
                   
                 SEQ ID 
               
               
                 Name 
                 Sequence 
                 NO: 
               
               
                   
               
               
                 
                   02 VH 
                 
                 EVQLVQSGAEVKKPGASVKVSCKASGYTFT   DFWVS   WVRQAPGQGLEWMG   E     
                 34 
               
               
                 (67F3G7 VH 
                     IYPNSGVSRYNEKFKG   RVTMTVDKSISTAYMELSRLRSDDTAVYYCTK   YF     
                   
               
               
                 V2) 
                     GYTYWFGY   WGQGTLVTVSS 
                   
               
               
                   
               
               
                 
                   02 VL 
                 
                 DIQMTQSPSSLSASVGDRVTITC   RASKSVSTYMH   WYQQKPGKQPKLLIYS 
                 43 
               
               
                 (67F3G7 VL 
                     ASHLES   GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC   QQSNELPVT   FGG 
                   
               
               
                 V4) 
                 GTKVEIK 
                   
               
               
                   
               
               
                 
                   06 VH 
                 
                 EVQLVESGGGLVKPGGSLRLSCAASGFTFS   NYWMT   WIRQAPGKGLEWVA   S     
                 48 
               
               
                 (89C10H8 VH 
                     ITNTGSSTFYPDSVKG   RFTISRDNAKNSLYLQMNSLRAEDTAVYYCTR   DT     
                   
               
               
                 V3) 
                     TIA PFDY WGQGTMVTVSS 
                   
               
               
                   
               
               
                 
                   06 VL 
                 
                 DIQMTQSPSSLSASVGDRVTITC   KASQNLNEYLN   WYQQKPGKAPKRLIY   K     
                 53 
               
               
                 (89C10H8 
                     TNTLQA   GIPSRFSGSGSGTDYTLTISSLQPEDFATYYC   SQYNSGNT   FGQ 
                   
               
               
                 VLV2) 
                 GTKLEIK 
                   
               
               
                   
               
               
                 
                   06a VH 
                 
                 EVQLVESGGGLVKPGGSLRLSCAASGFTFS   NYWMTW   IRQAPGKGLEWVA   S     
                 48 
               
               
                 (89C10H8 VH 
                     ITNTGSSTFYPDSVKG   RFTISRDNAKNSLYLQMNSLRAEDTAVYYCTR   DT     
                   
               
               
                 V3) 
                     TIA PFDY WGQGTMVTVSS 
                   
               
               
                   
               
               
                 
                   06a VL 
                 
                 DIQMTQSPSSLSASVGDRVTITC   KASQNLNEYLN   WYQQKPGKAPKRLIY   K     
                 56 
               
               
                   
                     TNTLQA   GIPSRFSGSGSGTDYTLTISSLQPEDFATYYC   SQY         SGN   T F GQ 
                   
               
               
                   
                 GTKLEIK 
                   
               
               
                   
               
               
                 
                   06a-DA VH 
                 
                 EVQLVESGGGLVKPGGSLRLSCAASGFTFS   NYWMT   WIRQAPGKGLEWVA   S     
                 57 
               
               
                   
                    RFTISRDNAKNSLYLQMNSLRAEDTAVYYCTR   DT     
                   
               
               
                   
                     TIAPFDY   WGQGTMVTVSS 
                   
               
               
                   
               
               
                 
                   06a-ES VH 
                 
                 EVQLVESGGGLVKPGGSLRLSCAASGFTFS   NYWMT   WIRQAPGKGLEWVA   S     
                 58 
               
               
                   
                    RFTISRDNAKNSLYLQMNSLRAEDTAVYYCTR   DT     
                   
               
               
                   
                     TIAPFDY   WGQGTMVTVSS 
                   
               
               
                   
               
               
                 
                   89C10H8 
                 
                 SITNTGSSTFYP   DA   VKG 
                 91 
               
               
                 
                   CDRH2 DA 
                 
                   
                   
               
               
                   
               
               
                 
                   89C10H8 
                 
                 SITNTGSSTFYP   ES   VKG 
                 92 
               
               
                 
                   CDRH2 ES 
                 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 9 
               
             
            
               
                   
               
               
                 VH/VL of the bifunctional molecules 
               
            
           
           
               
               
               
               
            
               
                   
                 Bifunctional molecule 
                 VH 
                 VL 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 LP008-02 
                 02  
                 VH 
                 02  
                 VL 
               
               
                   
                 LP008-06 
                 06  
                 VH 
                 06  
                 VL 
               
               
                   
                 LP008-06a 
                 06a 
                 VH 
                 06a  
                 VL 
               
               
                   
                 LP008-06a-DA 
                 06a-DA 
                 VH 
                 06a  
                 VL 
               
               
                   
                 LP008-06a-ES 
                 06a-ES 
                 VH 
                 06a 
                 VL 
               
               
                   
                   
               
            
           
         
       
     
     Besides the VH, the heavy chain of the bifunctional molecule further includes constant regions (with C-terminal K mutated to A), the (Gly 4 Ser) 4 Gly linker, and the N-terminus of the soluble extracellular domain of TGF-β RII. Their sequences are shown in Table 10. 
     
       
         
           
               
             
               
                 TABLE 10 
               
             
            
               
                   
               
               
                 Additional sequences of the heavy chain, and entire heavy/ 
               
               
                 light chains 
               
            
           
           
               
               
               
            
               
                   
                   
                 SEQ ID 
               
               
                 Name 
                 Sequence 
                 NO: 
               
               
                   
               
               
                 Heavy chain 
                 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV 
                 59 
               
               
                 constant 
                 HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEP 
                   
               
               
                 regions 
                 KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDILMISRTPEVICVVVDVS 
                   
               
               
                   
                 HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK 
                   
               
               
                   
                 EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC 
                   
               
               
                   
                 LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW 
                   
               
               
                   
                 QQGNVFSCSVMHEALHNHYTQKSLSLSPGA 
                   
               
               
                   
               
               
                 (Gly 4 Ser) 4 Gly 
                 GGGGSGGGGSGGGGSGGGGSG 
                 60 
               
               
                 linker 
                   
                   
               
               
                   
               
               
                 TGF-β RH 
                 IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSI 
                 61 
               
               
                 extracellular 
                 TSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIM 
                   
               
               
                 domain 
                 KEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD 
                   
               
               
                   
               
               
                 LP008-02 
                 EVQLVQSGAEVKKPGASVKVSCKASGYTFTDFWVSWVRQAPGQGLEWMGE 
                 62 
               
               
                 heavy chain 
                 IYPNSGVSRYNEKFKGRVTMTVDKSISTAYMELSRLRSDDTAVYYCTKYF 
                   
               
               
                   
                 GYTYWFGYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD 
                   
               
               
                   
                 YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY 
                   
               
               
                   
                 ICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK 
                   
               
               
                   
                 DILMISRTPEVICVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS 
                   
               
               
                   
                 TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV 
                   
               
               
                   
                 YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL 
                   
               
               
                   
                 DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAG 
                   
               
               
                   
                 GGGSGGGGSGGGGSGGGGSGIPPHVQKSVNNDMIVTDNNGAVKFPQLCKF 
                   
               
               
                   
                 CDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHD 
                   
               
               
                   
                 PKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEY 
                   
               
               
                   
                 NTSNPD 
                   
               
               
                   
               
               
                 LP008-06 
                 EVQLVESGGGLVKPGGSLRLSCAASGFTFSNYWMTWIRQAPGKGLEWVAS 
                 63 
               
               
                 heavy chain 
                 ITNTGSSTFYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCTRDT 
                   
               
               
                   
                 TIAPFDYWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY 
                   
               
               
                   
                 FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI 
                   
               
               
                   
                 CNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKD 
                   
               
               
                   
                 TLMISRTPEVICVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST 
                   
               
               
                   
                 YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY 
                   
               
               
                   
                 TLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD 
                   
               
               
                   
                 SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAGG 
                   
               
               
                   
                 GGSGGGGSGGGGSGGGGSGIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFC 
                   
               
               
                   
                 DVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDP 
                   
               
               
                   
                 KLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYN 
                   
               
               
                   
                 TSNPD 
                   
               
               
                   
               
               
                 LP008-06a 
                 EVQLVESGGGLVKPGGSLRLSCAASGFTFSNYWMTWIRQAPGKGLEWVAS 
                 64 
               
               
                 heavy chain 
                 ITNTGSSTFYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCTRDT 
                   
               
               
                   
                 TIAPFDYWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY 
                   
               
               
                   
                 FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI 
                   
               
               
                   
                 CNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKD 
                   
               
               
                   
                 TLMISRTPEVICVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST 
                   
               
               
                   
                 YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY 
                   
               
               
                   
                 TLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD 
                   
               
               
                   
                 SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAGG 
                   
               
               
                   
                 GGSGGGGSGGGGSGGGGSGIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFC 
                   
               
               
                   
                 DVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDP 
                   
               
               
                   
                 KLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYN 
                   
               
               
                   
                 TSNPD 
                   
               
               
                   
               
               
                 LP008-06a- 
                 EVQLVESGGGLVKPGGSLRLSCAASGFTFSNYWMTWIRQAPGKGLEWVAS 
                 65 
               
               
                 DA heavy 
                 ITNTGSSTFYPDAVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCTRDT 
                   
               
               
                 chain 
                 TIAPFDYWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY 
                   
               
               
                   
                 FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI 
                   
               
               
                   
                 CNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKD 
                   
               
               
                   
                 TLMISRTPEVICVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST 
                   
               
               
                   
                 YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY 
                   
               
               
                   
                 TLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD 
                   
               
               
                   
                 SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAGG 
                   
               
               
                   
                 GGSGGGGSGGGGSGGGGSGIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFC 
                   
               
               
                   
                 DVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDP 
                   
               
               
                   
                 KLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYN 
                   
               
               
                   
                 TSNPD 
                   
               
               
                   
               
               
                 LP008-06a- 
                 EVQLVESGGGLVKPGGSLRLSCAASGFTFSNYWMTWIRQAPGKGLEWVAS 
                 66 
               
               
                 ES heavy 
                 ITNTGSSTFYPESVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCTRDT 
                   
               
               
                 chain 
                 TIAPFDYWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY 
                   
               
               
                   
                 FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI 
                   
               
               
                   
                 CNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKD 
                   
               
               
                   
                 TLMISRTPEVICVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST 
                   
               
               
                   
                 YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY 
                   
               
               
                   
                 TLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD 
                   
               
               
                   
                 SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAGG 
                   
               
               
                   
                 GGSGGGGSGGGGSGGGGSGIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFC 
                   
               
               
                   
                 DVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDP 
                   
               
               
                   
                 KLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYN 
                   
               
               
                   
                 TSNPD 
                   
               
               
                   
               
               
                 LP008-02 
                 DIQMTQSPSSLSASVGDRVTITCRASKSVSTYMHWYQQKPGKQPKLLIYS 
                 67 
               
               
                 light chain 
                 ASHLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSNELPVTFGG 
                   
               
               
                   
                 GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV 
                   
               
               
                   
                 DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG 
                   
               
               
                   
                 LSSPVTKSFNRGEC 
                   
               
               
                   
               
               
                 LP008-06 
                 DIQMTQSPSSLSASVGDRVTITCKASQNLNEYLNWYQQKPGKAPKRLIYK 
                 68 
               
               
                 light chain 
                 TNTLQAGIPSRFSGSGSGTDYTLTISSLQPEDFATYYCSQYNSGNTFGQ 
                   
               
               
                   
                 GTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV 
                   
               
               
                   
                 DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG 
                   
               
               
                   
                 LSSPVTKSFNRGEC 
                   
               
               
                   
               
               
                 LP008-06a 
                 DIQMTQSPSSLSASVGDRVTITCKASQNLNEYLNWYQQKPGKAPKRLIYK 
                 69 
               
               
                 (&amp; DA/ES) 
                 TNTLQAGIPSRFSGSGSGTDYTLTISSLQPEDFATYYCSQYQS-GNTFGQ 
                   
               
               
                 light chain 
                 GTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV 
                   
               
               
                   
                 DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG 
                   
               
               
                   
                 LSSPVTKSFNRGEC 
               
               
                   
               
            
           
         
       
     
     Example 9: The Binding Affinity of the Bifunctional Molecules 
     The binding of the LP008-06, LP008-06a, LP008-06a-DA, and LP008-06a-ES bifunctional molecules to recombinant PD-L1 protein (human PD-L1-his tag) was tested with Biacore® using a capture method. 
     The bifunctional molecules were captured using Protein A chip. A serial dilution of human PD-L1-his tag protein was injected over captured antibody for 2 mins at a flow rate of 30 μl/min. The antigen was allowed to dissociate for 1500 s. All the experiments were carried out on a Biacore® T200. Data analysis was carried out using Biacore® T200 evaluation software. The results are shown in  FIG. 10  and Table 11 below. 
     
       
         
           
               
             
               
                 TABLE 11 
               
             
            
               
                   
               
               
                 Affinity testing by Biacore ® 
               
            
           
           
               
               
            
               
                   
                 Human PD-L1 
               
            
           
           
               
               
               
               
            
               
                 Abs 
                 ka (1/Ms) 
                 kd (1/s) 
                 KD (M) 
               
               
                   
               
               
                 LP008-06 
                 7.832E+5 
                 5.918E−5 
                 7.556E−11 
               
               
                 LP008-06a 
                 6.865E+5 
                 5.276E−5 
                 7.684E−11 
               
               
                 LP008-06a-DA 
                 6.613E+5 
                 4.795E−5 
                 7.252E−11 
               
               
                 LP008-06a-ES 
                 8.404E+5 
                 5.300E−5 
                 6.307E−11 
               
               
                   
               
            
           
         
       
     
     The binding of the LP008-02 to recombinant PD-L1 protein and human TGF-01 was tested with Biacore® using a capture method. 
     LP008-02 was captured using Protein A chip. A serial dilution of human PD-L1-his tag protein and human TGF-01 was injected over captured antibody for 2 mins at a flow rate of 30 μl/min. PD-L1 was allowed to dissociate for 680 s, and TGF-01 was allowed to dissociate for 1000 s. All the experiments were carried out on a Biacore T200. Data analysis was carried out using Biacore T200 evaluation software. The result are shown in  FIG. 11  and Table 12 below. 
     
       
         
           
               
             
               
                 TABLE 12 
               
             
            
               
                   
               
               
                 Affinity testing by Biacore ® 
               
            
           
           
               
               
               
            
               
                   
                   
                 LP008-02 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                 ka (1/Ms) 
                 kd (1/s) 
                 KD (M) 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 PD-L1 
                 2.528E+6 
                 1.044E−4 
                 4.130E−11 
               
               
                   
                 TGF-β1 
                 1.294E+8 
                 2.716E−4 
                 2.099E−12 
               
               
                   
                   
               
            
           
         
       
     
     Example 10: Functional Assay for PD-1/PD-L1 Blockade 
     The activities of the bifunctional molecules in blocking PD1/PD-L1 interaction were measured with a bioluminescent cell-based assay in this example. 
     In this assay, when PD1 effector cells and PD-L1 target cells are co-cultured, the PD-1/PD-L1 interaction inhibits TCR signaling and NFAT-RE-mediated luminescence. Addition of either an anti-PD-1 or anti-PD-L1 antibody that blocks the PD-1/PD-L1 interaction releases the inhibitory signal and results in TCR activation and NFAT-RE-mediated luminescence. 
     As shown in  FIG. 12 , LP008-02 and LP008-06a-ES blockaded PD1 and PD-L1 interaction with considerably higher activity than M7824 (M7824 EC 50 =0.8504 nM, LP008-02 EC 50 =0.3630 nM, LP008-06a-ES EC 50 =0.4553 nM). 
     Example 11: Functional Assay for TGF-β 
     This example used luciferase assay to evaluate the effect of LP008-02 and LP008-06a-ES on canonical TGF-β signaling. 
     Serial dilutions of M7824 (bifunctional anti-PD-L1/TGFβ Trap fusion protein, see, e.g., Knudson et al.,  Oncoimmunology.  2018; 7(5): e1426519), LP008-02 or LP008-06a-ES were incubated with SMAD luciferase reporter-transfected 293 cells for about 20 hours in the presence of recombinant human TGF-β. 
     As shown in  FIG. 13 , M7824, LP008-02 and LP008-06a-ES blocked TGF-β canonical signaling (IC50=0.06687 nM, IC50=0.07352 nM, IC50=0.07167 nM) in a TGF-β SMAD luciferase reporter assay in transfected 293 cells. 
     Example 12: Binding Activity to Human PD-L1 
     ELISA with Recombinant Human PD-L1 
     To evaluate the binding activity of M7824, LP008-02, and LP008-06a-ES, the bifunctional molecules were subjected to ELISA test. 
     Briefly, microtiter plates were coated with human PD-L1-His protein at 0.5 μg/ml in PBS, 100 μl/well at 4° C. overnight, then blocked with 150 μl/well of 1% BSA. Three-fold dilutions of M7824, LP008-02, and LP008-06a-ES starting from 1 μg/ml were added to each well and incubated for 1 hour at 37° C. The plates were washed with PBS/Tween and then incubate with Goat-anti-human IgG antibody conjugated with Horse Radish Peroxidase (HRP) for 30 mins at 37° C. After washing, the plates were developed with TMB substrate and analyzed by spectrophotometer at OD 450 nm. 
     As shown in  FIG. 14 , LP008-02 and LP008-06a-ES bound to human PD-L1 with considerably higher activity than M7824 (EC 50 =11.82 ng/ml, and EC 50 =14.36 ng/ml vs. EC 50 =23.68 ng/ml). 
     Cross Species Activity 
     To evaluate the binding of bispecific antibodies to Mouse PD-L1, Rat PD-L1, Cynomolgus PD-L1, the antibodies were tested with ELISA. 
     Briefly, microtiter plates were coated with mouse, rat and cynomolgus PD-L1 protein at 0.5 μg/ml in PBS, 100 μl/well at 4° C. overnight, then blocked with 150 μl/well of 1% BSA. Three-fold dilutions of bispecific antibodies starting from 1 μg/ml were added to each well and incubated for 1 hour at 37° C. The plates were washed with PBS/Tween and then incubate with Goat-anti-human IgG antibody conjugated with Horse Radish Peroxidase (HRP) for 30 mins at 37° C. After washing, the plates were developed with TMB substrate and analyzed by spectrophotometer at OD 450 nm. 
     LP008-02 and LP008-06a-ES were able to bind to cynomolgus PD-L1 with higher affinity, but only M7824 bound to rat and mouse PD-L1 ( FIG. 15  and Table 13). 
     
       
         
           
               
             
               
                 TABLE 13 
               
             
            
               
                   
               
               
                 Cross species activity of M7824, CZ010-02, and CZ010-06a-ES 
               
            
           
           
               
               
               
               
            
               
                 EC50 
                 Cynomolgus 
                 Rat 
                 Mouse 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 M7824 
                 25.60 
                 ng/ml 
                 14.25 
                 ng/ml 
                 10.60  
                 ng/ml 
               
            
           
           
               
               
               
               
               
            
               
                 LP008-02 
                 7.890 
                 ng/ml 
                 No binding 
                 No binding 
               
               
                 LP008-06a-ES 
                 10.92 
                 ng/ml 
                 No binding 
                 No binding 
               
               
                   
               
            
           
         
       
     
     Example 13: Binding Activity to Human TGF-β 
     ELISA by Using Recombinant Human TGF-β 
     To evaluate the binding activity of M7824, LP008-02, and LP008-06a-ES, these bifunctional molecules were subjected to ELISA test. 
     Briefly, microtiter plates were coated with human TGF-β protein at 1 μg/ml in PBS, 100 μl/well at 4° C. overnight, then blocked with 150 μl/well of 1% BSA. Three-fold dilutions of the M7824, LP008-02, and LP008-06a-ES bifunctional molecules starting from 10 μg/ml were added to each well and incubated for 1 hour at 37° C. The plates were washed with PBS/Tween and then incubate with Goat-anti-human IgG antibody conjugated with Horse Radish Peroxidase (HRP) for 30 mins at 37° C. After washing, the plates were developed with TMB substrate and analyzed by spectrophotometer at OD 450 nm. 
     As shown in  FIG. 16 , all of M7824, LP008-02, and LP008-06a-ES bound to human TGF-β with high activity (EC 50 =43.43 ng/ml, EC 50 =28.58 ng/ml, EC 50 =39.38 ng/ml). 
     Cross Species Activity 
     To evaluate the binding of bispecific antibodies to Mouse TGF-β, Rat TGF-β, Cynomolgus TGF-β, the bifunctional molecules were performed for the ELISA test. 
     Briefly, microtiter plates were coated with mouse, rat and cynomolgus TGF-β protein at 1 μg/ml in PBS, 100 μl/well at 4° C. overnight, then blocked with 150 μl/well of 1% BSA. Three-fold dilutions of bispecific antibodies starting from 10 μg/ml were added to each well and incubated for 1 hour at 37° C. The plates were washed with PBS/Tween and then incubate with Goat-anti-human IgG antibody conjugated with Horse Radish Peroxidase (HRP) for 30 mins at 37° C. After washing, the plates were developed with TMB substrate and analyzed by spectrophotometer at OD 450 nm. 
     All of the tested bifunctional molecules bound to cynomolgus, rat, and mouse TGF-β with high affinity ( FIG. 17  and Table 14). 
     
       
         
           
               
             
               
                 TABLE 14 
               
             
            
               
                   
               
               
                 Cross species activity of M7824, LP008-02, and LP008-06a-ES 
               
            
           
           
               
               
               
               
            
               
                 EC50 
                 Cynomolgus 
                 Rat 
                 Mouse 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 M7824 
                 39.66 
                 ng/ml 
                 123.9 
                 ng/ml 
                 46.17 
                 ng/ml 
               
               
                 LP008-02 
                 34.41 
                 ng/ml 
                 77.84 
                 ng/ml 
                 34.70 
                 ng/ml 
               
               
                 LP008-06a-ES 
                 58.35 
                 ng/ml 
                 120.7 
                 ng/ml 
                 58.26 
                 ng/ml 
               
               
                   
               
            
           
         
       
     
     Example 14: Efficacy in a MC38 Tumor Mouse Model 
     This example used a tumor mouse model to test the in vivo efficacy of the bifunctional molecules. 
     MC38 cells expressing human PD-L1 resuspended in PBS were seeded subcutaneously into right skin of B-hPD-L1 humanized mice at a concentration of 5×10 5  cells in a volume of 0.2 mL. When the average tumor volume reached approximately 50 mm 3 , 24 mice with an appropriate individual tumor volume were selected for the group, and the animals were randomly assigned to 4 experimental groups according to the tumor volume, with 6 animals in each group. On the 3rd days after anti-mCD20 mAbs injection, total human IgG, M7824, LP008-02 and LP008-06a-ES were administered 3 times a week by intraperitoneal injection. The dose was calculated based on the experimental animal&#39;s body weight at 10 μg/g. Mice weight and tumor size were tested twice a week. 
     The results are shown in  FIG. 18 . Bifunctional molecules LP008-02 and LP008-06a-ES exhibited better efficacy than M7824 in these animal models in terms of tumor growth inhibition. Furthermore, animal deaths were observed in both the IgG and M7824 groups, but not in the LP008-02 and LP008-06a-ES groups, demonstrating the greater safety profiles of the new bifunctional molecules. 
     Example 15. Modification of Bifunctional Molecules 
     This example tested certain modified bifunctional molecules (Table 15) for their in vivo efficacy of functional assay for TGF-β. Some of them included a linker sequence of TAGHTQTSTGGGAITTGTSGAGHGP (SEQ ID NO:87), HYP, and/or G4S (SEQ ID NO:86) repeats. These molecules are referred to as LP008-02-1 to LP008-02-7, respectively. 
                     TABLE 15                  Modified sequences design of linker and TGF-β RII                                 SEQ ID       Name   Sequences (not showing the antibody portion)   NO:               1     GGGGSGGGGSGGGGSGGGGSG   I PPHVQKSVNNDMIVTDNNGAVKFP QLCKFCDVRF     79             STCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILE                   DAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFS EEYNTSNPD                   2   GGGG   LCKFCDVRFSTCDNQKSCMSNCSIT     80             SICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPG                   ETFFMCSCSSDECNDNIIFS EEYNTSNPD                   3     GGGGSGGGGSGGGGSGGGGSGGGGS     QLCKFC     81             DVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHD                   FILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFS EEYNTSNPD                   4     GGGGSGGGGSGGGGSGGGGSGGGGS     QLCKFC     82             DVRFSTCDNOKSCMSNCSITSICEKNEVCVAVWRKNDENITLETVCHDPKLPYHD                   FILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFS                     5     SGGGGSGGGGSGGGGSGGGGSGGGGS     QLCKFCDVRFSTCDNQKSCMSNCS IT S     83             ICEKNEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGE                   TFFMCSCSSDECNDNIIFS                     6     SGGGGSGGGGSGGGGSGGGGSGGGGS     QLCKFCDVRFSTCDNQKSCMSNCS IT S     84             ICEKNEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGE                   TFFMCSCSSDECNDNIIFS EEYNTSNPD                   7     GGGGSGGGGSGGGGS     QLCKFCDVRFSTCDNOKSCMSNCSITSICEKNEVCVA     85             VWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDE                   CNDNIIFS EEYNTSNPD                    
ELISA with Recombinant Human TGF-β1
 
     To evaluate the binding activity of modified LP008-02 bifunctional molecules, these bifunctional molecules were tested with ELISA. 
     Briefly, microtiter plates were coated with human TGF-β1 protein (Acro, TG1-H4212) at 1 μg/ml in PBS, 100 μl/well at 4° C. overnight, then blocked with 150 μl/well of 1% BSA. Three-fold dilutions of modified LP008-02 bifunctional molecules starting from 30 nM were added to each well and incubated for 1 hour at 37° C. The plates were washed with PBS/Tween and then incubate with Goat-anti-human IgG (H+L) antibody conjugated with Horse Radish Peroxidase (HRP) for 30 mins at 37° C. After washing, the plates were developed with TMB substrate and analyzed by spectrophotometer at OD 450 nm. 
     As shown in  FIG. 19 , all of modified LP008-02 bifunctional molecules bound to human TGF-β1 with high activity, comparable to LP008-02-1. 
     TGF-β Functional Assay 
     Serial dilutions of modified LP008-02 bifunctional molecules were incubated with SMAD luciferase reporter-transfected 293 cells for about 22 hours in the presence of recombinant human TGF-β1. 
     As shown in  FIG. 20 , LP008-02-2, LP008-02-3, and LP008-02-4 effectively blocked TGF-β canonical signaling (IC50=0.1435 nM, IC50=0.1639 nM, IC50=0.1732 nM) in a TGF-β SMAD luciferase reporter assay in transfected 293 cells, which was compared with LP008-02-1. 
     Example 16. Comparison of the Bifunctional Molecules 
     The molecules 1-7 of Table 15 included different sequences at the N- and C-terminal ends of the ectodomain (SEQ ID NO:72). They were tested for stability and activity to evaluate the impact of these sequences. 
     Molecule 1 included the entire excellular portion of the protein (SEQ ID NO:61), which contained 25 amino acids (IPPHVQKSVNNDMIVTDNNGAVKFP, SEQ ID NO:89, or amino acids 24-48 of isoform B, SEQ ID NO:71) from the N-terminus of the extracellular domain, and the C-terminal fragment (EEYNTSNPD, SEQ ID NO:90). In addition, this molecules added a few G4S (SEQ ID NO:86) repeats in the linker. 
     Molecule 2, compared to Molecule 1, replaced the N-terminal portion (amino acids 24-48 of isoform B, SEQ ID NO:89) of the extracellular domain with an artificial linker TAGHTQTSTGGGAITTGTSGAGHGP (SEQ ID NO:87). This linker was modeled based on SEQ ID NO:89. The changes included (i) removal of the rigid di-peptide PP, (ii) removal of potential cleavage sites QK, N and K, (iii) inclusion of multiple glycine residues to increase flexibility, (iv) partial removal of hydrophobic residues (e.g., retaining only one I). These changes are illustrated in the table below. Molecule 2 also included a single G4S unit at the N-terminus. 
     
       
         
           
               
               
               
            
               
                 Original 
                 IPPHVQKSVNNDMIVTDNNGAVKFP 
                 SEQ ID NO: 89 
               
               
                   
               
               
                 Modified 
                 TAGHTQTSTGGGAITTGTSGAGHGP 
                 SEQ ID NO: 87 
               
            
           
         
       
     
     Molecule 3 included a longer G4S linker than Molecule 2. On top of Molecule 3, Molecule 4 had a deletion of the C-terminal fragment, EEYNTSNPD (SEQ ID NO:90). Molecule 5 replaced the artificial linker, SEQ ID NO:87, with a short linker HYP. Molecules 6 and 7 included different lengths of the G4S linker at the N-terminal side of the HYP linker. 
     The present disclosure is not to be limited in scope by the specific embodiments described which are intended as single illustrations of individual aspects of the disclosure, and any compositions or methods which are functionally equivalent are within the scope of this disclosure. It will be apparent to those skilled in the art that various modifications and variations can be made in the methods and compositions of the present disclosure without departing from the spirit or scope of the disclosure. Thus, it is intended that the present disclosure cover the modifications and variations of this disclosure provided they come within the scope of the appended claims and their equivalents. 
     All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.