Bifunctional molecules targeting pd-l1 and tgf-beta

Abstract

Provided are anti-PD-L1 antibodies with superior activity in blocking the interaction between PD-1 and PD-L1. Also provided are multifunctional molecules comprising the anti-PD-L1 antibodies or fragments thereof fused to the extracellular domain of human TGF-β receptor type 2.

Description

BACKGROUND

[0001] Exciting advances in cancer immunotherapy in recent years have led to a paradigm shift in oncology. The most notable results are T cell-based therapies, including immune checkpoint inhibitors (ICIs), genetically engineered T cells, and bispecific antibodies (BsAbs). T cells are a major class of immune surveillance and tumor eradication with high specificity and long-term memory. However, in the tumor microenvironment, T cells can be exhausted or become tolerant to tumor cells. T cell exhaustion is often 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 receptor.

[0002] Monoclonal antibody (mAb)-based therapies can counteract these checkpoint molecules, thus eliminating the brake that suppresses tumor-infiltrating T cells, resulting in significant clinical benefits in different malignancies. For example, blocking PD-1 / PD-L1 interactions can enhance immune normalization and enhance anti-cancer responses. However, a significant drawback of PD-1 / PD-L1 blockade is the inconsistency in homogenous study populations with similar tumor characteristics. In addition, PD-1 / PD-L1 blockade therapy can also cause certain inflammatory side effects in certain patients. The limitations of PD-1 / PD-L1 blockade monotherapy and the lack of promising alternatives make it necessary to seek combination therapy approaches that can activate anti-tumor immunity and improve therapeutic efficacy.

[0003] 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-beta (TGF-beta) receptor II, which acts as a “trap” for all three TGF-beta subtypes. The PD-L1 portion is based on avelumab, which has been approved for the treatment of Merkel cell carcinoma and urothelial carcinoma. However, current clinical data show that the use of M7824 is associated with undesirable skin growth, with an overall response rate of only 35-40% in a phase II trial in HPV-positive malignancies. Therefore, there is a need for improved therapies.

[0004] SUMMARY

[0005] In some embodiments, the present disclosure provides bifunctional molecules targeting PD-L1 protein and TGF-β. The disclosed PD-L1 targeting unit consists of an anti-PD-L1 antibody fused to the extracellular domain of human transforming growth factor-β (TGF-β) receptor II, which serves as a trap for TGF-β. Experimental data show that these new bifunctional molecules are more effective than the main candidate drug currently under clinical development, M7824.

[0006] Accordingly, according to one embodiment of the present disclosure, there is provided a multifunctional molecule comprising an anti-PD-L1 (Programmed Death-Ligand 1) antibody or fragment thereof and an extracellular domain of human TGF-βRII (TGF-β Receptor Type 2), wherein the anti-PD-L1 antibody or fragment thereof is specific to 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, the VH CDR2, the VH CDR3, the VL CDR1, the VL CDR2, and the VL CDR3 comprise the amino acid sequences of SEQ ID NOs: 7-12, or SEQ ID NOs: 13-18, respectively, or wherein the VH CDR1 comprises SEQ ID NO: 19, the VH CDR2 comprises SEQ ID NO: 20, 91, or 92, the VH CDR3 comprises SEQ ID NO: 21, the VL CDR1 comprises SEQ ID NO: 22, the VL CDR2 comprises SEQ ID NO: 23, and the VL CDR3 comprises SEQ ID NO: 24, or 93, and wherein the extracellular domain of human TGF-βRII comprises the amino acid sequence of SEQ ID NO: 72 and is fused to the anti-PD-L1 antibody or fragment thereof.

[0007] In one embodiment, there is provided an anti-PD-L1 (Programmed Death-Ligand 1) antibody or fragment thereof, which is specific for 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 comprise the amino acid sequences of SEQ ID NOs: 7-12, or SEQ ID NOs: 13-18, respectively, or wherein the VH CDR1 comprises SEQ ID NO: 19, the VH CDR2 comprises SEQ ID NO: 20, 91, or 92, the VH CDR3 comprises SEQ ID NO: 21, the VL CDR1 comprises SEQ ID NO: 22, the VL CDR2 comprises SEQ ID NO: 23, and the VL CDR3 comprises SEQ ID NO: 24, or 93.

[0008] Also provided is a multifunctional molecule comprising an antibody or antigen binding fragment thereof fused to the N-terminus of the amino acid sequence of SEQ ID NO: 72 via a peptide linker, wherein the peptide linker is (a) at least 30 amino acid residues in length, or (b) at least 25 amino acid residues in length and comprises an alpha-helical motif.

[0009] Also provided are uses and methods of treating cancer with any molecule of the present disclosure. BRIEF DESCRIPTION OF DRAWINGS

[0010] Figure 1 It is shown that 47C6A3, 67F3G7, and 89C10H8 are able to bind human PD-L1 with high affinity.

[0011] Figure 2 It is shown that 47C6A3, 67F3G7, and 89C10H8 antibodies can bind effectively to PD-L1 expressed on mammalian cells.

[0012] Figure 3 It is shown that 47C6A3, 67F3G7, and 89C10H8 antibodies can bind cynomolgus monkey PD-L1 with high affinity, but not rat or mouse PD-L1.

[0013] Figure 4 It is shown that 47C6A3, 67F3G7, and 89C10H8 can effectively inhibit the binding of human PD-L1 to human PD1.

[0014] Figure 5 Binding kinetics of 47C6A3, 67F3G7, and 89C10H8 to recombinant PD-L1 are shown.

[0015] Figure 6A -C shows that all the tested humanized antibodies can bind to human PD-L1 with efficiency comparable to the chimeric antibody.

[0016] Figure 7 It shows that the tested humanized antibodies can bind to PD-L1 expressed on mammalian cells with high efficiency, comparable to the chimeric antibody.

[0017] Figure 8A -C shows that some humanized antibodies can effectively inhibit the binding of human PD-L1 to human PD1.

[0018] Figure 9A -C shows that some humanized antibodies can effectively inhibit the binding of human PD-L1 to human CD80.

[0019] Figure 10 The binding kinetics of LP008-06, LP008-06a, LP008-06a-DA and LP008-06a-ES to recombinant human PD-L1 are shown.

[0020] Figure 11 The binding kinetics of LP008-02 to human PD-L1 and human TGF-β1 are shown.

[0021] Figure 12 It shows that LP008-02 and LP008-06a-ES can block PD1 and PD-L1 interaction with higher affinity than M7824.

[0022] Figure 13 It shows that M7824, LP008-02 and LP008-06a-ES can effectively block TGF-β classic signaling.

[0023] Figure 14 It shows that LP008-02 and LP008-06a-ES can bind to human PD-L1 with high affinity.

[0024] Figure 15 It shows that LP008-02 and LP008-06a-ES can bind to cynomolgus monkey PD-L1 with higher affinity, but cannot bind to rat PD-L1 or mouse PD-L1.

[0025] Figure 16 It shows that LP008-02 and LP008-06a-ES can bind to human TGF-β with efficiency comparable to M7824.

[0026] Figure 17Figure 8 shows that LP008-02 and LP008-06a-ES bind cynomolgus TGF-β, mouse TGF-β and rat TGF-β with comparable efficiency as M7824.

[0027] Figure 18A Figure 9 shows the pharmacodynamic effect of LP008-02 and LP008-06a-ES in an animal model.

[0028] Figure 19 Figure 10 shows that all tested modified bifunctional molecules bind human TGF-β with comparable efficiency as LP008-02-1.

[0029] Figure 20 Figure 11 shows that all tested modified bifunctional molecules effectively block TGF-β canonical signaling.

[0030] Figure 21 Figure 12 shows that all tested modified bifunctional molecules bind human TGF-β with comparable efficiency as LP008-02-1.

[0031] Figure 22 Figure 13 shows that all tested modified bifunctional molecules effectively block TGF-β canonical signaling.

[0032] Figure 23 Figure 14 shows that antibodies MPDL3280A, 47C6A3, Hu67F3G7-22 and Hu89C10H8-7 can block PD1 and PD-L1 interaction with high affinity.

[0033] DETAILED DESCRIPTION

[0034] DEFINITIONS

[0035] It should be noted that the terms "a" or "an" entity refers to one or more of that entity; for example, "an antibody," is understood to mean one or more antibodies. As such, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[0036] As used herein, "antibody" or "antigen binding polypeptide" refers to a polypeptide or polypeptide complex that specifically recognizes and binds an antigen. An antibody can be an intact antibody and any antigen binding fragment or single chain thereof. Thus, the term "antibody" includes any protein or peptide-containing molecule that comprises at least a portion of an immunoglobulin molecule that has the biological activity of binding to an 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 heavy chain variable region, a heavy chain or light chain constant region, a framework (FR) region, or any portion thereof, or at least a portion of a binding protein.

[0037] The terms "antibody fragment" or "antigen binding fragment" as used herein are portions of an antibody, such as F(ab')2, F(ab)2, Fab', Fab, Fv, scFv, and the like. Regardless of structure, the antibody fragment binds to the same antigen recognized by the whole antibody. The term "antibody fragment" includes aptamers, spiegelmers, and diabodies. The term "antibody fragment" also includes any synthetic or genetically engineered protein that functions like an antibody by forming a complex with a particular antigen.

[0038] The term "antibody" encompasses a wide variety of polypeptide classes that can be distinguished biochemically. Those skilled in the art will appreciate that the heavy chains are classified as gamma, mu, alpha, delta, epsilon, some of which are further divided into subclasses or isotypes, e.g., gamma l-gamma 4. It is the nature of this chain that determines the "class" of the antibody as IgG, IgM, IgA, IgG, or IgE. The immunoglobulin subclasses, isotypes, e.g., IgGl, IgG2, IgG3, IgG4, IgG5, etc., are well characterized and are known to impart functional specificity. Given the present disclosure, modifications of each of these classes and isotypes will be readily discernible by those skilled in the art and are therefore all within the scope of the present disclosure. All immunoglobulin classes are clearly within the scope of the present disclosure, and the following discussion will generally be directed to the IgG class of immunoglobulin molecules. With respect to IgG, the standard immunoglobulin molecule comprises two identical light chain polypeptides of molecular mass approximately 23,000 daltons, and two identical heavy chain polypeptides of molecular mass 53,000-70,000. These four chains are typically linked by disulfide bonds in a "Y" configuration, with the light chains encircling the heavy chains, starting at the mouth of the "Y" and continuing through the variable region.

[0039] "Specifically binds" or "has specificity for," generally means that an antibody binds via its antigen binding domain to an epitope, and that such binding requires some complementarity between the antigen binding domain and the epitope. According to this definition, an antibody is said to "specifically bind" to a certain epitope when it binds to that epitope via its antigen binding domain more readily than it binds to a random, unrelated epitope. The term "specificity" is used herein to qualify the relative avidity with which a particular antibody binds to a particular epitope. For example, antibody "A" can be considered to have a higher specificity for a given epitope than antibody "B," or antibody "A" can be considered to bind to epitope "C" with a higher specificity than it does to related epitope "D."

[0040] As used herein, the term "treatment" refers to 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 cancer progression. Beneficial or desired 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 disease or disorder as well as those in which the disease or disorder is to be prevented.

[0041] "Subject" or "individual" or "animal" or "patient" or "mammal" refers to any subject, especially a mammalian subject, in need of diagnosis, prognosis, or treatment. Mammalian subjects include humans, domestic animals, farm animals, zoo animals, sport animals, or pet animals, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cows, dairy cows, and the like.

[0042] As used herein, phrases such as "to a patient in need of treatment" or "a subject in need of treatment" include subjects, such as mammalian subjects, who would benefit from administration of an antibody or composition of the present application, for example, for detection, diagnostic procedures, and / or treatment.

[0043] Multifunctional molecules

[0044] As demonstrated by the appended experimental examples, the present inventors were able to identify a number of bifunctional fusion proteins comprising an anti-PD-L1 unit and a TGF-β targeting unit. For example, as shown in Example 14, both of the tested bifunctional proteins LP008-02 and LP008-06a-ES showed better efficacy than M7824 in the MC38 mouse model. M7824 is a PD-L1 / TGF-β dual targeting fusion protein, which is currently undergoing a phase II clinical trial in HPV-positive malignant tumor patients. The anti-PD-L1 unit of M7824 is based on avelumab, which is a leading PD-L1 antibody, already approved for the treatment of Merkel cell carcinoma and urothelial carcinoma. Thus, the superior performance of the newly disclosed bifunctional proteins compared to M7824 is surprising.

[0045] Furthermore, as shown in Example 12, the bifunctional proteins of the present disclosure have better species specificity. Unlike M7824, which also reacts with mouse and rat PD-L1, the new bifunctional proteins bind only to human and cynomolgus monkey PD-L1 in addition to having superior PD-L1 binding activity.

[0046] Accordingly, in one embodiment, the present disclosure provides a multifunctional molecule having at least an anti-PD-Ll unit and a TGF-β targeting unit. The anti-PD-Ll unit can comprise an anti-PD-Ll antibody or fragment of the present disclosure. The TGF-β targeting unit is preferably an extracellular domain of human transforming growth factor-beta (TGF-β) receptor II (TGF-βRII or TGFBR2).

[0047] TGF-βRII 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). The sequences are provided in Table A below.

[0048] Table A. Sequences related to TGF-βRII (underlined and bold: core ectodomain; underlined and italic: residues different between isoforms; underlined only: mutations)

[0049]

[0050]

[0051] In some embodiments, the TGF-βRII ectodomain includes the core ectodomain (SEQ ID NO: 72) as well as some flanking residues. For example, variant 1 (SEQ ID NO: 61), which was tested in Examples 8-16, includes an additional 25 residues N-terminal to the core ectodomain and nine residues C-terminal to the core ectodomain. Another variant, variant 2 (SEQ ID NO: 73), includes only the nine C-terminal flanking residues. Other variants, such as variants 4-7 (SEQ ID NOs: 75-78), include alternative linkers in place of part of the N-terminal sequence of SEQ ID NO: 61.

[0052] In some embodiments, the TGF-βRII ectodomain 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-βRII ectodomain does not include the last 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acids of SEQ ID NO: 61.

[0053] Another variant - variant 3 is based on variant 1 but includes at least amino acid substitutions of X positions within the N-terminal portion (SEQ ID NO: 88). These X positions are potential glycosylation sites. Thus, the substitutions are made with amino acids other than K, S, and N. Examples of substitutions are R, A, G, Q, I, L, D, or E, but are not limited thereto.

[0054] In some embodiments, the anti-PD-Ll unit consists of an anti-PD-Ll antibody or fragment thereof as further described below. The antibody or fragment can take any antibody form, such as a conventional full IgG form, a Fab fragment, a single chain fragment, or a single domain antibody, but is not limited thereto. When the antibody or fragment thereof has a light chain and a separate heavy chain, the TGF-βRII extracellular domain can be fused to 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-βRII extracellular domain can be fused closer to the light chain or the heavy chain.

[0055] In some embodiments, the TGF-βRII extracellular domain is fused to the N-terminus of a chain of the anti-PD-Ll unit. In some embodiments, the TGF-βRII extracellular domain is fused to the C-terminus of a chain of the anti-PD-Ll unit. In preferred embodiments, the TGF-βRII extracellular domain is fused to the C-terminus of a heavy chain of the anti-PD-Ll unit, optionally through a peptide linker (e.g., SEQ ID NO: 60, or one, two, or three GGGGS (SEQ ID NO: 86) repeats).

[0056] In some embodiments, the anti-PD-Ll unit includes a VH (heavy chain variable region) and a VL (light chain variable region). The VH and VL regions include a VH CDR1, a VH CDR2, a VH CDR3, a VL CDR1, a VL CDR2, and a VL CDR3, such as those shown in Tables 1A-1C.

[0057] 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). Exemplary humanized antibodies include antibodies having a VH of SEQ ID NO: 26, or 27, or 28 and a VL of SEQ ID NO: 30.

[0058] 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). Exemplary humanized antibodies include antibodies having a VH of SEQ ID NO: 34 and a VL of SEQ ID NO: 39, 40, or 43, a VH of SEQ ID NO: 35 and a VL of SEQ ID NO: 39, or a VH of SEQ ID NO: 37 and a 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.

[0059] 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, the VH CDR2 can include SITNTGSSTFYPDAVKG (SEQ ID NO: 91) or SITNTGSSTFYPESVKG (SEQ ID NO: 92). Alternatively, the VL CDR3 can be SQYQSGNT (SEQ ID NO: 93).

[0060] 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. 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), SITNTGSSTFYPDAVKG (SEQ ID NO: 91), DTTIAPFDY (SEQ ID NO: 21), KASQNLNEYLN (SEQ ID NO: 22), KTNTLQA (SEQ ID NO: 23), and SQYNSGNT (SEQ ID NO: 24), respectively. 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), SITNTGSSTFYPESVKG (SEQ ID NO: 92), DTTIAPFDY (SEQ ID NO: 21), KASQNLNEYLN (SEQ ID NO: 22), KTNTLQA (SEQ ID NO: 23), and SQYNSGNT (SEQ ID NO: 24), respectively.

[0061] 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 SQYQSGNT (SEQ ID NO: 93), respectively. 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), SITNTGSSTFYPDAVKG (SEQ ID NO: 91), DTTIAPFDY (SEQ ID NO: 21), KASQNLNEYLN (SEQ ID NO: 22), KTNTLQA (SEQ ID NO: 23), and SQYQSGNT (SEQ ID NO: 93), respectively. 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), SITNTGSSTFYPESVKG (SEQ ID NO: 92), DTTIAPFDY (SEQ ID NO: 21), KASQNLNEYLN (SEQ ID NO: 22), KTNTLQA (SEQ ID NO: 23), and SQYQSGNT (SEQ ID NO: 93), respectively.

[0062] Examples of such VH sequences are provided in SEQ ID NOs: 44 (mouse) and 45-49 (humanized) and 57-58 (humanized). Examples of such VL sequences are provided in SEQ ID NOs: 50 (mouse) and 51-55 (humanized) and 56 (humanized).

[0063] Exemplary humanized antibodies include antibodies having a VH of SEQ ID NO: 49 and a VL of SEQ ID NO: 52 or 54, or antibodies having a VH of SEQ ID NO: 48 and a VL of SEQ ID NO: 53 or 54. In one embodiment, a humanized antibody includes a VH of SEQ ID NO: 48 and a VL of SEQ ID NO: 53. In one embodiment, a humanized antibody includes a VH of SEQ ID NO: 48 and a VL of SEQ ID NO: 56. In one embodiment, a humanized antibody includes a VH of SEQ ID NO: 57 and a VL of SEQ ID NO: 56. In one embodiment, a humanized antibody includes a VH of SEQ ID NO: 58 and a VL of SEQ ID NO: 56.

[0064] In some embodiments, the antibody or fragment thereof further includes a heavy chain constant region (e.g., CH1, CH2, and / or CH3) and / or a light chain constant region (e.g., CL). An exemplary heavy chain constant region is provided in SEQ ID NO: 59, and an exemplary light chain constant region is provided in SEQ ID NO: 67 (residues 108-214).

[0065] TGF-βRII x antibody fusion

[0066] Testing using different fusion protein designs (e.g., Table 15) indicates that only the core extracellular domain of TGF-βRII (SEQ ID NO: 72) is necessary for activity. Furthermore, the extracellular domain of TGF-βRII should not be fused directly to the antibody. There should be sufficient distance, provided by a peptide linker.

[0067] With respect to the extracellular domain, the peptide linker (which can be a fully artificial linker, or include a portion of the extracellular fragment of the N-terminus of the extracellular domain SEQ ID NO: 89) should have a minimum length. If the distance is too short, the stability or activity of the fusion protein is reduced. In some embodiments, the minimum length 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 residues. In some embodiments, the linker is no 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.

[0068] In some embodiments, inclusion of a flexible linker, such as one or more G4S (SEQ ID NO: 86) units, is 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.

[0069] In some embodiments, it is shown (e.g., Example 17) that the native N-terminal fragment (IPPHVQKSVNNDMIVTDNNGAVKFP; SEQ ID NO: 89) can be replaced with an alternative peptide to increase stability without sacrificing or even increasing activity. In some embodiments, the alternative peptide is not identical to SEQ ID NO: 89, but has at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% sequence identity to SEQ ID NO: 99.

[0070] An exemplary alternative peptide is IPPHVQXXVNNDMIVTDNXGAVKFP (SEQ ID NO: 88), where X is any amino acid other than K, S, or N. In some embodiments, the substitution can be made to remove the rigid dipeptide PP, remove 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, does not contain the PP dipeptide, and does not have more than 3 hydrophobic amino acid residues selected from I, L, M, F, V, W, Y, and P. In some embodiments, the variant includes at least 5 G and does not have more than 1 hydrophobic amino acid residue selected from I, L, M, F, V, W, Y, and P.

[0071] In some embodiments, the peptide linker between the antibody or fragment thereof and the extracellular domain of TGF-bRII (SEQ ID NO: 72) includes a flexible linker. In some embodiments, the peptide linker includes an alternative peptide of SEQ ID NO: 89. In some embodiments, the peptide linker includes both a flexible linker and an alternative peptide. In some embodiments, the flexible linker is N-terminal to the alternative peptide. In some embodiments, the flexible linker is C-terminal to the alternative peptide.

[0072] In some embodiments, the multifunctional molecule does not include at least the entire sequence of EEYNTSNPD (SEQ ID NO: 90). The multifunctional molecule can have the entire SEQ ID NO: 90 removed from the extracellular domain of TGF-βRII. 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).

[0073] The antibody or antigen-binding fragment thereof of the multifunctional molecule can target any antigen. Non-limiting examples are PD-1, 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, CD4, CD2, CD8, CD47, and CD73. They can also be any of the antibodies or fragments disclosed herein.

[0074] The extracellular domain of TGF-βRII can be fused to any portion of the antibody or fragment. In some embodiments, the extracellular domain is fused to the C-terminus of the heavy or light chain of the antibody or fragment. In some embodiments, the extracellular domain is fused to the C-terminus of the Fc fragment of the antibody or fragment.

[0075] Anti-PD-L1 antibodies and fragments

[0076] Also provided are anti-PD-L1 antibodies and fragments that can be used as anti-PD-L1 units in multifunctional molecules, bispecific or multispecific antibodies, or alone in monospecific antibodies.

[0077] Exemplary mouse anti-PD-L1 antibodies have been prepared, as well as their humanized and improved antibodies, and tested in the accompanying experimental examples. All mouse antibodies (47C6A3, 67F3G7, and 89C10H8) and their corresponding humanized versions exhibit excellent binding affinity, cross-reactivity, and effectiveness in inhibiting PD-1 / PD-L1 binding.

[0078] Importantly, humanized 67F3G7 and 89C10H8 exhibited higher activity in blocking the interaction between PD-1 and PD-L1 than MPDL3280A (Atezolizumab) compared to MPDL3280A (see, e.g., Example 18). Furthermore, interestingly, all of the tested antibodies of the present disclosure showed lower hydrophobicity and lower viscosity than MPDL3280A. Higher hydrophobicity is known to reduce the solubility of a protein. Likewise, high viscosity is also a barrier for high concentration protein formulation development. Thus, these data suggest that the antibodies of the present invention are more suitable for making high concentration antibody formulations.

[0079] In addition, antigen-binding fragments of the antibodies of the present disclosure were included as a unit in a bifunctional fusion protein that further comprises a TGF-β targeting unit. The resulting bifunctional fusion protein exhibited better efficacy than M7824 in the MC38 mouse model. M7824 is a PD-L1 / TGF-β dual targeting fusion protein that is currently in Phase II clinical trial in HPV-positive malignancies. The anti-PD-L1 unit of M7824 is based on Avelumab, which is a leading PD-L1 antibody that has been approved for the treatment of Merkel cell carcinoma and urothelial carcinoma. Thus, these data demonstrate the unique advantage of the antibodies of the present disclosure in making bifunctional or multifunctional molecules.

[0080] In some embodiments, the anti-PD-L1 antibody or fragment comprises a VH (heavy chain variable region) and a VL (light chain variable region). The VH and VL regions comprise a VH CDR1, a VH CDR2, a VH CDR3, a VL CDR1, a VL CDR2, and a VL CDR3, e.g., as shown in Tables 1A-1C.

[0081] 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 NOs: 25 (mouse) and 26-28 (humanized). Examples of such VL sequences are provided in SEQ ID NOs: 29 (mouse) and 30 (humanized). An exemplary humanized antibody includes an antibody having a VH of SEQ ID NO: 26, or 27, or 28, and a VL of SEQ ID NO: 30.

[0082] 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 NOs: 31 (mouse) and 32-37 (humanized). Examples of such VL sequences are provided in SEQ ID NOs: 38 (mouse) and 39-43 (humanized). Exemplary humanized antibodies include antibodies having a VH of SEQ ID NO: 34 and a VL of SEQ ID NOs: 39, 40, or 43, a VH of SEQ ID NO: 35 and a VL of SEQ ID NO: 39, or a VH of SEQ ID NO: 37 and a 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.

[0083] 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, the VH CDR2 can include SITNTGSSTFYPDAVKG (SEQ ID NO: 91) or SITNTGSSTFYPESVKG (SEQ ID NO: 92). Alternatively, the VL CDR3 can be SQYQSGNT (SEQ ID NO: 93).

[0084] 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. 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), SITNTGSSTFYPDAVKG (SEQ ID NO: 91), DTTIAPFDY (SEQ ID NO: 21), KASQNLNEYLN (SEQ ID NO: 22), KTNTLQA (SEQ ID NO: 23), and SQYNSGNT (SEQ ID NO: 24), respectively. 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), SITNTGSSTFYPESVKG (SEQ ID NO: 92), DTTIAPFDY (SEQ ID NO: 21), KASQNLNEYLN (SEQ ID NO: 22), KTNTLQA (SEQ ID NO: 23), and SQYNSGNT (SEQ ID NO: 24), respectively.

[0085] 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 SQYQSGNT (SEQ ID NO: 93), respectively. 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), SITNTGSSTFYPDAVKG (SEQ ID NO: 91), DTTIAPFDY (SEQ ID NO: 21), KASQNLNEYLN (SEQ ID NO: 22), KTNTLQA (SEQ ID NO: 23), and SQYQSGNT (SEQ ID NO: 93), respectively. 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), SITNTGSSTFYPESVKG (SEQ ID NO: 92), DTTIAPFDY (SEQ ID NO: 21), KASQNLNEYLN (SEQ ID NO: 22), KTNTLQA (SEQ ID NO: 23), and SQYQSGNT (SEQ ID NO: 93), respectively.

[0086] Examples of such VH sequences are provided in SEQ ID NOs: 44 (mouse) and 45-49 (humanized) and 57-58 (humanized). Examples of such VL sequences are provided in SEQ ID NOs: 50 (mouse) and 51-55 (humanized) and 56 (humanized).

[0087] Exemplary humanized antibodies include antibodies having a VH of SEQ ID NO: 49 and a VL of SEQ ID NO: 52 or 54, or antibodies having a VH of SEQ ID NO: 48 and a VL of SEQ ID NO: 53 or 54. In one embodiment, a humanized antibody includes a VH of SEQ ID NO: 48 and a VL of SEQ ID NO: 53. In one embodiment, a humanized antibody includes a VH of SEQ ID NO: 48 and a VL of SEQ ID NO: 56. In one embodiment, a humanized antibody includes a VH of SEQ ID NO: 57 and a VL of SEQ ID NO: 56. In one embodiment, a humanized antibody includes a VH of SEQ ID NO: 58 and a VL of SEQ ID NO: 56.

[0088] In some embodiments, the antibody or fragment thereof further includes a heavy chain constant region (e.g., CH1, CH2, and / or CH3) and / or a light chain constant region (e.g., CL). An exemplary heavy chain constant region is provided in SEQ ID NO: 59, and an exemplary light chain constant region is provided in SEQ ID NO: 67 (residues 108-214).

[0089] It is contemplated that small changes (e.g., addition, deletion, or substitution of one amino acid) can be designed into these CDR sequences that can preserve the activity of the antibody or even improve them. Such modified CDR sequences are referred to as CDR variants. One of ordinary skill in the art will also appreciate that the antibodies disclosed herein can be modified such that their amino acid sequence is not identical to the naturally occurring binding polypeptide from which they are derived. For example, a polypeptide or amino acid sequence derived from a specified protein can be similar to the starting sequence, e.g., have a certain percentage of identity, e.g., it can 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 specified CDR sequence.

[0090] In certain embodiments, the antibody includes an amino acid sequence or one or more moieties that are not typically associated with antibodies. Exemplary modifications are described in more detail below. For example, the antibodies of the present disclosure can include a flexible linker sequence, or can be modified to add a functional moiety (e.g., PEG, a drug, a toxin, or a tag).

[0091] Polynucleotides encoding proteins and methods of making proteins

[0092] The present disclosure also provides isolated polynucleotides or nucleic acid molecules encoding the multifunctional proteins, antibodies, variants or derivatives thereof of the present disclosure. The polynucleotides of the present disclosure can 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 polynucleotides. Further, the polynucleotides of the present disclosure can encode a portion 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 polynucleotides.

[0093] Methods of making antibodies are well known in the art and are 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 to a particular antigen can be made by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to an antigen challenge, but which has been disabled in its endogenous loci. Exemplary techniques that can be used to make such antibodies are described in U.S. Patents 6,150,584, 6,458,592, 6,420,140, the entire contents of which are incorporated herein by reference.

[0094] Cancer treatment

[0095] As described herein, the antibodies, variants or derivatives of the present disclosure can be used in certain therapeutic and diagnostic methods.

[0096] The present invention further relates to multifunctional molecule or antibody-based therapies involving the administration of the multifunctional molecules and antibodies of the present invention to patients (e.g., animals, mammals, and humans) to treat one or more diseases or conditions described herein. Therapeutic compounds of the present disclosure include, but are not limited to, the antibodies of the present disclosure (including variants and derivatives thereof as described herein) and nucleic acids or polynucleotides encoding the antibodies of the present disclosure (including variants and derivatives thereof as described herein).

[0097] The antibodies of the present invention 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 anti-tumor T cell immunity. Results using small molecule inhibitors or monoclonal antibodies against PD-L1 in mouse tumor models suggest that targeting PD-L1 therapy is an important alternative and realistic approach to effectively control tumor growth. As shown in the experimental examples, anti-PD-L1 antibodies activated adaptive immune response mechanisms, which can improve the survival rate of cancer patients.

[0098] Accordingly, in some embodiments, methods of treating cancer in a patient in need thereof are provided. In one embodiment, the method entails administering to the patient an effective amount of a multifunctional molecule or antibody of the present disclosure. In some embodiments, at least one cancer cell (e.g., stromal cell) in the patient expresses, overexpresses, or is induced to express PD-L1. Induction of PD-L1 expression can be accomplished, for example, by administering a tumor vaccine or radiation therapy.

[0099] Tumors expressing PD-L1 protein include bladder cancer, non-small cell lung cancer, renal cancer, breast cancer, urethral cancer, colorectal cancer, head and neck cancer, squamous cell carcinoma, Merkel cell carcinoma, gastrointestinal cancer, gastric cancer, esophageal cancer, ovarian cancer, renal cancer, and small cell lung cancer. Accordingly, the antibodies of the present disclosure can be used to treat any one or more of such cancers.

[0100] The present disclosure also provides cell therapies, such as chimeric antigen receptor (CAR) T cell therapies. Suitable cells can be used that are contacted with (or alternatively engineered to express) an anti-PD-L1 antibody of the present disclosure. Following such contacting or engineering, the cells can be introduced into a cancer patient in need of treatment. The cancer patient can have any type of cancer disclosed herein. The cells (e.g., T cells) can be, for example, tumor infiltrating T lymphocytes, CD4+ T cells, CD8+ T cells, or combinations thereof, but are not limited thereto.

[0101] In some embodiments, the cells are isolated from the cancer patient themselves. In some embodiments, the cells are provided from a donor or cell bank. When the cells are isolated from the cancer patient, undesirable immune reactions can be minimized.

[0102] Other diseases or conditions associated with increased cell survival that can be treated, prevented, diagnosed, and / or prognosed with the antibodies of the present disclosure, or variants or derivatives thereof, include, but are not limited to, the progression of malignancies and related diseases, and / or metastasis, such as leukemias (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 (myelogenous) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom'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's tumor, leiomyosarcoma, rhabdomyosarcoma, 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 adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testicular tumor, lung cancer, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, angioma, melanoma, neuroblastoma, and retinoblastoma.

[0103] Compositions

[0104] 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 comprises a second anti-cancer agent (e.g., an immune checkpoint inhibitor).

[0105] In particular embodiments, 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" is generally a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.

[0106] The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which a therapeutic agent is administered. Such a pharmaceutical carrier can be a sterile liquid, 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, dry 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 sodium acetate, tri- sodium citrate, zwitterions such as buffering agents. Antimicrobial agents, such as benzyl alcohol or methyl parabens, can also be employed as preservatives. Chelating agents, such as ethylenediaminetetraacetic acid, can also be used to prevent the oxidation of the active ingredient. Saline, buffered saline, dextrose, and related sugar solutions, and glycols such as polyethylene glycol are exemplary aqueous excipients. These compositions can take the form of solutions, suspensions, emulsions, 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 glycerol, polyethylene glycol, fatty acids, fatty acid esters, wax, cocoa butter and the like. Oral formulations 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's Pharmaceutical Sciences by E. W. Martin. 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 parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials, made of glass or plastic, as is required.

[0107] In one 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 can also include a solubilizing agent and a local anesthetic such as lidocaine 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 ampule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed, for example, with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration. Example

[0108] Example 1: Generation of mouse monoclonal antibodies against human PD-L1

[0109] This example describes the production of anti-human PD-L1 mouse monoclonal antibodies using hybridoma technology.

[0110] Antigens: Human PDL1-Fc protein and human PD-L1 highly expressed on CHOK1 cell line (PDL1-CHOK1 cell line).

[0111] Immunization: To generate mouse monoclonal antibodies targeting human PD-L1, Balb / c mice and Wistar rats were first immunized with PD-L1-Fc protein. Subsequently, immunized mice and rats were boosted with PD-L1-Fc protein and CHO-K1 / PD-L1 stable cells, respectively. To select mice or rats producing antibodies that bind to PD-L1 protein, antibody titers of sera from immunized mice or rats were evaluated by ELISA. Briefly, microtiter plates were coated with 0.5 pg / ml of human PD-L1 protein in ELISA coating buffer, 100 mΐ / well overnight at 4°C, and then blocked with 150 mΐ / well of 1% BSA. Serum dilutions from immunized mice were added to each well and incubated at 37°C for 1-2 hours. Plates were washed with PBS / Tween and then incubated with anti-mouse IgG antibody conjugated with horseradish peroxidase (HRP) or anti-rat IgG antibody conjugated with HRP for 1 hour at 37°C. After washing, plates were developed with TMB substrate and analyzed with a spectrophotometer at OD 450 nm. After 3 rounds of immunization, immune responses to rhPD-L1 protein and FACS detection of CHO-K1 / PDL-1 stable cell line were also detected by serum ELISA, with CHO-K1 parental cell line as a negative control. After 3 rounds of immunization, mice showing sufficient titers of anti-PDL1-IgG were boosted with 25 pg of human PDL1-Fc protein. The resulting mice were used for fusion. Anti-PD-L1 IgG of hybridoma supernatants was detected by ELISA.

[0112] Cell fusion: Fusion was performed by electrofusion. Fusion cells were placed in 50 96-well plates for each fusion.

[0113] Screening: Supernatants were screened by ELISA against recombinant human (rh) PD-L1-Fc protein and counter-screening antigens. Then, CHO-K1 / PD-L1 stable cell line and rhPD-1-Fc protein were preliminarily screened by receptor blocking FACS, and positive supernatants were subjected to confirmatory screening.

[0114] Subcloning and screening: Positive primary clones from each fusion were subcloned by limiting dilution to ensure that subclones originated from a single parental cell. Subclones were screened in the same way as primary clones, and culture supernatants of positive clones were subjected to additional confirmatory screening by affinity ranking.

[0115] 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 listed in Table 1 below.

[0116] Table 1. Sequences of the variable regions of 47C6A3, 67F3G7, and 89C10H8

[0117]

[0118] Table 1A. CDR sequences of 47C6A3

[0119]

[0120]

[0121] Table 1B. CDR sequences of 67F3G7

[0122] 67F3G7 Sequence SEQ ID NO: CDRH1 DFWVS 13 CDRH2 EIYPNSGVSRYNEKFKG 14 CDRH3 YFGYTYWFGY 15 CDRL1 RASKSVSTYMH 16 CDRL2 SASHLES 17 CDRL3 QQSNELPVT 18

[0123] Table 1C. CDR sequences of 89C10H8

[0124]

[0125] Example 2: Binding activity to PD-L1 antigen

[0126] ELISA test

[0127] To evaluate the binding activity of hybridoma clones 47C6A3, 67F3G7, and 89C10H8, chimeric mAbs from these clones were tested in ELISA.

[0128] Briefly, microtiter plates were coated with 0.5 μg / ml human PD-L1-Fc protein in PBS, 100 μl / well at 4°C overnight, and then blocked with 150 μl / well of 1% BSA. Three-fold dilutions of 47C6A3, 67F3G7, and 89C10H8 antibodies starting at 10 μg / ml were added to each well and incubated at 37°C for 1 hour. The plates were washed with PBS / Tween and then incubated with mouse anti-human IgG Fab antibodies conjugated to horseradish peroxidase (HRP) at 37°C for 30 minutes. After washing, the plates were developed with TMB substrate and analyzed with a spectrophotometer at OD 450nm. Figure 1 As shown, 47C6A3, 67F3G7, and 89C10H8 bind to human PD-L1 with high affinity (EC 50 =10.24 ng / ml, EC of 67F3G7 50 =10.76ng / ml, EC of 89C10H850 =8.112ng / ml).

[0129] Cell-based binding: FACS was used to assess the binding activity of 47C6A3, 67F3G7, and 89C10H8 chimeric mAbs to CHOK1 cells overexpressing human PD-L1.

[0130] Briefly, PDL1-CHOK1 cells were first incubated with 3-fold serial dilutions of 47C6A3, 67F3G7, and 89C10H8 chimeric mAbs starting at 100 nM at 4°C for 40 min. 647 AffiniPure goat anti-human IgG (H+L) was added to each well and incubated at 4°C for 30 minutes. The samples were washed twice with FACS buffer. Alexa Fluor 500 was evaluated by FACSCanto. The mean fluorescence intensity (MFI) of 647. Figure 2 As shown, 47C6A3, 67F3G7, and 89C10H8 bound to PDL1-CHOK1 cells with high affinity (EC 50 =0.1476 nM, EC of 67F3G7 50 =0.1035 nM, EC of 89C10H8 50 =0.1696nM).

[0131] Cross-species activity

[0132] ELISA assays were performed to evaluate the binding of chimeric antibodies to human, mouse, rat, and cynomolgus monkey PD-L1, respectively.

[0133] Briefly, microtiter plates were coated with 0.5 μg / ml of human, mouse, rat, and cynomolgus monkey PD-L1 proteins in PBS, 100 μl / well at 4°C overnight, and then blocked with 150 μl / well of 1% BSA. Chimeric antibodies diluted threefold starting from 10 μg / ml were added to each well and incubated at 37°C for 1 hour. The plates were washed with PBS / Tween and then incubated with mouse anti-human IgG Fab antibodies conjugated to horseradish peroxidase (HRP) at 37°C for 30 minutes. After washing, the plates were developed with TMB substrate and analyzed spectrophotometrically at OD 450nm. 47C6A3, 67F3G7, and 89C10H8 antibodies bound to human and cynomolgus monkey PD-L1 but not to rat and mouse PD-L1 ( Figure 3 and Table 2 ).

[0134] Table 2. Cross-species activity of 47C6A3, 67F3G7, and 89C10H8

[0135] Human Cynomolgus monkey 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 .

[0136] Example 3. Blocking of PD-L1 binding to PD-1 by antibodies

[0137] To assess the blocking effect of 47C6A3, 67F3G7 and 89C10H8 chimeric mAbs on the binding of recombinant human PD-L1 to its receptor PD-1, an ELISA-based receptor blocking assay was used.

[0138] Briefly, microtiter plates were coated with 0.5 pg / ml of human PD-L1-Fc protein in PBS, 100 mI / well overnight at 4°C, then blocked with 150 mI / well of 1% BSA. 50 mI of biotin-labeled human PD-1-Fc protein and 50 mI of 47C6A3, 67F3G7 and 89C10H8 antibodies starting at 10 pg / ml in 3-fold dilutions were added to each well and incubated for 1 hour at 37°C. Plates were washed with PBS / Tween and then incubated with streptavidin HRP for 10 minutes at 37°C. After washing, plates were developed with TMB substrate and analyzed with a spectrophotometer at OD 450 nm. As shown in Figure 3, 47C6A3, 67F3G7 and 89C10H8 blocked the binding of human PD-1 to human PD-L1 with IC50 of 91.18 ng / ml, 139.8 ng / ml and 129.8 ng / ml, respectively. Figure 4 50 efficiently inhibit the binding of human PD-L1 to human PD1.

[0139] Example 4: Binding affinity of mAbs

[0140] The 47C6A3, 67F3G7 and 89C10H8 antibodies were tested for binding to recombinant PD-L1 protein (human PD-L1-his tag) using a capture method with Biacore. Protein A chips were used to capture 47C6A3, 67F3G7 and 89C10H8 mAbs. Serial dilutions of human PD-L1-his tag protein were injected over the captured antibodies at a flow rate of 30 mI / min for 2 min. The antigen was allowed to dissociate for 480-1500 s. All experiments were performed on a Biacore T200. Data analysis was performed using Biacore T200 evaluation software. Results are shown in Figure 4 and Table 3 below. Figure 5

[0141] Table 3. Affinity measured by Biacore

[0142]

[0143] Example 5. Humanization of mouse antibodies

[0144] ​​Humanized mAbs were created using the 47C6A3, 67F3G7, and 89C10H8 variable region genes. 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 to available databases of human Ig gene sequences to find the best overall matching human germline Ig gene sequences. For the light chain of 47C6A3, human Vk1-4 was the best fit germline, while 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, while 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, while for the heavy chain, the closest human match was the VH3-21 / JH3 gene.

[0145] For the VL of 47C6A3, human Vk1-4 was the best fit germline, while for the VH of 47C6A3, human VH1-2 was chosen as the backbone. The humanized variable domain sequence for 47C6A3 was then designed with CDRL1, L2, and L3 grafted onto the framework sequence of the Vk1-4 gene, and CDRH1, H2, and H3 grafted onto the framework sequence 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 with a human amino acid could affect binding and / or CDR conformation. In the case of the heavy chain, R, M, and I in the framework were of concern with respect to back mutations.

[0146] The humanized variable domain sequence for 67F3G7 was then designed with CDRL1, L2, and L3 grafted onto the framework sequence of the Vk1-39 / JK4 gene, and CDRH1, H2, and H3 grafted onto the framework sequence 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 with a 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 were of concern with respect to back mutations. In the case of the light chain, T, V, L, and Q in the framework were of concern with respect to back mutations.

[0147] The humanized variable domain sequence for 89C10H8 was then designed with CDRL1, L2, and L3 grafted onto the framework sequence of the Vk1-17 / JK2 gene, and CDRH1, H2, and H3 grafted onto the framework sequence 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 with a 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 were of concern with respect to back mutations. In the case of the light chain, Y, I, E, and F in the framework were of concern with respect to back mutations.

[0148] The amino acid and nucleotide sequences of some humanized antibodies are listed in Table 4 below.

[0149] Table 4. Humanized antibody sequences (underlined CDRs; bold / italics indicate reverse mutations)

[0150]

[0151]

[0152]

[0153] These genes were cloned into pcDNA3.4 vector and transfected into 293F cells. Antibodies were prepared according to the table below.

[0154] Humanized VH and VL genes were generated synthetically and then cloned into vectors containing human γ1 and human κ constant domains, respectively. Pairing of human VH and human VL generated 41 humanized antibodies (see Table 5).

[0155] Table 5. Humanized antibodies and their VH and VL regions A.47C6A3

[0156]

[0157] B.67F3G7

[0158]

[0159] C.89C10H8

[0160]

[0161] Example 6: Antigen Binding Properties of Humanized Antibodies

[0162] Binding to recombinant human PD-L1

[0163] To evaluate antigen binding activity, the humanized antibodies were subjected to ELISA testing. Briefly, microtiter plates were coated with 0.5 μg / ml human PD-L1-Fc protein in PBS, 100 μl / well at 4 ° C overnight, and 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 at 37 ° C for 1 hour. The plates were washed with PBS / Tween and then incubated with mouse anti-human IgGFab antibodies conjugated to horseradish peroxidase (HRP) at 37 ° C for 1 hour. After washing, the plates were developed with TMB substrate and analyzed with a spectrophotometer at OD 450nm. As shown in Figure 6, all humanized antibodies showed binding efficiency to human PD-L1 comparable to that of the chimeric antibodies.

[0164] To explore the binding kinetics of humanized antibodies, affinity ranking was performed 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 was comparable to that of chimeric antibody.

[0165] Table 6. Affinity ranking of humanized antibodies

[0166]

[0167]

[0168] Binding to human PD-L1 overexpressed on mammalian cells

[0169] To evaluate the antigen binding property, the binding of humanized antibodies to PD-L1 overexpressed on mammalian cells was analyzed by FACS. Briefly, PDL1-CHO K1 cells were first incubated with 3-fold serial dilutions of humanized antibodies starting at 15 μg / ml at 4°C for 40 min. After washing with PBS, Alexa 647 AffiniPure goat anti-human IgG (H+L) antibody was added to each well and incubated at 4°C for 30 min. The MFI of Alexa 647 was evaluated by FACSCanto. As shown, all humanized antibodies could bind to PD-L1 expressed on mammalian cells with high efficiency. Figure 7

[0170] Full kinetic affinity of humanized antibodies measured by Biacore

[0171] ​The binding of 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 a protein A chip. Serial dilutions of human PD-L1-his tag protein were injected over the captured antibodies at a flow rate of 30 μl / min for 2 minutes. Antigen dissociation was allowed for 1500 s. All experiments were performed on a Biacore T200. Data analysis was performed using Biacore T200 evaluation software, and the results are shown in Table 7 below.

[0172] Table 7 Affinity measured by Biacore

[0173]

[0174]

[0175] Example 7: Blocking of the binding of PDL1 to PD1 by humanized antibodies

[0176] Receptor blockade trial using recombinant human PD-L1

[0177] Human PD-L1 has two receptors, PD-1 and CD80. To investigate the blocking properties of humanized PD-L1 antibodies against these two proteins, a protein-based receptor blocking assay was employed.

[0178] Briefly, microtiter plates were coated with 0.5 pg / ml of human PD-L1-Fc protein in PBS, 100 mΐ / well overnight at 4°C, then blocked with 150 mΐ / well of 1% BSA for 2 hours at 37°C. To each well, 50 mΐ of biotin-labeled human PD-1-Fc or CD80-Fc protein and 50 mΐ of 3-fold dilutions of PD-L1 antibodies starting at 10 pg / ml were added and incubated for 1 hour at 37°C. Plates were washed with PBS / Tween, then incubated with streptavidin HRP for 10 minutes at 37°C. After washing, plates were developed with TMB substrate and analyzed with a spectrophotometer at OD 450 nm. As shown in Figure 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 effectively inhibited the binding of human PD-L1 to human PD1. In addition, 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 effectively inhibited the binding of human PD-L1 to human CD80 in a dose-dependent manner (Figure 9).

[0179] Example 8. Bispecific proteins targeting PD-L1 and TGF-β pathway

[0180] Bispecific recombinant anti-PD-L1 antibody and TGF-βRII fusion proteins were prepared and tested in this example.

[0181] The light chain of the molecule is the light chain of the anti-PDL1 mAb. The heavy chain is a fusion of the heavy chain of the anti-PDL1 mAb to the N-terminus of the TGF-βRII soluble extracellular domain through a flexible (Gly4Ser)4Gly linker. At the fusion junction, the C-terminal lysine residue of the antibody heavy chain was mutated to alanine to reduce potential proteolytic cleavage.

[0182] In some examples, potential modification sites in the CDRs were mutated to similar amino acids. The sequences of the antibody portion of the bispecific molecules are shown in Table 8 below.

[0183] Table 8. Sequences of variable regions of antibody portion of bispecific molecules

[0184]

[0185]

[0186] Table 9. VH / VL of bifunctional molecules

[0187] 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 .

[0188] In addition to the VH, the heavy chain of the bifunctional molecule also includes a constant region (with the C-terminal K mutated to A), a (Gly4Ser)4Gly linker, and the N-terminus of the TGF-βRII soluble extracellular domain. The sequences are shown in Table 10.

[0189] Table 10. Other sequences of heavy chains, and entire heavy / light chains

[0190]

[0191]

[0192]

[0193] Example 9: Binding affinity of bifunctional molecules

[0194] The binding of 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 by Biacore using the capture method.

[0195] Bifunctional molecules were captured using a Protein A chip. Serial dilutions of human PD-L1-his tagged protein were injected over the captured antibody at a flow rate of 30 μl / min for 2 minutes. Antigen dissociation was allowed for 1500 seconds. All experiments were performed on a Biacore T200. Data analysis was performed using Biacore T200 evaluation software. Results are shown in Figure 10 and Table 11 below.

[0196] Table 11. Affinity test by Biacore

[0197]

[0198]

[0199] The binding of LP008-02 to recombinant PD-L1 protein and human TGF-β1 was tested by Biacore using a capture method.

[0200] LP008-02 was captured using a protein A chip. Serial dilutions of human PD-L1-his tagged protein and human TGF-β1 were injected over the captured antibody at a flow rate of 30 μl / min for 2 minutes. PD-L1 was allowed to dissociate for 680 s and TGF-β1 was allowed to dissociate for 1000 s. All experiments were performed on a Biacore T200. Data analysis was performed using Biacore T200 evaluation software. Results are shown in Figure 11 and Table 12 below.

[0201] Table 12. Affinity test on Biacore

[0202]

[0203] Example 10: Functional assay of PD-1 / PD-L1 blockade

[0204] In this example, the activity of the bifunctional molecules in blocking PD1 / PD-L1 interaction was determined by a bioluminescent cell-based test.

[0205] In this assay, PD-1 / PD-L1 interaction inhibits TCR signaling and NFAT RE-mediated luminescence when PD1 effector cells are co-cultured with PD-L1 target cells. Addition of anti-PD-1 or anti-PD-L1 antibodies that block PD-1 / PD-L1 interaction will release the inhibitory signal and lead to TCR activation and NFAT RE-mediated luminescence.

[0206] As shown in Figure 12 , LP008-02 and LP008-06a-ES block PD1 and PD-L1 interaction with much 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).

[0207] Example 11: Functional assay of TGF-β

[0208] This example used a luciferase assay to assess the effect of LP008-02 and LP008-06a-ES on canonical TGF-β signaling.

[0209] Series dilutions of M7824 (dual function anti-PD-L1 / TGF Trap fusion protein, see e.g. Knudson et al., Oncoimmunology. 2018; 7(5):e1426519), LP008-02 or LP008-06a-ES were incubated with SBE luciferase reporter transfected 293 cells in the presence of recombinant human TGF-β for about 20 hours.

[0210] As shown in the TGF-β SBE luciferase reporter assay system constructed in 293 cells, M7824, LP008-02 and LP008-06a-ES blocked TGF-β canonical signaling (IC50 = 0.06687 nM, IC50 = 0.07352 nM, IC50 = 0.07167 nM). Figure 13

[0211] Example 12: Binding activity to human PD-L1

[0212] ELISA of recombinant human PD-L1

[0213] To assess the binding activity of M7824, LP008-02 and LP008-06a-ES, the dual function molecules were tested in ELISA.

[0214] Briefly, microtiter plates were coated with 0.5 pg / ml human PD-L1 -His protein in PBS, 100 mΐ / well overnight at 4°C, then blocked with 150 mΐ / well of 1% BSA. Three-fold dilutions of M7824, LP008-02 and LP008-06a-ES starting from 1 pg / ml were added to each well and incubated for 1 hour at 37°C. Plates were washed with PBS / Tween and then incubated with goat anti-human IgG antibody conjugated with horseradish peroxidase (HRP) for 30 minutes at 37°C. After washing, plates were developed with TMB substrate and analyzed with a spectrophotometer at OD 450 nm.

[0215] As shown in the ELISA of recombinant human PD-L1, LP008-02 and LP008-06a-ES bound to human PD-L1 with significantly higher activity than M7824 (EC 50 = 11.82 ng / ml and EC 50 = 14.36 ng / ml vs. EC 50 = 23.68 ng / ml). Figure 14 Cross-species activity

[0216]

[0217] ​​To assess the binding of the bispecific antibodies to mouse PD-L1, rat PD-L1, cynomolgus PD-L1, the antibodies were tested using an ELISA.

[0218] Briefly, microtiter plates were coated with 0.5 pg / ml of mouse, rat and cynomolgus PD-L1 protein in PBS, 100 mΐ / well overnight at 4°C, then blocked with 150 mΐ / well of 1% BSA. Three-fold dilutions of the bispecific antibodies starting from 1 pg / ml were added to each well and incubated for 1 hour at 37°C. Plates were washed with PBS / Tween and then incubated with goat anti-human IgG antibody conjugated with horseradish peroxidase (HRP) for 30 minutes at 37°C. After washing, plates were developed with TMB substrate and analyzed with a spectrophotometer at OD 450 nm.

[0219] LP008-02 and LP008-06a-ES were able to bind cynomolgus PD-L1 with higher affinity than M7824, but only M7824 was able to bind rat and mouse PD-L1 (Table 13). Figure 15 and Table 13).

[0220] Table 13. Cross-species activity of M7824, CZ010-02 and CZ010-06a-ES

[0221] EC50 Cynomolgus monkey 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 .

[0222] Example 13: Binding activity to human TGF-β

[0223] ELISA using recombinant human TGF-β

[0224] To assess the binding activity of M7824, LP008-02 and LP008-06a-ES to human TGF-β, these bifunctional molecules were tested in an ELISA.

[0225] Briefly, microtiter plates were coated with 1 pg / ml of human TGF-β protein in PBS, 100 mΐ / well overnight at 4°C, then blocked with 150 mΐ / well of 1% BSA. Three-fold dilutions of M7824, LP008-02 and LP008-06a-ES bifunctional molecules starting from 10 pg / ml were added to each well and incubated for 1 hour at 37°C. Plates were washed with PBS / Tween and then incubated with goat anti-human IgG antibody conjugated with horseradish peroxidase (HRP) for 30 minutes at 37°C. After washing, plates were developed with TMB substrate and analyzed with a spectrophotometer at OD 450 nm.

[0226] As Figure 16As shown, M7824, LP008-02 and LP008-06a-ES all bind human TGF-β with high activity (EC 50 = 43.43 ng / ml, EC 50 = 28.58 ng / ml, EC 50 = 39.38 ng / ml).

[0227] Cross-species activity

[0228] To assess the binding of the bispecific antibodies to mouse, rat and cynomolgus TGF-β, the bifunctional molecules were tested in ELISA.

[0229] Briefly, microtiter plates were coated with 100 μΐ / well of 1 μg / ml mouse, rat and cynomolgus TGF-β protein in PBS overnight at 4°C and then blocked with 150 μΐ / well of 1% BSA. Three-fold dilutions of the 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 incubated with goat anti-human IgG antibody conjugated with horseradish peroxidase (HRP) for 30 minutes at 37°C. After washing, the plates were developed with TMB substrate and analyzed with a spectrophotometer at OD 450 nm.

[0230] All tested bifunctional molecules bind cynomolgus, rat and mouse TGF-β with high activity (EC Figure 17 and Table 14).

[0231] Table 14. Cross-species activity of M7824, LP008-02 and LP008-06a-ES

[0232] EC50 Cynomolgus monkey 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 .

[0233] Example 14: Efficacy in MC38 tumor mouse model

[0234] This example uses a tumor mouse model to test the in vivo efficacy of the bifunctional molecules.

[0235] MC38 cells expressing human PD-L1 resuspended in PBS were inoculated subcutaneously into the right flank of B-hPD-L1 humanized mice at a concentration of 5 x 10 5 cells in a volume of 0.2 mL when the average tumor volume reached approximately 55 mm 3At this time, 24 mice with appropriate individual tumor volumes were selected as a group, and the animals were randomly divided into 4 experimental groups according to the tumor volume, 6 in each group. After injection of anti-mCD20 mAb, total human IgG, M7824, LP008-02 and LP008-06a-ES were administered 3 times a week by intraperitoneal injection. The dose was calculated according to the body weight of the experimental animals at 10 μg / g. The body weight and tumor size of the mice were tested twice a week.

[0236] The results are shown in Figure 18. In these animal models, the bifunctional molecules LP008-02 and LP008-06a-ES showed better efficacy than M7824 in terms of tumor growth inhibition. In addition, animal deaths were observed in both the IgG and M7824 groups, but not in the LP008-02 and LP008-06a-ES groups, indicating that the new bifunctional molecules have better safety.

[0237] Example 15. Modification of bifunctional molecules

[0238] This example tested the in vitro efficacy of certain modified bifunctional molecules (Table 15) on the TGF-β functional assay. Some of them include linker sequences with TAGHTQTSTGGGAITTGTSGAGHGP (SEQ ID NO: 87), HYP and / or G4S (SEQ ID NO: 86) repeats. These molecules are called LP008-02-1 to LP008-02-7, respectively.

[0239] Table 15. Design of modified sequences of linkers and TGF-βRII

[0240]

[0241] ELISA of recombinant human TGF-β1

[0242] To evaluate the binding activity of the modified LP008-02 bifunctional molecules, these bifunctional molecules were tested with ELISA.

[0243] Briefly, microtiter plates were coated with 100 μl / well of 1 μg / ml human TGF-β1 protein (Acro, TG1-H4212) in PBS 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 at 37°C for 1 hour. The plates were washed with PBS / Tween and then incubated with goat anti-human IgG (H+L) antibody conjugated with horseradish peroxidase (HRP) for 30 minutes at 37°C. After washing, the plates were developed with TMB substrate and analyzed with a spectrophotometer at OD 450 nm.

[0244] As shown in Figure 19 Figure 8, all modified LP008-02 bifunctional molecules bind human TGF-β1 with high activity, comparable to LP008-02-1.

[0245] TGF-β function assay

[0246] Serial dilutions of modified LP008-02 bifunctional molecules were incubated with SBE luciferase reporter transfected 293 cells in the presence of recombinant human TGF-β1 for about 22 hours.

[0247] As shown in Figure 20 Figure 8, all modified LP008-02 bifunctional molecules bind human TGF-β1 with high activity, comparable to LP008-02-1.

[0248] Example 16. Comparison of bifunctional molecules

[0249] Molecules 1-7 of Table 15 comprise different sequences at the N- and C-terminus of the extracellular domain (SEQ ID NO: 72). Their stability and activity were tested to assess the influence of these sequences.

[0250] Molecule 1 (LP008-02-1) comprises the entire extracellular part of the protein (SEQ ID NO: 61), which contains a 25 amino acid stretch from the N-terminus of the extracellular domain (IPPHVQKSVNNDMIVTDNNGAVKFP, SEQ ID NO: 89, or amino acids 24-48 of the B subtype, SEQ ID NO: 71) and a C-terminal fragment (EEYNTSNPD, SEQ ID NO: 90). In addition, the molecule adds several G4S (SEQ ID NO: 86) repeats in the linker.

[0251] Molecule 2 (LP008-02-2) has the N-terminal portion of the extracellular domain (amino acids 24-48 of subtype B, SEQ ID NO: 89) replaced with an artificial linker TAGHTQTSTGGGAITTGTSGAGHGP (SEQ ID NO: 87) compared to molecule 1. This linker is modeled based on SEQ ID NO: 89. Changes include: (i) removal of the rigid dipeptide 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., only one I is retained). These changes are shown in Table 16 below. Molecule 2 also includes a single G4S unit at the N-terminus.

[0252] Table 16. Artificial linker

[0253] Name Sequence SEQ ID NO: Original IPPHVQKSVNNDMIVTDNNGAVKFP 89 Modified TAGHTQTSTGGGAITTGTSGAGHGP 87 .

[0254] Molecule 3 (LP008-02-3) includes a longer G4S linker than molecule 2. Above molecule 3, molecule 4 (LP008-02-4) has a deletion of the C-terminal fragment EEYNTSNPD (SEQ ID NO: 90). Molecule 5 (LP008-02-5) replaces the artificial linker SEQ ID NO: 87 with a short linker HYP. Molecules 6 (LP008-02-6) and 7 (LP008.02-7) include G4S linkers of different lengths on the N-terminal side of the HYP linker.

[0255] Example 17. Binding activity and stability of bifunctional molecules

[0256] This example uses SEC-HPLC and CE-SDS to assess the stability of some modified bifunctional molecules, including LP008-02-1 and four further modified molecules, LP008-02-2, LP008-02-3, LP008-02-6 and LP008-02-7.

[0257] The 5 sequences were expressed in CHO-K1 cells by polyethylenimine (PEI)-mediated transient transfection, and the supernatant was harvested after 10 days. The bifunctional molecules were purified from the culture supernatant by protein A, followed by Superdex 200 pg purification, with purity levels greater than 99% as detected by SEC-HPLC (Table 17).

[0258] Table 17. SEC-HPLC and CE-SDS results for day 0 test articles

[0259]

[0260] To evaluate the binding activity of the modified LP008-02 bifunctional molecules, these bifunctional molecules were tested by ELISA.

[0261] Briefly, microtiter plates were coated with 1 μg / ml human TGF-β1 protein (Acro, TG1-H4212) in PBS, 100 μl / well at 4°C overnight, and then blocked with 150 μl / well of 1% BSA. Four-fold serial dilutions of the modified LP008-02 bifunctional molecule starting at 30 nM were added to each well and incubated for 1 hour. The plates were washed with PBS / Tween and then incubated with goat anti-human IgG Fc antibody conjugated to horseradish peroxidase (HRP) for 30 minutes. After washing, the plates were incubated with TMB substrate for color development and analyzed by spectrophotometer at OD 450 nm.

[0262] like Figure 21 As shown, all other modified LP008-02 bifunctional molecules bound to human TGF-β1 with high activity, comparable to LP008-02-1.

[0263] To evaluate the effect of the modified LP008-02 bifunctional molecule on canonical TGF-β signaling, the modified bifunctional molecule was tested using a luciferase assay. Serial dilutions of the bifunctional molecule were incubated with 293 cells transfected with the SBE luciferase reporter in the presence of recombinant human TGF-β for 24 hours. Figure 22 As shown, in the TGF-β SBE luciferase reporter assay system constructed in 293 cells, LP008-02-2, LP008-02-3, LP008-02-6 and LP008-02-7, like LP008-02-1, effectively blocked TGF-β canonical signaling (IC50 = 0.04231 nM, IC50 = 0.0527 nM, IC50 = 0.09616 nM, and IC50 = 0.1962 nM).

[0264] The bifunctional molecule was dissolved in two buffers for antibody stability testing. Buffer information is as follows: Buffer A: 20 mM sodium acetate, 250 mM sorbitol, 0.02% polysorbate 80, pH 4.9; Buffer B: 20 ​​mM His / HisHCl, 250 mM trehalose, pH 5.4.

[0265] Preparations samples at 3.0 mg / ml were incubated at 40°C and then tested by SEC-HPLC and CE-SDS at day 0 and day 14, respectively. As shown in Table 18, the stability of LP008-02-2, LP008-02-3, LP008-02-6 and LP008-02-7 formulated in buffer A and buffer B was higher than LP008-02-1 in SEC-HPLC, non-reduced CE-SDS and reduced CE-SDS.

[0266] Table 18. SEC-HPLC and CE-SDS results of test articles at day 14

[0267]

[0268]

[0269] Accordingly, this example shows that the modified bifunctional molecules LP008-02-2, LP008-02-3, LP008.02-6 and LP008-02-7 exhibit similar activity as LP008-02-1 but their stability is significantly higher than LP008-021. Replacing the N-terminal portion of TGF-βRII (IPPHVQKSVNNDMIVTDNNGAVKFP, SEQ ID NO: 89) in LP008-02-1 with an artificial linker (e.g. TAGHTQTSTGGGAITTGTSGAGHGP (SEQ ID NO: 87) or HYP) leads to a significant improvement in stability.

[0270] Example 18. High concentration formulation of anti-PD-Ll antibodies

[0271] This example uses HIC-HPLC and viscosity tests to evaluate the development potential and risk of high concentration anti-PD-Ll molecule formulations.

[0272] Four anti-PD-Ll molecules were expressed in CHO-K1 or 293F cells by transient transfection. The constant region of the heavy chain is human IgGl (N297A)-Fc. Purified MPDL3280A (Atezolizumab), 47C6A3, Hu67F3G7-22 and Hu89C10H8-7 antibodies were tested by HIC-HPLC and the ammonium sulfate concentration corresponding to the hydrophobic elution time was obtained for predicting the solubility range of these molecules. As shown in Table 19, the ammonium sulfate concentration corresponding to the hydrophobic elution time of MPDL3280A, 47C6A3, Hu67F3G7-22 and Hu89C10H8-7 was 0.41 M, 0.78 M, 0.97 M and 1.10 M, respectively. All the newly developed antibodies have lower hydrophobicity than the reference antibody MPDL3280A.

[0273] Table 19. Antibody hydrophobicity from HIC-HPLC testing

[0274] Sample HIC (M) MPDL3280A 0.41 47C6A3 0.78 Hu67F3G7-22 0.97 Hu89C10H8-7 1.10 .

[0275] The activity of anti-PD-L1 antibodies in blocking the PD1 / PD-L1 interaction was then measured using a bioluminescent cell-based assay. In this assay, when PD1 effector cells are co-cultured with PD-L1 target cells, the PD-1 / PD-L1 interaction inhibits TCR signaling and NFAT RE-mediated luminescence. Addition of anti-PD-1 or anti-PD-L1 antibodies that block the PD-1 / PD-L1 interaction will release the inhibitory signal and lead to TCR activation and NFAT-RE-mediated luminescence. Figure 23 As shown, MPDL3280A, 47C6A3, Hu67F3G7-22, and Hu89C10H8-7 blocked the interaction between PD1 and PD-L1 with considerable activity (MPDL3280AEC 50 =0.1327nM, 47C6A3 EC 50 =0.1501nM, Hu67F3G7-22 EC 50 =0.1034nM, Hu89C10H8-7 EC 50 =0.2138nM).

[0276] MPDL3280A and Hu67F3G7-22 with human IgG1 Fc were expressed in CHO-K1 cells by transient transfection. The purified MPDL3280A-hIgG1 Fc and Hu67F3G7-22-hIgG1 Fc antibodies were tested by HIC-HPLC, and the ammonium sulfate concentration corresponding to the hydrophobic elution time was obtained to predict the solubility range of the two molecules. As shown in Table 20, the ammonium sulfate concentration corresponding to the hydrophobic elution time for MPDL3280A-hIgG1 Fc and Hu67F3G7-22-hIgG1 Fc was 0.42M and 0.99M, respectively. Similarly, for the same Fc fragment, Hu67F3G7-22 exhibited lower hydrophobicity than MPDL3280A.

[0277] Table 20. Results of HIC-HPLC test

[0278]

[0279] To further confirm the solubility and viscosity properties of the antibodies, two purified candidates were directly concentrated in phosphate buffer (containing 60 mM NaCl) by ultrafiltration. During the ultrafiltration process, the concentration, SEC-HPLC, and viscosity properties were measured at different stages. As shown in Table 21, the viscosity of MPDL3280A-hlgGl Fc was much higher than that of Hu 67F3G7-22-hlgGl Fc at similar concentrations. For high-concentration formulations, antibodies with lower viscosity are generally superior to those with higher viscosity. Therefore, the Hu 67F3G7-22 antibody has higher potential than MPDL3280A as a therapeutic protein.

[0280] Table 21. Results of solubility test

[0281]

[0282] ***

[0283] The scope of the disclosure is not limited to the specific embodiments described, which are intended as single illustrations of various aspects of the disclosure and any compositions or methods equivalent thereto are within the scope of the disclosure. It will be apparent to those skilled in the art that various modifications and variations can be made to 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.

[0284] 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. SEQUENCE LISTING <110> LEPU BIOTECH, INC. <120> bifunctional molecules targeting PD-L1 and TGF-β <130> 20F-1637-WOP3 <150> PCT / CN2020 / 105286 <151> 2020-07-28 <150> PCT / CN2021 / 098476 <151> 2021-06-04 <160> 93 <170> PatentIn version 3.5 <210> 1 <211> 120 <212> PRT <213> artificial sequence <220> <223> synthetic <400> 1 Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Asp 20 25 30 Tyr Ala Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp 35 40 45 Met Gly Tyr Ile Ile Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe 65 70 75 80 Leu Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Arg Ser Thr Met Ile Ala Thr Asn Trp Phe Ala Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ala 115 120 <210> 2 <211> 107 <212> PRT <213> artificial sequence <220> <223> synthetic <400> 2 Asp Ile Val Met Thr Gin Ser His Lys Phe Met Ser Thr Ser Val Val 1 5 10 15 Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gin Asp Val Ser Leu Ala 20 25 30 Val Ala Trp Tyr Gin Gin Lys Pro Gly Gin Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Ala Leu Thr Ile Ser Ser Val Gin Ala 65 70 75 80 Glu Asp Leu Ala Leu Tyr Tyr Cys Gin Gin His Tyr Ile Thr Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 3 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 3 Gln Val Lys Leu Leu Gin Ser Gly Ala Ala Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Asp Phe 20 25 30 Trp Val Ser Trp Val Lys Gin Ser His Glu Lys Ser Leu Glu Trp He 35 40 45 Gly Glu He Tyr Pro Asn Ser Gly Val Ser Arg Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Arg Ala Thr Met Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Leu Glu Leu Ser Arg Leu Thr Ser Glu Asp Ser Ala He Tyr Tyr Cys 85 90 95 Thr Lys Tyr Phe Gly Tyr Thr Tyr Trp Phe Gly Tyr Trp Gly Gin Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 4 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 4 Asp Thr Val Leu Thr Gin Ser Pro Ala Leu Ala Val Ser Leu Gly Gin 1 5 10 15 Arg He Thr He Ser Cys Arg Ala Ser Lys Ser Val Ser Thr Tyr Met 20 25 30 His Trp Tyr Gin Gin Arg Ser Gly Leu Gin Pro Lys Leu Leu He Tyr 35 40 45 Ser Ala Ser His Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asp Pro Val Glu Ala Asp 65 70 75 80 Asp Ile Ala Asn Tyr Tyr Cys Gln Gln Ser Asn Glu Leu Pro Val Thr 85 90 95 Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 5 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 5 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Thr Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Trp Met Thr Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ser Ile Thr Asn Thr Gly Ser Ser Thr Phe Tyr Pro Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Arg Ser Thr Leu Phe 65 70 75 80 Leu Gin He Asn Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Thr Arg Asp Thr Thr He Ala Pro Phe Asp Tyr Trp Gly Gin Gly Val 100 105 110 Met Val Thr Val Ser Ser 115 <210> 6 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 6 Asp He Gin Met Thr Gin Ser Pro Ser Phe Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr He Thr Tyr Lys Ala Ser Gin Asn Leu Asn Glu Tyr 20 25 30 Leu Asn Trp Tyr Gin Gin Lys Leu Gly Glu Ala Pro Lys Arg Leu He 35 40 45 Tyr Lys Thr Asn Thr Leu Gin Ala Gly He Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly He Asp Tyr Thr Leu Thr He Ser Ser Leu Gin Pro 65 70 75 80 Glu Asp Val Ala Thr Tyr Phe Cys Ser Gin Tyr Asn Ser Gly Asn Thr 85 90 95 Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 <210> 7 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 7 Ser Asp Tyr Ala Trp Asn 1 5 <210> 8 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 8 Tyr Ile Ile Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 <210> 9 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 9 Ser Thr Met Ile Ala Thr Asn Trp Phe Ala Tyr 1 5 10 <210> 10 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 10 Lys Ala Ser Gln Asp Val Ser Leu Ala Val Ala 1 5 10 <210> 11 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 11 Trp Ala Ser Thr Arg His Thr 1 5 <210> 12 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 12 Gln Gln His Tyr Ile Thr Pro Trp Thr 1 5 <210> 13 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 13 Asp Phe Trp Val Ser 1 5 <210> 14 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 14 Glu Ile Tyr Pro Asn Ser Gly Val Ser Arg Tyr Asn Glu Lys Phe Lys 1 5 10 15 Gly <210> 15 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 15 Tyr Phe Gly Tyr Thr Tyr Trp Phe Gly Tyr 1 5 10 <210> 16 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 16 Arg Ala Ser Lys Ser Val Ser Thr Tyr Met His 1 5 10 <210> 17 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 17 Ser Ala Ser His Leu Glu Ser 1 5 <210> 18 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 18 Gln Gln Ser Asn Glu Leu Pro Val Thr 1 5 <210> 19 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 19 Asn Tyr Trp Met Thr 1 5 <210> 20 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 20 Ser lie Thr Asn Thr Gly Ser Ser Thr Phe Tyr Pro Asp Ser Val Lys 1 5 10 15 Gly <210> 21 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 21 Asp Thr Thr lie Ala Pro Phe Asp Tyr 1 5 <210> 22 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 22 Lys Ala Ser Gin Asn Leu Asn Glu Tyr Leu Asn 1 5 10 <210> 23 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 23 Lys Thr Asn Thr Leu Gin Ala 1 5 <210> 24 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 24 Ser Gin Tyr Asn Ser Gly Asn Thr 1 5 <210> 25 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 25 Asp Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gin 1 5 10 15 Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Asp 20 25 30 Tyr Ala Trp Asn Trp Ile Arg Gin Phe Pro Gly Asn Lys Leu Glu Trp 35 40 45 Met Gly Tyr Ile Ile Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gin Phe Phe 65 70 75 80 Leu Gin Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Arg Ser Thr Met Ile Ala Thr Asn Trp Phe Ala Tyr Trp Gly Gin 100 105 110 Gly Thr Leu Val Thr Val Ser Ala 115 120 <210> 26 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 26 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp 20 25 30 Tyr Ala Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ile Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Thr Met Ile Ala Thr Asn Trp Phe Ala Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 27 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 27 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp 20 25 30 Tyr Ala Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ile Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Thr Met Ile Ala Thr Asn Trp Phe Ala Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 28 <211> 120 <212> PRT <213> artificial sequence <220> <223> synthetic <400> 28 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp 20 25 30 Tyr Ala Trp Asn Trp Ile Arg Gin Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45 Met Gly Tyr He He Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg He Thr He Ser Arg Asp Thr Ser Lys Asn Gin Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Thr Met He Ala Thr Asn Trp Phe Ala Tyr Trp Gly Gin 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 29 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 29 Asp He Val Met Thr Gin Ser His Lys Phe Met Ser Thr Ser Val Val 1 5 10 15 Asp Arg Val Ser He Thr Cys Lys Ala Ser Gin Asp Val Ser Leu Ala 20 25 30 Val Ala Trp Tyr Gin Gin Lys Pro Gly Gin Ser Pro Lys Leu Leu lie 35 40 45 Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Ala Leu Thr lie Ser Ser Val Gin Ala 65 70 75 80 Glu Asp Leu Ala Leu Tyr Tyr Cys Gin Gin His Tyr lie Thr Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu lie Lys 100 105 <210> 30 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 30 Asp lie Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr lie Thr Cys Lys Ala Ser Gin Asp Val Ser Leu Ala 20 25 30 Val Ala Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Lys Leu Leu lie 35 40 45 Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Ile Thr Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 31 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 31 Gln Val Lys Leu Leu Gln Ser Gly Ala Ala Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Asp Phe 20 25 30 Trp Val Ser Trp Val Lys Gln Ser His Glu Lys Ser Leu Glu Trp Ile 35 40 45 Gly Glu Ile Tyr Pro Asn Ser Gly Val Ser Arg Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Arg Ala Thr Met Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Leu Glu Leu Ser Arg Leu Thr Ser Glu Asp Ser Ala Ile Tyr Tyr Cys Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Ile Thr Pro Trp85 90 95 Thr Leu Val Thr Val Ser Ser 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 32 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 32 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Phe 20 25 30 Trp Val Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Glu Ile Tyr Pro Asn Ser Gly Val Ser Arg Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Phe Gly Tyr Thr Tyr Trp Phe Gly Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 33 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 33 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Phe 20 25 30 Trp Val Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Glu Ile Tyr Pro Asn Ser Gly Val Ser Arg Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Arg Val Thr Met Thr Val Asp Lys Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Phe Gly Tyr Thr Tyr Trp Phe Gly Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 34 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 34 Glu Val Gin Leu Val Gin Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Phe 20 25 30 Trp Val Ser Trp Val Arg Gin Ala Pro Gly Gin Gly Leu Glu Trp Met 35 40 45 Gly Glu He Tyr Pro Asn Ser Gly Val Ser Arg Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Arg Val Thr Met Thr Val Asp Lys Ser He Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Lys Tyr Phe Gly Tyr Thr Tyr Trp Phe Gly Tyr Trp Gly Gin Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 35 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 35 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Asp Phe 20 25 30 Trp Val Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Tyr Pro Asn Ser Gly Val Ser Arg Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Arg Val Thr Met Thr Val Asp Lys Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Lys Tyr Phe Gly Tyr Thr Tyr Trp Phe Gly Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 36 <211> 119 <212> PRT <213> artificial sequence <220> <223> synthetic <400> 36 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Asp Phe 20 25 30 Trp Val Ser Trp Val Arg Gin Ala Pro Gly Gin Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Tyr Pro Asn Ser Gly Val Ser Arg Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Arg Val Thr Met Thr Val Asp Lys Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Phe Gly Tyr Thr Tyr Trp Phe Gly Tyr Trp Gly Gin Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 37 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 37 Glu Val Gin Leu Val Gin Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Asp Phe 20 25 30 Trp Val Ser Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp He 35 40 45 Gly Glu He Tyr Pro Asn Ser Gly Val Ser Arg Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Arg Val Thr Met Thr Val Asp Lys Ser He Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Lys Tyr Phe Gly Tyr Thr Tyr Trp Phe Gly Tyr Trp Gly Gin Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 38 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 38 Asp Thr Val Leu Thr Gin Ser Pro Ala Leu Ala Val Ser Leu Gly Gin 1 5 10 15 Arg He Thr He Ser Cys Arg Ala Ser Lys Ser Val Ser Thr Tyr Met 20 25 30 His Trp Tyr Gin Gin Arg Ser Gly Leu Gin Pro Lys Leu Leu He Tyr 35 40 45 Ser Ala Ser His Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asp Pro Val Glu Ala Asp 65 70 75 80 Asp Ile Ala Asn Tyr Tyr Cys Gln Gln Ser Asn Glu Leu Pro Val Thr 85 90 95 Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 39 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 39 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Lys Ser Val Ser Thr Tyr 20 25 30 Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser His Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Asn Glu Leu Pro Val 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 40 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 40 Asp Thr Val Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Lys Ser Val Ser Thr Tyr 20 25 30 Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser His Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Asn Glu Leu Pro Val 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 41 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 41 Asp Thr Val Leu Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Lys Ser Val Ser Thr Tyr 20 25 30 Met His Trp Tyr Gin Gin Lys Pro Gly Lys Gin Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser His Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gin Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gin Gin Ser Asn Glu Leu Pro Val 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 42 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 42 Asp Thr Val Leu Thr Gin Ser Pro Ser Leu Ser Ala Ser Val Gly Asp 1 5 10 15 Arg Val Thr Ile Thr Cys Arg Ala Ser Lys Ser Val Ser Thr Tyr Met 20 25 30 His Trp Tyr Gin Gin Lys Pro Gly Lys Gin Pro Lys Leu Leu Ile Tyr 35 40 45 Ser Ala Ser His Leu Gin Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gin Pro Gin 65 70 75 80 Asp Phe Ala Thr Tyr Tyr Cys Gin Gin Ser Asn Gin Leu Pro Val Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 43 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 43 Asp Ile Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Lys Ser Val Ser Thr Tyr 20 25 30 Met His Trp Tyr Gin Gin Lys Pro Gly Lys Gin Pro Lys Leu Leu lie 35 40 45 Tyr Ser Ala Ser His Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr lie Ser Ser Leu Gin Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gin Gin Ser Asn Glu Leu Pro Val 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu lie Lys 100 105 <210> 44 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 44 Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Arg 1 5 10 15 Ser Leu Thr Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Trp Met Thr Trp lie Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ser lie Thr Asn Thr Gly Ser Ser Thr Phe Tyr Pro Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Arg Ser Thr Leu Phe 65 70 75 80 Leu Gln Ile Asn Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Thr Arg Asp Thr Thr Ile Ala Pro Phe Asp Tyr Trp Gly Gln Gly Val 100 105 110 Met Val Thr Val Ser Ser 115 <210> 45 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 45 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Trp Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Thr Asn Thr Gly Ser Ser Thr Phe Tyr Pro Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Thr Thr lie Ala Pro Phe Asp Tyr Trp Gly Gin Gly Thr 100 105 110 Met Val Thr Val Ser Ser 115 <210> 46 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 46 Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Trp Met Thr Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ser lie Thr Asn Thr Gly Ser Ser Thr Phe Tyr Pro Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Thr Thr lie Ala Pro Phe Asp Tyr Trp Gly Gin Gly Thr 100 105 110 Met Val Thr Val Ser Ser 115 <210> 47 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 47 Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Trp Met Thr Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ser lie Thr Asn Thr Gly Ser Ser Thr Phe Tyr Pro Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Asp Thr Thr lie Ala Pro Phe Asp Tyr Trp Gly Gin Gly Thr 100 105 110 Met Val Thr Val Ser Ser 115 <210> 48 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 48 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Trp Met Thr Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ser Ile Thr Asn Thr Gly Ser Ser Thr Phe Tyr Pro Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Asp Thr Thr Ile Ala Pro Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Met Val Thr Val Ser Ser 115 <210> 49 <211> 118 <212> PRT <213> artificial sequence <220> <223> synthetic <400> 49 Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Trp Met Thr Trp He Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ser He Thr Asn Thr Gly Ser Ser Thr Phe Tyr Pro Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr He Ser Arg Asp Asn Ala Lys Ser Ser Leu Tyr 65 70 75 80 Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Asp Thr Thr He Ala Pro Phe Asp Tyr Trp Gly Gin Gly Thr 100 105 110 Met Val Thr Val Ser Ser 115 <210> 50 <211> 106 <212> PRT <213> artificial sequence <220> <223> synthetic <400> 50 Asp Ile Gin Met Thr Gin Ser Pro Ser Phe Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gin Asn Leu Asn Glu Tyr 20 25 30 Leu Asn Trp Tyr Gin Gin Lys Leu Gly Glu Ala Pro Lys Arg Leu Ile 35 40 45 Tyr Lys Thr Asn Thr Leu Gin Ala Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Ile Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gin Pro 65 70 75 80 Glu Asp Val Ala Thr Tyr Phe Cys Ser Gin Tyr Asn Ser Gly Asn Thr 85 90 95 Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 <210> 51 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 51 Asp Ile Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gin Asn Leu Asn Glu Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile 35 40 45 Tyr Lys Thr Asn Thr Leu Gln Ala Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Ser Gln Tyr Asn Ser Gly Asn Thr 85 90 95 Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 52 <211> 106 <212> PRT <213> artificial sequence <220> <223> synthetic <400> 52 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Leu Asn Glu Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile 35 40 45 Tyr Lys Thr Asn Thr Leu Gln Ala Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Ser Gln Tyr Asn Ser Gly Asn Thr 85 90 95 Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 53 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 53 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Leu Asn Glu Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile 35 40 45 Tyr Lys Thr Asn Thr Leu Gln Ala Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Ser Gln Tyr Asn Ser Gly Asn Thr 85 90 95 Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 54 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 54 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Leu Asn Glu Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile 35 40 45 Tyr Lys Thr Asn Thr Leu Gln Ala Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Ile Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Ser Gln Tyr Asn Ser Gly Asn Thr 85 90 95 Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 55 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 55 Asp Ile Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gin Asn Leu Asn Glu Tyr 20 25 30 Leu Asn Trp Tyr Gin Gin Lys Leu Gly Glu Ala Pro Lys Arg Leu Ile 35 40 45 Tyr Lys Thr Asn Thr Leu Gin Ala Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Ile Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gin Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Phe Cys Ser Gin Tyr Asn Ser Gly Asn Thr 85 90 95 Phe Gly Gin Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 56 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 56 Asp Ile Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Leu Asn Glu Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile 35 40 45 Tyr Lys Thr Asn Thr Leu Gln Ala Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Ser Gln Tyr Gln Ser Gly Asn Thr 85 90 95 Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 57 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 57 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Trp Met Thr Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ser lie Thr Asn Thr Gly Ser Ser Thr Phe Tyr Pro Asp Ala Val 50 55 60 Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Asp Thr Thr lie Ala Pro Phe Asp Tyr Trp Gly Gin Gly Thr 100 105 110 Met Val Thr Val Ser Ser 115 <210> 58 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 58 Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Trp Met Thr Trp lie Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ser lie Thr Asn Thr Gly Ser Ser Thr Phe Tyr Pro Glu Ser Val 50 55 60 Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Asp Thr Thr lie Ala Pro Phe Asp Tyr Trp Gly Gin Gly Thr 100 105 110 Met Val Thr Val Ser Ser 115 <210> 59 <211> 330 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 59 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gin Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gin Thr 65 70 75 80 Tyr lie Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met lie Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gin Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro lie Glu Lys Thr lie Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gin Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp He Ala Val Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gin Gin Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Ala 325 330 <210> 60 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 60 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser Gly 20 <210> 61 <211> 136 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 61 Ile Pro Pro His Val Gin Lys Ser Val Asn Asn Asp Met lie Val Thr 1 5 10 15 Asp Asn Asn Gly Ala Val Lys Phe Pro Gin Leu Cys Lys Phe Cys Asp 20 25 30 Val Arg Phe Ser Thr Cys Asp Asn Gin Lys Ser Cys Met Ser Asn Cys 35 40 45 Ser lie Thr Ser lie Cys Glu Lys Pro Gin Glu Val Cys Val Ala Val 50 55 60 Trp Arg Lys Asn Asp Glu Asn lie Thr Leu Glu Thr Val Cys His Asp 65 70 75 80 Pro Lys Leu Pro Tyr His Asp Phe lie Leu Glu Asp Ala Ala Ser Pro 85 90 95 Lys Cys lie Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met 100 105 110 Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn lie lie Phe Ser Glu 115 120 125 Glu Tyr Asn Thr Ser Asn Pro Asp 130 135 <210> 62 <211> 606 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 62 Glu Val Gin Leu Val Gin Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Phe 20 25 30 Trp Val Ser Trp Val Arg Gin Ala Pro Gly Gin Gly Leu Glu Trp Met 35 40 45 Gly Glu He Tyr Pro Asn Ser Gly Val Ser Arg Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Arg Val Thr Met Thr Val Asp Lys Ser He Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Lys Tyr Phe Gly Tyr Thr Tyr Trp Phe Gly Tyr Trp Gly Gin Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 60 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 450 455 460 Gly Gly Gly Gly Ser Gly lie Pro Pro His Val Gin Lys Ser Val Asn 465 470 475 480 Asn Asp Met lie Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gin 485 490 495 Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gin Lys 500 505 510 Ser Cys Met Ser Asn Cys Ser lie Thr Ser lie Cys Glu Lys Pro Gin 515 520 525 Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn lie Thr Leu 530 535 540 Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe lie Leu 545 550 555 560 Glu Asp Ala Ala Ser Pro Lys Cys lie Met Lys Glu Lys Lys Lys Pro 565 570 575 Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp 580 585 590 Asn lie lie Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp 595 600 605 <210> 63 <211> 605 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 63 Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Trp Met Thr Trp He Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ser He Thr Asn Thr Gly Ser Ser Thr Phe Tyr Pro Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr He Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Asp Thr Thr He Ala Pro Phe Asp Tyr Trp Gly Gin Gly Thr 100 105 110 Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295 300 Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu 340 345 350 Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gin Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly Ala 435 440 445 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 450 455 460 Gly Gly Gly Ser Gly lie Pro Pro His Val Gin Lys Ser Val Asn Asn 465 470 475 480 Asp Met lie Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gin Leu 485 490 495 Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gin Lys Ser 500 505 510 Cys Met Ser Asn Cys Ser lie Thr Ser lie Cys Glu Lys Pro Gin Glu 515 520 525 Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn lie Thr Leu Glu 530 535 540 Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe lie Leu Glu 545 550 555 560 Asp Ala Ala Ser Pro Lys Cys lie Met Lys Glu Lys Lys Lys Pro Gly 565 570 575 Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn 580 585 590 lie lie Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp 595 600 605 <210> 64 <211> 605 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 64 Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Trp Met Thr Trp He Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ser He Thr Asn Thr Gly Ser Ser Thr Phe Tyr Pro Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr He Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Asp Thr Thr He Ala Pro Phe Asp Tyr Trp Gly Gin Gly Thr 100 105 110 Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295 300 Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu 340 345 350 Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gin Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly Ala 435 440 445 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 450 455 460 Gly Gly Gly Ser Gly lie Pro Pro His Val Gin Lys Ser Val Asn Asn 465 470 475 480 Asp Met lie Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gin Leu 485 490 495 Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gin Lys Ser 500 505 510 Cys Met Ser Asn Cys Ser lie Thr Ser lie Cys Glu Lys Pro Gin Glu 515 520 525 Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn lie Thr Leu Glu 530 535 540 Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe lie Leu Glu 545 550 555 560 Asp Ala Ala Ser Pro Lys Cys lie Met Lys Glu Lys Lys Lys Pro Gly 565 570 575 Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn 580 585 590 lie lie Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp 595 600 605 <210> 65 <211> 605 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 65 Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Trp Met Thr Trp He Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ser He Thr Asn Thr Gly Ser Ser Thr Phe Tyr Pro Asp Ala Val 50 55 60 Lys Gly Arg Phe Thr He Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Asp Thr Thr He Ala Pro Phe Asp Tyr Trp Gly Gin Gly Thr 100 105 110 Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295 300 Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu 340 345 350 Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gin Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly Ala 435 440 445 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 450 455 460 Gly Gly Gly Ser Gly Ile Pro Pro His Val Gln Lys Ser Val Asn Asn 465 470 475 480 Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu 485 490 495 Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser 500 505 510 Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu 515 520 525 Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu 530 535 540 Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu 545 550 555 560 Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly 565 570 575 Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn 580 585 590 Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp 595 600 605 <210> 66 <211> 605 <212> PRT <213> 人工序列 <220> <223> 合成的 <400> 66 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Trp Met Thr Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ser Ile Thr Asn Thr Gly Ser Ser Thr Phe Tyr Pro Glu Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Asp Thr Thr Ile Ala Pro Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295 300 Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu 340 345 350 Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gin Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly Ala 435 440 445 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 450 455 460 Gly Gly Gly Ser Gly lie Pro Pro His Val Gin Lys Ser Val Asn Asn 465 470 475 480 Asp Met lie Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gin Leu 485 490 495 Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gin Lys Ser 500 505 510 Cys Met Ser Asn Cys Ser lie Thr Ser lie Cys Glu Lys Pro Gin Glu 515 520 525 Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn lie Thr Leu Glu 530 535 540 Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe lie Leu Glu 545 550 555 560 Asp Ala Ala Ser Pro Lys Cys lie Met Lys Glu Lys Lys Lys Pro Gly 565 570 575 Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn 580 585 590 lie lie Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp 595 600 605 <210> 67 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 67 Asp Ile Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Lys Ser Val Ser Thr Tyr 20 25 30 Met His Trp Tyr Gin Gin Lys Pro Gly Lys Gin Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser His Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gin Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gin Gin Ser Asn Glu Leu Pro Val 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gin Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gin Trp Lys Val Asp Asn Ala Leu Gin Ser Gly Asn Ser Gin 145 150 155 160 Glu Ser Val Thr Glu Gin Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gin Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 68 <211> 213 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 68 Asp He Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr He Thr Cys Lys Ala Ser Gin Asn Leu Asn Glu Tyr 20 25 30 Leu Asn Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Lys Arg Leu He 35 40 45 Tyr Lys Thr Asn Thr Leu Gin Ala Gly He Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr lie Ser Ser Leu Gin Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Ser Gin Tyr Asn Ser Gly Asn Thr 85 90 95 Phe Gly Gin Gly Thr Lys Leu Glu lie Lys Arg Thr Val Ala Ala Pro 100 105 110 Ser Val Phe lie Phe Pro Pro Ser Asp Glu Gin Leu Lys Ser Gly Thr 115 120 125 Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys 130 135 140 Val Gin Trp Lys Val Asp Asn Ala Leu Gin Ser Gly Asn Ser Gin Glu 145 150 155 160 Ser Val Thr Glu Gin Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser 165 170 175 Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala 180 185 190 Cys Glu Val Thr His Gin Gly Leu Ser Ser Pro Val Thr Lys Ser Phe 195 200 205 Asn Arg Gly Glu Cys 210 <210> 69 <211> 213 <212> PRT <213> Artificial sequences <220> <223> Synthetic <400> 69 Asp Ile Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gin Asn Leu Asn Glu Tyr 20 25 30 Leu Asn Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile 35 40 45 Tyr Lys Thr Asn Thr Leu Gin Ala Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gin Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Ser Gin Tyr Gin Ser Gly Asn Thr 85 90 95 Phe Gly Gin Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro 100 105 110 Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gin Leu Lys Ser Gly Thr 115 120 125 Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys 130 135 140 Val Gin Trp Lys Val Asp Asn Ala Leu Gin Ser Gly Asn Ser Gin Glu 145 150 155 160 Ser Val Thr Glu Gin Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser 165 170 175 Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala 180 185 190 Cys Glu Val Thr His Gin Gly Leu Ser Ser Pro Val Thr Lys Ser Phe 195 200 205 Asn Arg Gly Glu Cys 210 <210> 70 <211> 592 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 70 Met Gly Arg Gly Leu Leu Arg Gly Leu Trp Pro Leu His Ile Val Leu 1 5 10 15 Trp Thr Arg Ile Ala Ser Thr Ile Pro Pro His Val Gin Lys Ser Asp 20 25 30 Val Glu Met Glu Ala Gin Lys Asp Glu Ile Ile Cys Pro Ser Cys Asn 35 40 45 Arg Thr Ala His Pro Leu Arg His Ile Asn Asn Asp Met Ile Val Thr 50 55 60 Asp Asn Asn Gly Ala Val Lys Phe Pro Gin Leu Cys Lys Phe Cys Asp 65 70 75 80 Val Arg Phe Ser Thr Cys Asp Asn Gin Lys Ser Cys Met Ser Asn Cys 85 90 95 Ser He Thr Ser He Cys Glu Lys Pro Gin Glu Val Cys Val Ala Val 100 105 110 Trp Arg Lys Asn Asp Glu Asn He Thr Leu Glu Thr Val Cys His Asp 115 120 125 Pro Lys Leu Pro Tyr His Asp Phe He Leu Glu Asp Ala Ala Ser Pro 130 135 140 Lys Cys He Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met 145 150 155 160 Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn He He Phe Ser Glu 165 170 175 Glu Tyr Asn Thr Ser Asn Pro Asp Leu Leu Leu Val He Phe Gin Val 180 185 190 Thr Gly He Ser Leu Leu Pro Pro Leu Gly Val Ala He Ser Val He 195 200 205 He He Phe Tyr Cys Tyr Arg Val Asn Arg Gin Gin Lys Leu Ser Ser 210 215 220 Thr Trp Glu Thr Gly Lys Thr Arg Lys Leu Met Glu Phe Ser Glu His 225 230 235 240 Cys Ala Ile Ile Leu Glu Asp Asp Arg Ser Asp Ile Ser Ser Thr Cys 245 250 255 Ala Asn Asn Ile Asn His Asn Thr Glu Leu Leu Pro Ile Glu Leu Asp 260 265 270 Thr Leu Val Gly Lys Gly Arg Phe Ala Glu Val Tyr Lys Ala Lys Leu 275 280 285 Lys Gln Asn Thr Ser Glu Gin Phe Glu Thr Val Ala Val Lys Ile Phe 290 295 300 Pro Tyr Glu Glu Tyr Ala Ser Trp Lys Thr Glu Lys Asp Ile Phe Ser 305 310 315 320 Asp Ile Asn Leu Lys His Glu Asn Ile Leu Gin Phe Leu Thr Ala Glu 325 330 335 Glu Arg Lys Thr Glu Leu Gly Lys Gin Tyr Trp Leu Ile Thr Ala Phe 340 345 350 His Ala Lys Gly Asn Leu Gin Gin Tyr Leu Thr Arg His Val Ile Ser 355 360 365 Trp Glu Asp Leu Arg Lys Leu Gly Ser Ser Leu Ala Arg Gly Ile Ala 370 375 380 His Leu His Ser Asp His Thr Pro Cys Gly Arg Pro Lys Met Pro Ile 385 390 395 400 Val His Arg Asp Leu Lys Ser Ser Asn Ile Leu Val Lys Asn Asp Leu 405 410 415 Thr Cys Cys Leu Cys Asp Phe Gly Leu Ser Leu Arg Leu Asp Pro Thr 420 425 430 Leu Ser Val Asp Asp Leu Ala Asn Ser Gly Gln Val Gly Thr Ala Arg 435 440 445 Tyr Met Ala Pro Glu Val Leu Glu Ser Arg Met Asn Leu Glu Asn Val 450 455 460 Glu Ser Phe Lys Gln Thr Asp Val Tyr Ser Met Ala Leu Val Leu Trp 465 470 475 480 Glu Met Thr Ser Arg Cys Asn Ala Val Gly Glu Val Lys Asp Tyr Glu 485 490 495 Pro Pro Phe Gly Ser Lys Val Arg Glu His Pro Cys Val Glu Ser Met 500 505 510 Lys Asp Asn Val Leu Arg Asp Arg Gly Arg Pro Glu Ile Pro Ser Phe 515 520 525 Trp Leu Asn His Gln Gly Ile Gln Met Val Cys Glu Thr Leu Thr Glu 530 535 540 Cys Trp Asp His Asp Pro Glu Ala Arg Leu Thr Ala Gln Cys Val Ala 545 550 555 560 Glu Arg Phe Ser Glu Leu Glu His Leu Asp Arg Leu Ser Gly Arg Ser 565 570 575 Cys Ser Glu Glu Lys Ile Pro Glu Asp Gly Ser Leu Asn Thr Thr Lys 580 585 590 <210> 71 <211> 567 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 71 Met Gly Arg Gly Leu Leu Arg Gly Leu Trp Pro Leu His Ile Val Leu 1 5 10 15 Trp Thr Arg Ile Ala Ser Thr Ile Pro Pro His Val Gln Lys Ser Val 20 25 30 Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro 35 40 45 Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln 50 55 60 Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro 65 70 75 80 Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr 85 90 95 Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile 100 105 110 Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys 115 120 125 Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn 130 135 140 Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp Leu 145 150 155 160 Leu Leu Val Ile Phe Gln Val Thr Gly Ile Ser Leu Leu Pro Pro Leu 165 170 175 Gly Val Ala Ile Ser Val Ile Ile Ile Phe Tyr Cys Tyr Arg Val Asn 180 185 190 Arg Gln Gln Lys Leu Ser Ser Thr Trp Glu Thr Gly Lys Thr Arg Lys 195 200 205 Leu Met Glu Phe Ser Glu His Cys Ala Ile Ile Leu Glu Asp Asp Arg 210 215 220 Ser Asp Ile Ser Ser Thr Cys Ala Asn Asn Ile Asn His Asn Thr Glu 225 230 235 240 Leu Leu Pro lie Glu Leu Asp Thr Leu Val Gly Lys Gly Arg Phe Ala 245 250 255 Glu Val Tyr Lys Ala Lys Leu Lys Gin Asn Thr Ser Glu Gin Phe Glu 260 265 270 Thr Val Ala Val Lys lie Phe Pro Tyr Glu Glu Tyr Ala Ser Trp Lys 275 280 285 Thr Glu Lys Asp lie Phe Ser Asp lie Asn Leu Lys His Glu Asn lie 290 295 300 Leu Gin Phe Leu Thr Ala Glu Glu Arg Lys Thr Glu Leu Gly Lys Gin 305 310 315 320 Tyr Trp Leu lie Thr Ala Phe His Ala Lys Gly Asn Leu Gin Glu Tyr 325 330 335 Leu Thr Arg His Val lie Ser Trp Glu Asp Leu Arg Lys Leu Gly Ser 340 345 350 Ser Leu Ala Arg Gly lie Ala His Leu His Ser Asp His Thr Pro Cys 355 360 365 Gly Arg Pro Lys Met Pro lie Val His Arg Asp Leu Lys Ser Ser Asn 370 375 380 Ile Leu Val Lys Asn Asp Leu Thr Cys Cys Leu Cys Asp Phe Gly Leu 385 390 395 400 Ser Leu Arg Leu Asp Pro Thr Leu Ser Val Asp Asp Leu Ala Asn Ser 405 410 415 Gly Gln Val Gly Thr Ala Arg Tyr Met Ala Pro Glu Val Leu Glu Ser 420 425 430 Arg Met Asn Leu Glu Asn Val Glu Ser Phe Lys Gln Thr Asp Val Tyr 435 440 445 Ser Met Ala Leu Val Leu Trp Glu Met Thr Ser Arg Cys Asn Ala Val 450 455 460 Gly Glu Val Lys Asp Tyr Glu Pro Pro Phe Gly Ser Lys Val Arg Glu 465 470 475 480 His Pro Cys Val Glu Ser Met Lys Asp Asn Val Leu Arg Asp Arg Gly 485 490 495 Arg Pro Glu Ile Pro Ser Phe Trp Leu Asn His Gln Gly Ile Gln Met 500 505 510 Val Cys Glu Thr Leu Thr Glu Cys Trp Asp His Asp Pro Glu Ala Arg 515 520 525 Leu Thr Ala Gln Cys Val Ala Glu Arg Phe Ser Glu Leu Glu His Leu 530 535 540 Asp Arg Leu Ser Gly Arg Ser Cys Ser Glu Glu Lys Ile Pro Glu Asp 545 550 555 560 Gly Ser Leu Asn Thr Thr Lys 565 <210> 72 <211> 102 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 72 Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln 1 5 10 15 Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro 20 25 30 Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr 35 40 45 Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile 50 55 60 Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys 65 70 75 80 Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn 85 90 95 Asp Asn Ile Ile Phe Ser 100 <210> 73 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 73 Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln 1 5 10 15 Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro 20 25 30 Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr 35 40 45 Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile 50 55 60 Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys 65 70 75 80 Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn 85 90 95 Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp 100 105 110 <210> 74 <211> 136 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <220> <221> misc_feature <222> (7)..(8) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (19)..(19) <223> Xaa can be any naturally occurring amino acid <400> 74 Ile Pro Pro His Val Gin Xaa Xaa Val Asn Asn Asp Met lie Val Thr 1 5 10 15 Asp Asn Xaa Gly Ala Val Lys Phe Pro Gin Leu Cys Lys Phe Cys Asp 20 25 30 Val Arg Phe Ser Thr Cys Asp Asn Gin Lys Ser Cys Met Ser Asn Cys 35 40 45 Ser lie Thr Ser lie Cys Glu Lys Pro Gin Glu Val Cys Val Ala Val 50 55 60 Trp Arg Lys Asn Asp Glu Asn lie Thr Leu Glu Thr Val Cys His Asp 65 70 75 80 Pro Lys Leu Pro Tyr His Asp Phe lie Leu Glu Asp Ala Ala Ser Pro 85 90 95 Lys Cys lie Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met 100 105 110 Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn lie lie Phe Ser Glu 115 120 125 Glu Tyr Asn Thr Ser Asn Pro Asp 130 135 <210> 75 <211> 136 <212> PRT <213> Artificial sequences <220> <223> Synthetic <400> 75 Thr Ala Gly His Thr Gin Thr Ser Thr Gly Gly Gly Ala lie Thr Thr 1 5 10 15 Gly Thr Ser Gly Ala Gly His Gly Pro Gin Leu Cys Lys Phe Cys Asp 20 25 30 Val Arg Phe Ser Thr Cys Asp Asn Gin Lys Ser Cys Met Ser Asn Cys 35 40 45 Ser lie Thr Ser lie Cys Glu Lys Pro Gin Glu Val Cys Val Ala Val 50 55 60 Trp Arg Lys Asn Asp Glu Asn lie Thr Leu Glu Thr Val Cys His Asp 65 70 75 80 Pro Lys Leu Pro Tyr His Asp Phe lie Leu Glu Asp Ala Ala Ser Pro 85 90 95 Lys Cys lie Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met 100 105 110 Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn lie lie Phe Ser Glu 115 120 125 Glu Tyr Asn Thr Ser Asn Pro Asp 130 135 <210> 76 <211> 127 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 76 Thr Ala Gly His Thr Gin Thr Ser Thr Gly Gly Gly Ala lie Thr Thr 1 5 10 15 Gly Thr Ser Gly Ala Gly His Gly Pro Gin Leu Cys Lys Phe Cys Asp 20 25 30 Val Arg Phe Ser Thr Cys Asp Asn Gin Lys Ser Cys Met Ser Asn Cys 35 40 45 Ser lie Thr Ser lie Cys Glu Lys Pro Gin Glu Val Cys Val Ala Val 50 55 60 Trp Arg Lys Asn Asp Glu Asn lie Thr Leu Glu Thr Val Cys His Asp 65 70 75 80 Pro Lys Leu Pro Tyr His Asp Phe lie Leu Glu Asp Ala Ala Ser Pro 85 90 95 Lys Cys lie Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met 100 105 110 Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn lie lie Phe Ser 115 120 125 <210> 77 <211> 105 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 77 His Tyr Pro Gin Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys 1 5 10 15 Asp Asn Gin Lys Ser Cys Met Ser Asn Cys Ser He Thr Ser He Cys 20 25 30 Glu Lys Pro Gin Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu 35 40 45 Asn He Thr Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His 50 55 60 Asp Phe He Leu Glu Asp Ala Ala Ser Pro Lys Cys He Met Lys Glu 65 70 75 80 Lys Lys Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp 85 90 95 Glu Cys Asn Asp Asn He He Phe Ser 100 105 <210> 78 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 78 His Tyr Pro Gin Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys 1 5 10 15 Asp Asn Gin Lys Ser Cys Met Ser Asn Cys Ser He Thr Ser He Cys 20 25 30 Glu Lys Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu 35 40 45 Asn Ile Thr Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His 50 55 60 Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu 65 70 75 80 Lys Lys Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp 85 90 95 Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn 100 105 110 Pro Asp <210> 79 <211> 157 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 79 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser Gly Ile Pro Pro His Val Gln Lys Ser Val Asn Asn 20 25 30 Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu 35 40 45 Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser 50 55 60 Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu 65 70 75 80 Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu 85 90 95 Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu 100 105 110 Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly 115 120 125 Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn 130 135 140 Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp 145 150 155 <210> 80 <211> 141 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 80 Gly Gly Gly Gly Ser Thr Ala Gly His Thr Gln Thr Ser Thr Gly Gly 1 5 10 15 Gly Ala Ile Thr Thr Gly Thr Ser Gly Ala Gly His Gly Pro Gln Leu 20 25 30 Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser 35 40 45 Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu 50 55 60 Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu 65 70 75 80 Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu 85 90 95 Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly 100 105 110 Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn 115 120 125 Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp 130 135 140 <210> 81 <211> 161 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 81 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Ala Gly His Thr Gin Thr 20 25 30 Ser Thr Gly Gly Gly Ala Ile Thr Thr Gly Thr Ser Gly Ala Gly His 35 40 45 Gly Pro Gin Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp 50 55 60 Asn Gin Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu 65 70 75 80 Lys Pro Gin Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn 85 90 95 Ile Thr Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp 100 105 110 Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys 115 120 125 Lys Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu 130 135 140 Cys Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro 145 150 155 160 Asp <210> 82 <211> 152 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 82 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Ala Gly His Thr Gln Thr 20 25 30 Ser Thr Gly Gly Gly Ala Ile Thr Thr Gly Thr Ser Gly Ala Gly His 35 40 45 Gly Pro Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp 50 55 60 Asn Gln Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu 65 70 75 80 Lys Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn 85 90 95 Ile Thr Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp 100 105 110 Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys 115 120 125 Lys Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu 130 135 140 Cys Asn Asp Asn Ile Ile Phe Ser 145 150 <210> 83 <211> 131 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 83 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 Gly Gly Gly Gly Ser His Tyr Pro Gin Leu Cys Lys Phe Cys Asp Val 20 25 30 Arg Phe Ser Thr Cys Asp Asn Gin Lys Ser Cys Met Ser Asn Cys Ser 35 40 45 Ile Thr Ser Ile Cys Glu Lys Pro Gin Glu Val Cys Val Ala Val Trp 50 55 60 Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp Pro 65 70 75 80 Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys 85 90 95 Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met Cys 100 105 110 Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser 115 120 125 130 <210> 84​ <211> 140 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 84 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 Gly Gly Gly Gly Ser His Tyr Pro Gin Leu Cys Lys Phe Cys Asp Val 20 25 30 Arg Phe Ser Thr Cys Asp Asn Gin Lys Ser Cys Met Ser Asn Cys Ser 35 40 45 Ile Thr Ser Ile Cys Glu Lys Pro Gin Glu Val Cys Val Ala Val Trp 50 55 60 Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp Pro 65 70 75 80 Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys 85 90 95 Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met Cys 100 105 110 Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu Glu 115 120 125 Tyr Asn Thr Ser Asn Pro Asp 130 135 140 <210> 85 <211> 129 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 85 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser His 1 5 10 15 Tyr Pro Gin Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp 20 25 30 Asn Gin Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu 35 40 45 Lys Pro Gin Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn 50 55 60 Ile Thr Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp 65 70 75 80 Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys 85 90 95 Lys Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu 100 105 110 Cys Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro 115 120 125 Asp <210> 86 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 86 Gly Gly Gly Gly Ser 1 5 <210> 87 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 87 Thr Ala Gly His Thr Gin Thr Ser Thr Gly Gly Gly Ala He Thr Thr 1 5 10 15 Gly Thr Ser Gly Ala Gly His Gly Pro 20 25 <210> 88 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <220> <221> misc_feature <222> (7)..(8) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (19)..(19) <223> Xaa can be any naturally occurring amino acid <400> 88 Ile Pro Pro His Val Gin Xaa Xaa Val Asn Asn Asp Met He Val Thr 1 5 10 15 Asp Asn Xaa Gly Ala Val Lys Phe Pro 20 25 <210> 89 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 89 Ile Pro Pro His Val Gin Lys Ser Val Asn Asn Asp Met lie Val Thr 1 5 10 15 Asp Asn Asn Gly Ala Val Lys Phe Pro 20 25 <210> 90 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 90 Glu Glu Tyr Asn Thr Ser Asn Pro Asp 1 5 <210> 91 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 91 Ser lie Thr Asn Thr Gly Ser Ser Thr Phe Tyr Pro Asp Ala Val Lys 1 5 10 15 Gly <210> 92 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 92 Ser lie Thr Asn Thr Gly Ser Ser Thr Phe Tyr Pro Glu Ser Val Lys 1 5 10 15 Gly <210> 93 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 93 Ser Gin Tyr Gin Ser Gly Asn Thr 1 5

Claims

1. A multifunctional molecule comprising an anti-PD-L1 (programmed death ligand 1) antibody and the extracellular domain of human TGF-βRII (TGF-β receptor type 2), wherein the anti-PD-L1 antibody is specific for human PD-L1 protein and comprises a heavy chain variable region (VH) comprising VH CDR1, VH CDR2 and VH CDR3, and a light chain variable region (VL) comprising VL CDR1, VL CDR2 and VL CDR3, comprising a light chain comprising the VL and a light chain constant region, and a heavy chain comprising the VH, a heavy chain constant region, a peptide linker, and the TGF-βRII extracellular domain; wherein the TGF-βRII extracellular domain is fused to the C-terminus of the heavy chain of the anti-PD-L1 antibody or a fragment thereof via a peptide linker; wherein the C-terminal lysine of the heavy chain constant region is mutated to alanine; wherein the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 are: (1) the amino acid sequence of SEQ ID NO: 13-18; or (2) the amino acid sequences of SEQ ID NOs: 19, 20, 21, 22, 23, and 24; or (3) the amino acid sequences of SEQ ID NOs: 19, 92, 21, 22, 23, and 93; or (4) the amino acid sequences of SEQ ID NOs: 19, 20, 21, 22, 23, and 93; or (5) amino acid sequences of SEQ ID NOs: 19, 91, 21, 22, 23, and 93; The human TGF-βRII extracellular domain is any one of the amino acid sequences of SEQ ID NO: 61, 75-78, and is fused with the anti-PD-L1 antibody.

2. The multifunctional molecule according to claim 1, wherein the VH is an amino acid sequence selected from the group consisting of SEQ ID NOs: 44-49 and 57-58, and the VL is an amino acid sequence selected from the group consisting of SEQ ID NOs: 50-56.

3. The multifunctional molecule according to claim 2, wherein the VH is the amino acid sequence of SEQ ID NO: 48, 57 or 58, and the VL is the amino acid sequence of SEQ ID NO: 53 or 56.

4. The multifunctional molecule according to claim 1, wherein VH CDR1, VH CDR2, VH CDR3, VL CDR1, VLCDR2 and VL CDR3 are the amino acid sequences of SEQ ID NOs: 19, 92, 21, 22, 23 and 93, respectively.

5. The multifunctional molecule according to claim 4, wherein the VH has the amino acid sequence of SEQ ID NO: 58, and the VL has the amino acid sequence of SEQ ID NO:

56.

6. The multifunctional molecule according to claim 1, wherein the heavy chain constant region is the amino acid sequence of SEQ ID NO:

59.

7. The multifunctional molecule of claim 1, wherein the peptide linker comprises a flexible linker and / or an alternative peptide to IPPHVQKSVNNDMIVTDNNGAVKFP (SEQ ID NO: 89), wherein the alternative peptide is different from SEQ ID NO:

89.

8. The multifunctional molecule of claim 7, wherein the surrogate peptide comprises the amino acid sequence of IPPHVQXXVNNDMIVTDNXGAVKFP (SEQ ID NO: 88), wherein X is any amino acid except K, S or N.

9. The multifunctional molecule according to claim 7, wherein the flexible linker comprises S and at least 50% G.

10. The multifunctional molecule according to claim 9, wherein the flexible linker comprises one or more GGGGS (SEQ ID NO: 86) units.

11. The multifunctional molecule according to claim 7, which does not at least comprise the entire sequence of EEYNTSNPD (SEQ ID NO: 90).

12. A cell comprising one or more polynucleotides encoding the multifunctional molecule of any one of claims 1-11.

13. One or more polynucleotides encoding the multifunctional molecule of any one of claims 1-11.

14. A composition comprising the multifunctional molecule according to any one of claims 1 to 11 and a pharmaceutically suitable carrier.

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