Anti-tumor immune checkpoint regulator antagonists

Abstract

The present application discloses an anti-tumor antagonist that specifically binds to an immune checkpoint regulator. The present application also discloses a method for treating a proliferative disease using the anti-tumor antagonist.

Description

[0001] This application claims priority to U.S. Provisional Patent Application No. 62 / 691,658, filed June 29, 2018, and U.S. Provisional Patent Application No. 62 / 823,989, filed March 26, 2019, the contents of which are expressly incorporated herein by reference. Technical Field

[0002] This application relates primarily to cancer treatment, and more particularly to bispecific inhibitors capable of modulating pathways associated with tumorigenesis and tumor immunity. Background Technology

[0003] The inability of the host to eliminate cancer cells remains a major problem. Despite the increasing number of approved therapeutic monoclonal antibodies for treating various cancers, resistance to these antibodies is frequently observed due to the many different molecular pathways involved in cancer growth and metastasis. Although the immune system is the primary mechanism for cancer prevention, cancer cells can resist immune surveillance. Natural control mechanisms limiting T cell activation have been identified to prevent collateral damage caused by unrestrained T cell activity. Tumor cells have utilized this process to evade the immune response. Restoring the ability of immune effector cells, particularly T cells, to recognize and eliminate cancer is a primary goal of immunotherapy.

[0004] There is a need for improved therapeutic binding antagonists or antibodies, as well as methods for using such agents to treat cancer and chronic viral infections. Summary of the Invention

[0005] One aspect of this application relates to a bispecific antitumor antagonist comprising a first targeting domain that specifically binds to an immune checkpoint modulator; a second targeting domain in the form of scFv that specifically binds to TIGIT; and an immunoglobulin scaffold structurally connected to the first and second targeting domains, wherein the first targeting domain is located at the N-terminus of the antagonist, and wherein the second targeting domain is located at the C-terminus of the antagonist and is connected to the immunoglobulin scaffold via a linker. In some embodiments, the linker comprises four or six copies of the amino acid sequence G4S (4x G4S and 6x G4S, respectively).

[0006] In some implementations, the first targeting domain specifically binds to PD-1, PD-L1, or LAG-3.

[0007] Another aspect of this application relates to humanized anti-LAG-3 antibodies that inhibit the binding of ligands to LAG-3.

[0008] Another aspect of this application relates to a method for treating proliferative cell diseases. The method includes administering an effective amount of the bispecific antitumor antagonist or anti-LAG-3 antibody of this application to a subject in need. Attached Figure Description

[0009] Figure 1 The complementarity-determining region (CDR) sequences of certain anti-TIGIT mAb (monoclonal antibody) are shown. The frame region (FR) sequences flanking the anti-TIGIT CDR sequence are also shown. Figure 39A It is listed as SEQ ID NO:216-262.

[0010] Figure 2A-2C Several implementation schemes of the variable domain sequence of the anti-TIGIT antibody are shown.

[0011] Figure 3 The CDR sequences for some anti-PD-1 mAb are shown. The FR sequences flanking the anti-PD-1 CDR sequences are also shown. Figure 39B It is listed as SEQ ID NO: 263-292.

[0012] Figures 4A-4B Several implementation schemes of the variable domain sequence of the anti-PD-1 antibody are shown.

[0013] Figure 5 The image shows some CDR sequences for anti-PD-L1 mAb. The FR sequences flanking the anti-PD-L1 CDR sequences are shown. Figure 39C It is listed as SEQ ID NO: 293-315.

[0014] Figures 6A-6C Several implementation schemes of the variable domain sequence of the anti-PD-L1 antibody are shown.

[0015] Figures 7A-7C Three exemplary bispecific antitumor antagonists are shown, namely Bi-TPM-93 ( Figure 7A Bi-TPM-94A Figure 7B ) and Bi-TPM-94B ( Figure 7C ).

[0016] Figure 8 summarizes the... Figures 7A to 7C The arrangement of functional domains in the bispecific antagonist is shown in the figure.

[0017] Figures 9A to 9B It shows the corresponding Figures 7A-7C The heavy chain (HC) and light chain (LC) amino acid sequences of the bispecific antagonist are shown in the figure.

[0018] Figure 10The blocking assay is shown, which demonstrates that Bi-TPM-94A (IC50 = 1.5 nM) blocks the interaction between PD-1 and its ligand PD-L1 better than Bi-TPM-93 (IC50 = 0.83 nM).

[0019] Figure 11 Non-reducing PAGE analysis of transient expression of Bi-TPM-94A and Bi-TPM-94B in human embryonic kidney (HEK)293 cells is shown.

[0020] Figure 12 Size exclusion ultra-high performance liquid chromatography (SE-UHPLC) analysis is shown, which illustrates the species heterogeneity in Bi-TPM-93 and Bi-TPM-94, which is eliminated by linker modification in Bi-TPM-94B.

[0021] Figure 13A The results showed that Bi-TPM-94A and Bi-TPM-94B had a stronger binding affinity for PD-1 than the baseline anti-PD-1 antibody. Figure 13B The results showed that Bi-TPM-94A and Bi-TPM-94B had a stronger binding affinity for TIGIT than the baseline anti-TIGIT antibody.

[0022] Figures 14A-14B The results showed that Bi-TPM-94A and Bi-TPM-94B effectively blocked the binding of TIGIT to its ligand human PVR (CD155). Figure 14A And it blocked the binding of PD-1 to its ligand PD-L1. Figure 14B ).

[0023] Figure 15 The results of the ELISA assay demonstrate that Bi-TPM-94A and Bi-TPM-94B simultaneously bind to PD-1 and TIGIT. A 96-well plate coated with huPD-1-Fc was incubated with serially diluted samples of Bi-TPM-94A and Bi-TPM-94B, followed by incubation with His-tagged huTIGIT protein. The bound molecules were then detected using HRP-conjugated anti-His-tag Abs and TMB substrates.

[0024] Figures 16A-16B Showing individuals or combinations of parental anti-PD-1 and anti-TIGIT antibodies, as well as negative controls, from human PBMCs (donor 287) with Bi-TPM-94B. Figure 16A Donor 401 Figure 16B Increased IFN-γ secretion.

[0025] Figures 17A-17BThe results showed that Bi-TPM-94B enhanced the levels of donor 287 PBMCs to a greater extent than individual or combination of parental anti-PD-P1 and anti-TIGIT antibodies, as well as the negative control. Figure 17A ) and donor 401 PBMC ( Figure 17B The proliferation of primary human T cells.

[0026] Figure 18 These are pharmacokinetic curves, showing that Bi-TPM-94A and Bi-TPM-94B have similar in vivo half-lives (T1) after intravenous injection into 6-10 week old female CD1 mice. 1 / 2 The bispecific antagonist was recovered from serum collected at different time points after injection and analyzed by ELISA.

[0027] Figure 19A The heavy chain CDR sequences corresponding to anti-LAG-3mAb 2L2A.1, 2L2A.6, 2L27B and 3L1A are shown. Figure 19B The light chain CDR sequences corresponding to anti-LAG-3 mAbs 2L2A.1, 2L2A.6, 2L27B, and 3L1A are shown. The FR sequence flanking the anti-LAG-3 CDR sequence is... Figure 39C It is listed in SEQ ID NO: 316-337.

[0028] Figure 20 The VH and VL sequences of anti-LAG-3mAb 2L2A.1, 2L2A.6, 2L27B and 3L1A are shown.

[0029] Figures 21A-21B The results of the assay confirm the ability of the anti-LAG-3 mAb to block LAG-3 binding.

[0030] Figure 22 The results of a cell-based blocking assay are shown, which measures the ability of anti-LAG-3 mAb 2L2A.1, baseline (BM) anti-LAG-3 mAb, and chimeric 2L2A antibodies (SEQ ID NO: 203 and 204) to block the interaction between LAG-3-muFc and its major ligand (the major histocompatibility complex (MHC) antigen expressed on Raji cells). Assay data were used to calculate the described IC50 values ​​(nm).

[0031] Figures 23A-23B The resistance to LAG-3 2L2A.1 mAb to human LAG-3-His (as determined by surface plasmon resonance (SPR)) is shown. Figure 23A ) or human LAG-3-mIgG2a ( Figure 23B Affinity analysis of ) and corresponding binding affinity constants.

[0032] Figure 24 The binding of the anti-LAG-3 mAb variant 2L2A.1 or the baseline antibody (BM) to LAG-3 is shown, including the half-maximum effective concentration (EC50) generated between baseline and maximum. 50 ).

[0033] Figure 25 The image shows a non-denaturing polyacrylamide gel (PAGE) analysis of the humanized anti-LAG-3 mAb variant 2L2A.1 transiently expressed by HEK293. The positive control was HybPL1 (1PL11 CDRg-VH; 1PL25 CDRg-VL).

[0034] Figure 26 FACS analysis showed that LAG-3 and PD-1 were activated in human CD3. + Co-expression in T cells.

[0035] Figures 27A-27B The IFN-γ generation was shown to originate from two donors (donor 0105, ...). Figure 27A Donor 0817 Figure 27B PBMCs were stimulated with or without staphylococcal enterotoxin B (SEB; lanes 2-4) in 96-well plates. Following stimulation, donor PBMCs were incubated with either no antibody (lanes 1 and 2), anti-LAG-3 baseline antibody (BM) (lane 3), or anti-LAG-3 mAb 2L2A.1. The results of this assay indicated that 2L2A.1 induced greater IFN-γ production in both donor PBMCs compared to the anti-LAG-3 baseline antibody.

[0036] Figures 28A-28C Displaying PBMCs (donor 223) from three donors, stimulated with SEB (lanes 2-4) or without SEB stimulation (channel 1). Figure 28A ; Donor 224, Figure 28B ; Donor 225, Figure 28C Increased IFN-γ production. In addition, donor PBMCs were incubated with the following antibodies: antibody-free (lanes 1 and 2), anti-LAG-3 baseline antibody (lane 3), or anti-LAG-3 mAb2L2A.1.

[0037] Figures 29A-29C The anti-LAG-3 mAb 2L2A.1 showed a greater enhancement of the response from donor 223 than the baseline anti-LAG-3 antibody. Figure 29A ), donor 224 ( Figure 29B ) and donor 225 ( Figure 29C The proliferation of primary human T cells.

[0038] Figures 30A-30BTwo exemplary bispecific antitumor antagonists, Bi-LT-1, are shown. Figure 30A ) and Bi-LT-3( Figure 30B ).

[0039] Figure 31 Summarize Figures 30A to 30B The arrangement of functional domains in the bispecific antagonist is shown in the figure.

[0040] Figure 32 The display corresponds to Figures 30A-30B The heavy chain (HC) and light chain (LC) amino acid sequences of the bispecific antagonist are shown in the figure.

[0041] Figures 33A-33B The results of a cell-based blocking assay are shown, which measures the blocking effect of bispecific antagonists LT-1 and LT-3, as well as bispecific antagonists Bi-TPM-94B or anti-LAG-3 baseline mAb, on the interaction between LAG-3-muFc and its major ligand, the major histocompatibility complex (MHC) antigen expressed on Raji cells. Figure 33A Or block the interaction between TIGIT and its ligand, namely human PVR (CD 155). Figure 33B The measurement data is used to calculate the IC shown. 50 Value (nm).

[0042] Figure 34 The results of the ELISA assay demonstrate the simultaneous binding of LAG-3 and TIGIT via Bi-LT-1, Bi-LT-3, or parental anti-LAG-3 mAb. A 96-well plate coated with LAG-3-muFc was incubated with serially diluted samples of Bi-LT-1, Bi-LT-3, or parental anti-LAG-3 mAb, followed by incubation with His-tagged huTIGIT protein. The binding molecules were then detected using HRP-conjugated anti-His-tag HRP and TMB substrates. The assay data were used to calculate the EC50 values ​​shown. 50 Value (nm).

[0043] Figures 35A-35D This shows that after injection into the tail vein of 6-10 week old female CD1 mice, the corresponding parental anti-LAG-3 mAb ( Figure 35A ), anti-LAG-3 benchmark mAb( Figure 35B ), Bi-LT-1 ( Figure 35C ) or Bi-LT-3( Figure 35D Pharmacokinetic curves and in vivo half-life (T) 1 / 2 Antibodies and bispecific antagonists were recovered from serum collected at different time points after injection and analyzed by ELISA. Parental anti-LAG-3 mAb, Bi-LT-1, and Bi-LT-3 T...1 / 2 For 5 to 6 days, the T-cell antibody against LAG-3BMmAb... 1 / 2 The duration was 2 days (mice 3), 3 days, or 7 days (mice 4).

[0044] Figure 36A Size exclusion chromatography (SEC) curves of Bi-LT-1 and Bi-LT-3 are shown, demonstrating their homogeneity and good stability at 4°C after 7 days. Figure 36B Size exclusion ultra-high performance liquid chromatography (SE-UHPLC) analysis demonstrated the species homogeneity of purified Bi-LT-1 and Bi-LT-3 of protein A, as reflected in the following: compared to the levels of the dimer species on days 0 and 7 (Bi-LT-1 98.4%, 98.3%; Bi-LT-3 98.4%, 98.1%), the levels of the high molecular weight (HMW) species on days 0 and 7 (Bi-LT-1 1.3%, 1.5%; Bi-LT-3 1.3%, 1.4%) and the low molecular weight (LMW) species on days 0 and 7 (Bi-LT-1 0.2%, 0.2%; Bi-LT-3 0.3%, 0.5%) were lower.

[0045] Figures 37A-37B Displayed in SHP-77 cells ( Figure 37A ) or H358 cells ( Figure 37B In the presence of staphylococcal enterotoxin B (SEB; lanes 2-8) or without SEB stimulation (lane 1), human PBMCs incubated with the following antibodies generated: no antibody (lanes 1, 2); human IgG (lane 3); parental anti-TIGIT B21-35 mAb (lane 4); parental anti-LAG-3 2L2A.1 mAb (lane 5); parental monoclonal antibodies, namely anti-TIGIT B21-35 and anti-LAG-3 2L2A.1 mAb (lane 6); Bi-LT-1 (lane 7); and Bi-LT-3 (lane 8).

[0046] Figure 38 Demonstrated proliferation of CD4 T cells from SEB-stimulated human PBMCs in the presence of SHP-77 cells (lanes 2-8) and combinations of human IgG control (lane 3), anti-TIGIT mAb B21-35 (lane 4), anti-LAG-3 mAb (lane 5), anti-TIGIT mAb and anti-LAG-3 mAb (lane 6), Bi-LT-1 (lane 7), or Bi-LT-3 (lane 8).

[0047] Figure 39A The display corresponds to Figure 1The framework region (FR) for anti-TIGIT CDR in the middle. Figure 39B The display corresponds to Figure 3 FR against PD-1CDR. Figure 39C Showing the corresponding Figure 5 FRs and corresponding to anti-PD-L1 CDRs in Figure 19A and 19B FR against LAG-3CDR. Detailed Implementation

[0048] definition

[0049] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed methods and compositions pertain. It must be noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include the plural forms unless the context clearly indicates otherwise. Thus, for example, as “a peptide” includes “one or more” peptides or “a plurality of” peptides as referred to. Regarding the teachings in this application, any published patent or patent application publications described herein are expressly incorporated herein by reference.

[0050] As used herein, the term “TIGIT” refers to any form of TIGIT and its variants that retain at least a portion of the activity of TIGIT. Unless otherwise indicated, such as by specifically referring to human TIGIT, TIGIT includes the natural sequence TIGIT of all mammalian species, such as humans, dogs, cats, horses, and cattle.

[0051] As used herein, the term “PD-1” refers to any form of PD-1 and its variants that retain at least a portion of PD-1 activity. Unless otherwise specified, such as by specifically referring to human PD-1, PD-1 includes the natural sequence PD-1 of all mammalian species, such as humans, dogs, cats, horses, and cattle.

[0052] As used herein, the term “PD-L1” refers to any form of PD-L1 and its variants that retain at least a portion of the activity of PD-L1. Unless otherwise indicated, such as by specifically referring to human PD-L1, PD-L1 includes the natural sequence PD-L1 of all mammalian species, such as humans, dogs, cats, horses, and cattle.

[0053] The term "agonist" refers to a substance that promotes (i.e., induces, causes, enhances, or increases) the biological activity or action of another molecule. The term agonist includes substances that bind to receptors, such as antibodies, and substances that promote receptor function without binding to them (e.g., by activating related proteins).

[0054] The terms "antagonist" or "inhibitor" refer to substances that prevent, block, inhibit, neutralize, or reduce the biological activity or action of another molecule, such as a receptor or ligand. Antagonists can be monospecific or bispecific antibodies.

[0055] As used herein, the term "antibody" refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen through one or more immunoglobulin variable regions. An antibody can be a complete antibody, an antigen-binding fragment, or a single chain thereof. The term "antibody" encompasses a wide variety of polypeptides that can be distinguished biochemically. Those skilled in the art will understand that heavy chains are classified as alfa, delta, epsilon, gamma, and mu, or α, δ, ε, γ, and μ, with some subclasses (e.g., γ1–γ4). The properties of this chain determine the "class" of the antibody, namely IgG, IgM, IgA, IgG, or IgE, respectively. Immunoglobulin subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, etc., have been well characterized and are known to have functional specificity. Given this disclosure, the modified forms of each of these classes and isotypes are readily identifiable to those skilled in the art and are therefore within the scope of this disclosure. All immunoglobulin classes are within the scope of this disclosure; the following discussion will generally refer to the IgG class of immunoglobulin molecules.

[0056] The antibodies disclosed herein include, but are not limited to, polyclonal, monoclonal, multispecific, bispecific, human, humanized, primate-derived, chimeric, and single-chain antibodies. The antibodies disclosed herein can be derived from any animal source, including birds and mammals. Preferably, the antibodies are from humans, mice, rats, donkeys, rabbits, goats, guinea pigs, camels, llamas, horses, or chickens. In some embodiments, the variable region may originate from marine organisms (condricthoids) (e.g., from sharks).

[0057] The terms "antibody fragment" and "antigen-binding fragment" are used to refer to a portion of an antibody, such as F(ab')2, F(ab)2, Fab', Fab, Fv, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv), fragments containing VL or VH domains, fragments generated from Fab expression libraries, and anti-Id antibodies. Regardless of structure, antibody fragments bind to the same antigen recognized by the intact antibody. The term "antibody fragment" includes DART and biantibodies. The term "antibody fragment" also includes any synthetic or genetically engineered protein containing a variable region of an immunoglobulin that functions like an antibody by forming a complex with a specific antigen. A "single-chain variable region" or "scFv" refers to a fusion protein of the variable region of the heavy chain (VH) and light chain (VL) of an immunoglobulin. In some aspects, said region is linked to a short linker peptide of 10 to about 25 amino acids. The linker can be enriched with glycine for flexibility and serine or threonine for solubility, and can connect to the N-terminus of VH or the C-terminus of VL, or vice versa. Despite the removal of the constant region and the introduction of the linker, this protein retains the specificity of the original immunoglobulin. Regarding IgG, the standard immunoglobulin molecule contains two identical light chain polypeptides with a molecular weight of approximately 23,000 Daltons and two identical heavy chain polypeptides with molecular weights of 53,000–70,000. The four chains are typically linked by disulfide bonds in a “Y” configuration, with the light chain linking to the heavy chain from the “Y” and extending through the variable region.

[0058] Both the light and heavy chains are divided into structurally and functionally homologous regions. The terms "constant" and "variable" are used functionally. In this respect, it will be understood that the variable domains of the light (VL) and heavy (VH) chain portions determine antigen recognition and specificity. Conversely, the constant domains of the heavy chain (CH1, CH2, or CH3) and light chain (CL) endow important biological properties such as secretion, placental migration, Fc receptor binding, complement binding, etc. By convention, the numbering of constant domains in conventional antibodies increases with their distance from the antigen-binding site or N-terminus of the antibody. In conventional antibodies, the N-terminal portion is the variable region, while the C-terminal portion is the constant region. The CH3 and CL domains actually contain the carboxyl terms of the heavy and light chains, respectively.

[0059] As described above, the variable region allows antibodies to selectively recognize and specifically bind to epitopes on antigens. That is, the VL and VH domains of the antibody, or a subset of the antibody's complementarity-determining regions (CDRs), combine to form a variable region defining a three-dimensional antigen-binding site. This quaternary antibody structure forms the antigen-binding site located at the ends of each arm of each Y-configuration. More specifically, this antigen-binding site is defined by three CDRs (i.e., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3) on each of the VH and VL chains. In some cases, such as certain immunoglobulin molecules derived from camel species or engineered based on camel immunoglobulins, or immunoglobulin molecules that consist only of a heavy chain lacking a light chain or only of a light chain lacking a heavy chain, the immunoglobulin molecule may be composed of a heavy chain lacking only a light chain.

[0060] In naturally occurring antibodies, the six chain-receptor domains (CDRs) present in each antigen-binding domain are short, discontinuous amino acid sequences. Because the antibody exhibits its three-dimensional conformation in an aqueous environment, these CDRs are specifically localized to form the antigen-binding domain. The remaining amino acids in the antigen-binding domain, referred to as the "framework" region, exhibit less intermolecular variability. The framework region primarily adopts a β-sheet conformation, and the CDRs form loops that are connected and, in some cases, form part of the β-sheet structure. Thus, the framework region acts as a scaffold that positions the CDRs in the correct orientation through interchain non-covalent interactions. The antigen-binding domain formed by the localized CDRs defines a surface complementary to an epitope on an immunoreactive antigen. This complementary surface facilitates non-covalent binding of the antibody to its associated epitope. Because it has been precisely defined, those skilled in the art can readily identify the amino acids containing the CDRs and framework region for any given heavy or light chain variable region.

[0061] As used herein, the terms “VH1” and “VH2” refer to the immunoglobulin heavy chain variable domains corresponding to two different binding specificities. Similarly, the terms “VL1” and “VL2” refer to the light chain variable domains corresponding to two different binding specificities. When used together, it should be understood that the VH1 and VL1 regions define a common binding specificity, and the VH2 and VL2 domains define another binding specificity.

[0062] As used herein, the term “frame region (FR)” refers to variable domain residues other than CDR residues. Each variable domain typically has four FRs flanking the corresponding CDR. For example, a VH domain typically has four HFRs: HFR1, HFR2, HFR3, and HFR4, which are located flanking the three HCDRs in the configuration HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4. Similarly, an LH domain typically has four LFRs, which are located flanking the three LCDRs in the configuration LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4. Exemplary FRs that can be used in the antagonists described herein are summarized in Figures 39A-39C middle.

[0063] Light chains are classified as kappa or lambda (κ, λ). Each heavy chain class can bind to either a κ or λ light chain. Typically, when immunoglobulins are produced by hybridomas, B cells, or genetically engineered host cells, the light and heavy chains are covalently bonded to each other, and the "tail" portions of the two heavy chains are bonded to each other via covalent disulfide bonds or non-covalent bonds. In the heavy chain, the amino acid sequence extends from the N-terminus of the Y-configuration forked end to the C-terminus at the bottom of each chain.

[0064] As used herein, the terms "light chain constant region" or "CL" may be used interchangeably when referring to an amino acid sequence derived from the antibody light chain. Preferably, the light chain constant region includes at least one of a constant κ domain or a constant λ domain.

[0065] As used herein, the term "heavy chain constant region" includes an amino acid sequence derived from the immunoglobulin heavy chain. A polypeptide containing a heavy chain constant region comprises at least one of the following: a CH1 domain, a hinge (e.g., upper, middle, and / or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof. For example, an antigen-binding polypeptide used in this disclosure may comprise a polypeptide chain having a CH1 domain; a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, and a CH2 domain; a polypeptide chain comprising a CH1 domain and a CH3 domain; a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, and a CH3 domain; or a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, a CH2 domain, and a CH3 domain. In some embodiments, the polypeptide of this disclosure comprises a polypeptide chain having a CH3 domain. Furthermore, antibodies used in this disclosure may lack at least a portion of the CH2 domain (e.g., all or part of the CH2 domain). It should be understood that the constant regions of heavy chains can be modified so that their amino acid sequences differ from those of naturally occurring immunoglobulin molecules. For example, the inventors of this application have discovered that the Fc loop in the CH3 domain can tolerate or adapt to significant insertions (e.g., greater than 100 aa).

[0066] The heavy chain constant region of the antibody disclosed herein can be derived from different immunoglobulin molecules. For example, the heavy chain constant region of a polypeptide may include a CH1 domain derived from an IgG1 molecule and a hinge region derived from an IgG3 molecule. In another example, the heavy chain constant region may include a hinge region that is partially derived from an IgG1 molecule and partially derived from an IgG3 molecule. In yet another example, the heavy chain portion may include a chimeric hinge that is partially derived from an IgG1 molecule and partially derived from an IgG4 molecule.

[0067] "Light chain-heavy chain pair" refers to a set of light and heavy chains that can form dimers through disulfide bonds between the CL domain of the light chain and the CH1 domain of the heavy chain.

[0068] The subunit structures and three-dimensional conformations of the constant regions of various immunoglobulin classes are well known. As used herein, the term "VH domain" includes the N-terminal variable domain of the immunoglobulin heavy chain, while the term "CH1 domain" includes the first (mostly N-terminal) constant region domain of the immunoglobulin heavy chain. The CH1 domain is adjacent to the VH domain and is located at the N-terminus of the hinge region of the immunoglobulin heavy chain molecule.

[0069] As used herein, the term "CH2 domain" encompasses the portion of a heavy chain molecule extending from approximately residue 244 to residue 360, for example, in an antibody, using a conventional numbering scheme (residues 244 to 360, Kabat numbering system; and residues 231-340, EU numbering system). The CH2 domain is unique in that it is not tightly paired with another domain. Instead, two N-linked branched carbohydrate chains are inserted between the two CH2 domains of the intact native IgG molecule. The CH3 domain extends from the CH2 domain of the IgG molecule to the C-terminus and contains approximately 108 residues.

[0070] As used herein, the term "hinge region" refers to the portion of a heavy chain molecule that connects the CH1 domain to the CH2 domain. This hinge region contains approximately 25 residues and is flexible, thus allowing the two N-terminal antigen-binding regions to move independently. The hinge region can be subdivided into three distinct domains: the upper, middle, and lower hinge domains.

[0071] As used herein, the term "disulfide bond" refers to a covalent bond formed between two sulfur atoms. The amino acid cysteine ​​contains a thiol group that can form a disulfide bond or bridge with another thiol group. In most naturally occurring IgG molecules, the CH1 and CL regions are linked by disulfide bonds, and the two heavy chains are linked by two disulfide bonds at positions corresponding to 239 and 242 (using the Kabat numbering system (positions 226 or 229, EU numbering system)).

[0072] As used herein, a “variant” of an antibody, antibody fragment, or antibody domain means that the antibody, antibody fragment, or antibody domain: (1) has a sequence identity of at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% with respect to the original antibody, antibody fragment, or antibody domain, and (2) specifically binds to the same target that specifically binds to the original antibody, antibody fragment, or antibody domain. It should be understood that when sequence identity is expressed in the form of “at least x% identical” or “at least x% identity,” such embodiments include any and all numerical percentages equal to or higher than the lower limit. Furthermore, it should be understood that the presence of an amino acid sequence in this application should be interpreted as additional disclosure or inclusion of an amino acid sequence having an identity of at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

[0073] As used herein, the phrase “humanized antibody” refers to an antibody derived from a non-human antibody, typically a mouse monoclonal antibody. Alternatively, a humanized antibody may be derived from a chimeric antibody that retains or substantially retains the antigen-binding properties of the parental non-human antibody, but exhibits reduced immunogenicity compared to the parental antibody when administered to humans.

[0074] As used herein, the phrase "chimeric antibody" refers to an antibody in which the immunoreactive region or site is derived from or derived from a first species and the constant region (which may be complete, partial, or modified according to this disclosure) is derived from a second species. In some embodiments, the target binding region or site will be derived from a non-human source (e.g., mouse or primate), and the constant region is human.

[0075] The scope of multispecific antibodies in this application includes various compositions and methods, including asymmetric IgG-like antibodies (e.g., triomab / quadroma, TrionPharma / Fresenius Biotech); knot-into-hole antibodies (Genentech); cross-linked monoclonal antibodies (Cross MAb, Roche); electrostatically matched antibodies (AMGEN); LUZ-Y (Genentech); chain-exchange engineered domain (SEED) bodies (EMD Serono; biolononic, Merus); Fab-exchange antibodies (Genmab); symmetric IgG antibodies (e.g., dual-targeting (DT)-Ig (GSK / Domantis); dual-targeting antibodies (Genentech); cross-linked monoclonal antibodies (Karmanos Cancer Center); mAb2 (F-star); Cov X-body (Cov X / Pfizer); dual-variable domain (DVD)-Ig fusion protein (Abbott); and IgG-like bispecific antibodies (Eli). Lilly); Ts2Ab (Medimmune / AZ); BsAb (ZymoGenetics); HERCULES (Biogen Idec, TvAb, Roche); scFv / Fc fusion; SCORPION (EmergentBioSolutions / Trubion, ZymoGenetics / BMS); Fc-DART (bi-affinity repositioning technology), MacroGenics; Bis(scFv)2-Fabs (National Research Center for Antibody Medicine) Medicine); F(ab)2 fusion protein (Medarex / AMGEN); bi-acting or Bis-Fab (Genentech); Dock-and-Lock (DNL, ImmunoMedics); Fab-Fv (UCB-Celltech); scFv-based antibodies and bi-antibody-based antibodies (e.g., bispecific T-cell conjugates (BiTEs, Micromet); tandem biantibodies (Tandab, Affimed); DARTs (MacroGenics); single-chain biantibodies; TCR-like antibodies (AIT, Receptor Logics);Human serum albumin scFv fusion protein (Merrimack), COMBODIES (Epigen Biotech), and IgG / no n-IgG fusion proteins (e.g., immune cytokines (EMDSerono, Philogen, ImmunGene, ImmunoMedics)).

[0076] "Specific binding" or "specificity" generally refers to an antibody binding to an epitope via its antigen-binding domain, and binding requires a certain complementarity between the antigen-binding domain and the epitope. According to this definition, an antibody is said to "specifically bind" to an epitope when it binds more readily to that epitope via its antigen-binding domain than to a random, unrelated epitope. The term "specificity" is used herein to define the relative affinity of an antibody for a given epitope. For example, antibody "A" can be considered to have higher specificity for a given epitope than antibody "B," or antibody "A" can be said to bind to epitope "C" with higher specificity than to the associated epitope "D." In some embodiments, if the antibody or antibody fragment binds to the antigen with an affinity (IQ) equal to or less than 10... -6 M, equal to or less than 10 -7 M, equal to or less than 10 -8 M, equal to or less than 10 -9 M or equal to or less than 10 -10 The dissociation constant of M (K) d If an antibody or antibody fragment forms a complex, then the antibody or antibody fragment is "specific" to the antigen.

[0077] As used herein, the phrase "chimeric antibody" refers to an antibody in which the immunoreactive region or site is derived from or derived from a first species and the constant region (which may be complete, partial, or modified according to this disclosure) is derived from a second species. In some embodiments, the target binding region or site will be derived from a non-human source (e.g., mouse or primate), and the constant region will be human.

[0078] The term "antagonist antibody" refers to an antibody that binds to a target and prevents or reduces the biological effects of that target. In some embodiments, the term may refer to an antibody that prevents a target it binds to, such as TIGIT, from performing its biological functions.

[0079] As used herein, “anti-PD-1 antagonist antibody” refers to an antibody capable of inhibiting PD-1 biological activity and / or PD-1-mediated downstream events. Anti-PD-1 antagonist antibodies include antibodies that block, antagonize, inhibit, or reduce (in any degree including significantly reducing) PD-1 biological activity (including PD-1-mediated downstream events, such as PD-1 binding and downstream signaling, inhibition), T cell proliferation, T cell activation, IFN secretion, IL-2 secretion, TNF secretion, IL-10 induction, and inhibition of antitumor immune responses. For the purposes of this invention, it will be clearly understood that the term “anti-PD-1 antagonist antibody” (interchangeably referred to as “antagonist PD-1 antibody,” “antagonist anti-PD-1 antibody,” or “PD-1 antagonist antibody”) encompasses all previously identified terms, headings, functional states, and characteristics such that PD-1 itself, the biological activity of PD-1, or the consequences of that biological activity are substantially ineffective, reduced, or neutralized to any meaningful degree. In some implementations, anti-PD-1 antagonist antibodies bind to PD-1 and upregulate anti-tumor immune responses.

[0080] As used herein, “anti-PD-L1 antagonist antibody” refers to an antibody capable of inhibiting the biological activity of PD-L1 and / or PD-L1-mediated downstream events. Anti-PD-L1 antagonist antibodies include antibodies that block, antagonize, inhibit, or reduce (to any extent, including significantly) the biological activity of PD-L1, including PD-L1-mediated downstream events such as PD-L1 binding and downstream signaling, inhibition of T cell proliferation, inhibition of T cell activation, inhibition of IFN secretion, inhibition of IL-2 secretion, inhibition of TNF secretion, induction of IL-10, and inhibition of anti-tumor immune responses. For the purposes of this invention, the term "anti-PD-L1 antagonist" (which may be interchangeably referred to as "antagonist PD-L1 antibody," "antagonist anti-PD-L1 antibody," or "PD-L1 antagonist antibody") will be understood to encompass all previously identified terms, headings, functional states, and characteristics that substantially inactivate, reduce, or neutralize PD-L1 itself, the biological activity of PD-L1, or the result of such biological activity to any meaningful extent. In some embodiments, the anti-PD-L1 antagonist antibody binds to PD-L1 and upregulates the antitumor immune response.

[0081] The phrase "immune checkpoint modulator" refers to a class of functional agents that inhibit or stimulate signal transduction through immune checkpoints. "Immune checkpoint modulators" include receptors and their associated ligands, which together provide a means of inhibiting or stimulating signal transduction pathways that would otherwise lead to T cell activation. Exemplary immune checkpoint modulators include, but are not limited to, TIGIT and its CD155 ligand PVR; PD-1 and its ligands PD-L1 and PD-L2; CTLA-4 and its ligands B7-1 and B7-2; TIM-3 and its ligand Galectin-9; LAG-3 and its ligands, including sinusoidal endothelial cell lectin (LSECtin) and Galectin-3; CD122 and its CD122R ligand; CD70, B7H3, B and T lymphocyte attenuators (BTLA), and VISTA.

[0082] The phrases “checkpoint regulator antagonist,” “immune checkpoint binding antagonist,” and “immune checkpoint antagonist” are used interchangeably in this document. They refer to a class of agents that interfere with (or inhibit) the activity of immune checkpoint regulators, thereby blocking or inhibiting signal transduction through checkpoint regulator receptors by binding to the regulators or their ligands. By inhibiting this signal transduction, immunosuppression can be reversed, thereby restoring or enhancing T-cell immunity against cancer cells. Immune checkpoint regulator antagonists include antibody fragments, peptide inhibitors, dominant and negative peptides, and small molecule drugs, which can be in standalone form or as part of fusion proteins or conjugates.

[0083] The phrases “immune checkpoint binding agonist” and “immune checkpoint agonist” are used interchangeably here when referring to agents that stimulate the activity of immune checkpoint regulators, thereby stimulating signaling through checkpoint regulator receptors due to their binding to checkpoint regulators or their ligands. Stimulation of this signaling can rebuild or enhance T-cell immunity against cancer cells. Exemplary immune checkpoint regulator agonists include, but are not limited to, members of the tumor necrosis factor (TNF) receptor superfamily, such as CD27, CD40, OX40 (CD134), glucocorticoid-induced TNFR family-associated protein (GITR), and 4-1BB (CD137) and their ligands. Other checkpoint regulator agonists belong to the B7-CD28 superfamily, including CD28 and ICOS.

[0084] The phrase "dominant-negative protein" or "dominant-negative peptide" refers to a protein or peptide derived from a wild-type protein that has been genetically modified so that the modified protein or peptide interferes with the function of the endogenous wild-type protein from which it is derived through mutation and / or deletion.

[0085] The phrase "small molecule drug" refers to a molecular entity that is typically organic or organometallic, but not a polymer, possesses pharmaceutical activity, and has a molecular weight less than about 2 kDa, less than about 1 kDa, less than about 900 Da, less than about 800 Da, or less than about 700 Da. This term covers most pharmaceutical compounds other than proteins or nucleic acids that are called "drugs," although small peptides or nucleic acid analogs can also be considered small molecule drugs. Examples include chemotherapeutic anticancer drugs and enzyme inhibitors. Small molecule drugs can be synthetic, semi-synthetic (i.e., synthesized from naturally occurring precursors), or biologically derived.

[0086] When describing the arrangement of polypeptide domains with hyphens between the various domains (e.g., CH2-CH3), it should be understood that the order of the listed domains is from the amino terminus to the carboxyl terminus.

[0087] "Specific binding" or "specificity" generally refers to an antibody binding to an epitope through its antigen-binding domain, and this binding requires a certain complementarity between the antigen-binding domain and the epitope. According to this definition, an antibody is said to "specifically bind" to an epitope when it binds more readily to that epitope through its antigen-binding domain than to a random, unrelated epitope. This article uses the term "specificity" to define the relative affinity of an antibody for a given epitope. For example, antibody "A" can be considered to have higher specificity for a given epitope than antibody "B," or antibody "A" can be said to bind to epitope "C" with higher specificity than to the related epitope "D."

[0088] The term "immunoconjugate" refers to an antibody fused to an inhibitory peptide or small molecule drug via a covalent bond. The peptide or small molecule drug may be linked to the C-terminus of a constant heavy chain or the N-terminus of a variable light chain and / or heavy chain.

[0089] "Connectors" can be used to stably covalently link peptides or small molecule drugs, such as maytansinoid, to antitumor antagonists. While the compound or antibody remains active, the connector may be sensitive to or substantially resistant to acid-induced cleavage, light-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage. Suitable connectors are well known in the art and include, for example, disulfide groups, thioether groups, acid-labile groups, light-labile groups, peptidase-labile groups, and esterase-labile groups. Connectors also include charged connectors and their hydrophilic forms, as described herein and known in the art. Immunoconjugates may further comprise a flexible 3-15 amino acid peptide (or spacer) between the antitumor antagonist and the peptide and / or small molecule drug.

[0090] As used herein, the term "immunoglobulin scaffold" refers to any amino acid polymer exhibiting properties that support the desired function of an antagonist, including increased antibody specificity, enhanced antibody function, or support for antibody structure and stability. An immunoglobulin scaffold may have one or more immunoglobulin constant regions, including CH1, CH2, and / or CH3 regions from the immunoglobulin heavy chain and / or CL regions from the immunoglobulin light chain. Binding domains of donor peptides can be grafted onto the immunoglobulin scaffold to impart binding specificity to the donor peptide on the scaffold.

[0091] As used herein, the phrase "multispecific inhibitor" refers to a molecule comprising at least two targeting domains with different binding specificities. In some embodiments, a multispecific inhibitor is a polypeptide comprising a scaffold and two or more immunoglobulin antigen-binding domains targeting different antigens or epitopes. In some embodiments, a multispecific inhibitor is a bispecific antibody.

[0092] As used herein, the phrase "bispecific" refers to a molecule comprising at least two targeting domains with different binding specificities. Each targeting domain is capable of specifically binding to a target molecule and, upon binding, inhibits the biological function of the target molecule. In some embodiments, a bispecific checkpoint regulator antagonist is a polymer molecule having two or more peptides. In some embodiments, the targeting domain comprises an antigen-binding domain or CDR of an antibody. In some embodiments, a bispecific inhibitor is a bispecific antibody.

[0093] The terms "bispecific antibody" and "bispecific antagonist" are used interchangeably herein and refer to antibodies that can specifically bind to two different antigens (or epitopes). In some embodiments, a bispecific antibody is a full-length antibody that binds to one antigen (or epitope) on one of its two binding arms (a pair of HC / LC) and to a different antigen (or epitope) on its second arm (another pair of HC / LC). In these embodiments, the bispecific antibody has two distinct antigen-binding arms (on both the specificity and CDR sequences) and is monovalent for each antigen it binds to.

[0094] In other embodiments, a bispecific antibody is a full-length antibody capable of binding two different antigens (or epitopes) in each of its two binding arms (two pairs of HC / LC). In these embodiments, the bispecific antibody has two identical antigen-binding arms, the same specificity, and the same CDR sequence, and is bivalent for each antigen it binds to.

[0095] Exemplary bispecific antibodies may include asymmetric IgG-like antibodies (e.g., triomab / quadroma, Trion Pharma / Fresenius Biotech); buttonhole antibodies (Genentech); cross-linked monoclonal antibodies (Roche); electrostatically matched antibodies (AMGEN); LUZ-Y (Genentech); chain-exchange engineered domain (SEED) bodies (EMD Serono; biolonic, Merus); Fab-exchange antibodies (Genmab); symmetric IgG antibodies (e.g., dual-targeting (DT)-Ig (GSK / Domantis); two-in-one antibodies (Genentech); cross-linked monoclonal antibodies (Karmanos Cancer Center); mAb2 (F-star); Cov X-body (Cov X / Pfizer); dual variable domain (DVD)-Ig fusion proteins (Abbott); and IgG-like bispecific antibodies (Eli). Lilly); Ts2Ab (Medimmune / AZ); BsAb (ZymoGenetics); HERCULES (BiogenIdec, TvAb, Roche); scFv / Fc fusion protein; SCORPION (Emergent) BioSolutions / Trubion, ZymoGenetics / BMS; Dual Affinity Redirecting Technology (Fc-DART), MacroGenics; Bis(scFv)2-Fabs (National Research Center for Antibody Medicine); F(ab)2 Fusion Protein (Medarex / AMGEN); Bi-acting or Bis-Fab (Genentech); Dock-and-Lock Bodies (DNL, ImmunoMedics); Fab-Fv (UCB-Celltech); scFv-based Antibodies and Biantibody-based Antibodies (e.g., Bispecific T-cell conjugates (BiTEs, Micromet); Tandem Biantibodies (Tandab, Affimed); DARTs (MacroGenics); Single-chain Biantibodies; TCR-like Antibodies (AIT, Receptor Logics); Human Serum Albumin scFv Fusion Protein (Merrimack); COMBODIES (Epigen Biotech); and IgG / non-IgG fusion proteins (e.g., immune cytokines) (EMDSerono, Philogen, ImmunGene, ImmunoMedics).

[0096] The terms “treat” and “treatment” refer to the reduction of one or more symptoms associated with a proliferative disorder; prevention or delay of the onset of one or more symptoms of a proliferative disorder; and / or reduction of the severity or frequency of one or more symptoms of a proliferative disorder.

[0097] The phrases “patients in need,” “patients in need of treatment,” or “subjects in need of treatment” include subjects, such as mammalian subjects, who benefit from receiving the antitumor antagonist for the treatment of proliferative diseases as disclosed in this disclosure.

[0098] The terms “therapeutic effective amount,” “pharmacologically effective amount,” and “physiologically effective amount” are used interchangeably and refer to the amount of an antitumor antagonist required to deliver a threshold level of active antagonist in the bloodstream or in the target tissue. The precise amount will depend on many factors, such as the specific active agent, the composition and physical properties of the composition, the intended patient population, patient considerations, etc., and can be readily determined by those skilled in the art based on the information provided herein or other information available in relevant literature.

[0099] The terms “improvement,” “increase,” or “decrease” as used herein refer to values ​​or parameters measured relative to baseline, such as measurements in the same person prior to initiation of the treatment described herein or in control individuals (or multiple control individuals) in the absence of the treatment described herein.

[0100] A “control individual” is an individual with the same proliferative cellular disease as the individual being treated and of the same age as the individual being treated to ensure comparability of disease stages between the treated and control individuals. The individual being treated (also known as a “patient” or “subject”) can be a fetus, infant, child, adolescent, or adult with a proliferative cellular disease.

[0101] The term "cell proliferative disorder" refers to a disease characterized by abnormal cell proliferation. A proliferative disorder does not imply any limitation on the cell growth rate, but rather indicates a loss of normal control over growth and cell division. Therefore, in some implementations, cells with a proliferative disorder may have the same cell division rate as normal cells, but do not respond to signals that limit this growth. Within the scope of "cell proliferative disorders" are neoplasms, cancers, or tumors.

[0102] The terms "cancer" or "tumor" refer to any of a variety of malignant tumors characterized by proliferating cells with the ability to invade surrounding tissues and / or metastasize to new colonization sites, and include leukemia, lymphoma, carcinoma, melanoma, sarcoma, germ cell tumor, and blastoma. Exemplary cancers treated with the methods of this disclosure include brain cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, head and neck cancer, kidney cancer, lung cancer, non-small cell lung cancer, mesothelioma, ovarian cancer, prostate cancer, gastric cancer, uterine cancer, leukemia, and medulloblastoma.

[0103] The term "leukemia" refers to a progressive malignant disease of the blood-forming organs, typically characterized by the abnormal proliferation and development of white blood cells and their precursors in the blood and bone marrow. Exemplary leukemias include, for example, acute non-lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, acute promyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia, leukocytic leukemia, basophilic leukemia, blastic leukemia, bovine leukemia, chronic myeloid leukemia, keratinocyte leukemia, embryonic leukemia, eosinophilic leukemia, Gross's leukemia, hairy cell leukemia, hemoblastic leukemia, and hemocytoblastic leukemia. Leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenic leukemia, lymphocytic leukemia, lymphoblastic leukemia, lymphoid leukemia, lymphosarcoma cell leukemia, mast cell leukemia, megakaryocytic leukemia, micromyeloblastic leukemia, monocytic leukemia, myeloblastic leukemia, myelocytic leukemia, myeloid granulocytic leukemia Leukemia, myelomonocyticle leukemia, Neghly type leukemia, plasma cell leukemia, plasma cell leukemia, promyelocytic leukemia, Rydell cell leukemia, Schilling's leukemia, stem cell leukemia, sub-leukemia, and undifferentiated cell leukemia.

[0104] The term "cancer" refers to the malignant growth of epithelial cells that readily infiltrate surrounding tissues and cause metastasis. Exemplary cancers include, for example, acinar carcinoma, alveolar carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, adenomatous carcinoma, adrenocortical carcinoma, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, basal cell carcinoma, basement membrane carcinoma, basal squamous cell carcinoma, bronchoalveolar carcinoma, bronchial carcinoma, bronchogenic carcinoma, cerebral carcinoma, cholangiocarcinoma, choriocarcinoma, colloid carcinoma, comedo carcinoma, body carcinoma, cribriform carcinoma, armored carcinoma, skin cancer, cylindrical carcinoma, cylindrical cell carcinoma, ductal carcinoma, dura mater carcinoma, embryonal carcinoma, brain-like carcinoma, epithelial carcinoma, epithelial adenocarcinoma, exophytic carcinoma, ulcerative carcinoma, fibroma, gelatinous carcinoma, colloid carcinoma, giant cell carcinoma, and adenoid carcinoma. Carcinoma, adenocarcinoma, granular cell carcinoma, pilonidal stromal carcinoma, hemorrhoidal carcinoma, hepatocellular carcinoma, Hütter cell carcinoma, clear membrane carcinoma, herpesoid carcinoma, embryonal carcinoma, carcinoma in situ, intraepithelial carcinoma, intraepithelial carcinoma, Crompcher carcinoma, Kurzki cell carcinoma, large cell carcinoma, enticular carcinoma, lenticular carcinoma, liposomal carcinoma, lymphoepithelial carcinoma, medullary carcinoma, melanoma, nevus-like carcinoma, mucinous carcinoma, mucinous cell carcinoma, mucinous epidermal carcinoma, mucinous carcinoma. Muciparum, mucinous carcinoma (carcinoma mucocellulare), nasopharyngeal carcinoma, oat cell carcinoma, ossifying carcinoma, osteoid carcinoma, papillary carcinoma, portal vein carcinoma, invasive precarcinoma, spiculated cell carcinoma, pustular carcinoma, renal cell carcinoma, reserve cell carcinoma, sarcoma carcinoma, Schneider's carcinoma, sclerosing carcinoma, scrotal carcinoma, signet ring cell carcinoma, simple carcinoma, small cell carcinoma, solanaceous carcinoma, spherical cell carcinoma, spindle cell carcinoma, cavernous carcinoma, squamous carcinoma, squamous cell carcinoma, linear carcinoma, telangiectatic carcinoma, telangiectatic carcinoma, transitional cell carcinoma, nodular carcinoma, verrucous carcinoma, and choriocarcinoma.

[0105] The term "sarcoma" refers to a tumor composed of material similar to embryonic connective tissue and typically consists of tightly packed cells embedded in fibrous or homogeneous material. Exemplary sarcomas include, for example, chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, abemethyst sarcoma, adipose sarcoma, liposarcoma, alveolar soft tissue sarcoma, ameloblastic sarcoma, botryoid sarcoma, green algae sarcoma, choriocarcinoma, embryonal sarcoma, Wilns' tumor sarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma, granulocyte sarcoma, Hodgkin's sarcoma, idiopathic multicolor hemorrhagic sarcoma, B-cell immunoblastic sarcoma, lymphoma (such as lymphoma), Jensen's sarcoma, Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma, leukocyte sarcoma, malignant mesothelioma sarcoma, peritoneal sarcoma, reticulum cell sarcoma, Rouss sarcoma, serous sarcoma, and synovial sarcoma.

[0106] The term "melanoma" refers to a tumor originating from the melanocyte system of the skin and other organs. Melanomas include, for example, angular melanoma, ameliorative melanoma, benign juvenile melanoma, Cloudman melanoma, S91 melanoma, Harding-Passey melanoma, juvenile melanoma, tonsillar malignant melanoma, malignant melanoma, nodular melanoma, sublingual melanoma, and superficial melanoma.

[0107] Other cancers include, for example, Hodgkin's disease, multiple myeloma, neuroblastoma, breast cancer, ovarian cancer, lung cancer, rhabdomyosarcoma, essential thrombocytosis, essential macroglobulinemia, small cell lung cancer, primary brain tumor, stomach cancer, colon cancer, malignant pancreatic insulinoma, malignant carcinoid, precancerous skin lesions, testicular cancer, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, cervical cancer, endometrial cancer, and adrenocortical carcinoma.

[0108] I. Checkpoint modifiers and antagonists

[0109] In one aspect, this application provides an antitumor antagonist comprising an immunoglobulin scaffold having (1) a pair of arms comprising a variable domain region specifically binding to a first immune checkpoint regulator, and (2) a single chain (scFv) specifically binding to a second immune checkpoint regulator.

[0110] In another aspect, this application provides an antitumor antagonist comprising an immunoglobulin scaffold structurally linked to a first immune checkpoint modulator antagonist and a second immune checkpoint modulator antagonist in the form of scFv.

[0111] In both aspects, the immunoglobulin scaffold may include one or more immunoglobulin constant regions, such as IgGCH1, CH2, and / or CH3. In some embodiments, the immunoglobulin scaffold is an Fc (hinge-CH2-CH3).

[0112] In some embodiments, the antitumor antagonist includes an immunoglobulin scaffold, wherein the N-terminus of the antagonist includes a first immune checkpoint modulator antagonist structurally linked thereto, wherein the first immune checkpoint modulator antagonist specifically binds to PD-1, PD-L1, LAG-3, or TIGIT; and a second immune checkpoint modulator antagonist located at the C-terminus of the antagonist as a scFv that specifically binds to PD-1, PD-L1, LAG-3, or TIGIT.

[0113] In some embodiments, the scFv includes a linker connecting the heavy chain variable region to the light chain variable region. In one embodiment, the linker includes an amino acid sequence comprising 3, 4, 5, 6, 7, 8, 9, or 10 copies of the amino acid sequence G4S. In another embodiment, the linker includes an amino acid sequence shown in any of SEQ ID NO: 188-191. In a more specific embodiment, the linker includes the amino acid sequence of SEQ ID NO: 191.

[0114] In one embodiment, the anti-TIGIT scFv comprises one or more heavy chain CDRs selected from SEQ ID NO: 1-25 and one or more light chain CDRs selected from SEQ ID NO: 26-47.

[0115] In another embodiment, the anti-TIGIT scFv comprises a heavy chain / light chain variable region, wherein the scFv has a heavy chain variable region (HCVR) and a light chain variable region (LCVR), the heavy chain variable region (HCVR) having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with an amino acid sequence selected from SEQ ID NO: 48, 50, 52, 54, 56, 58, 60, 62, 64, and 66, and the light chain variable region (LCVR) having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with an LCVR having an amino acid sequence selected from SEQ ID NO: 49, 51, 53, 55, 57, 59, 61, 63, and 67.

[0116] In a more specific embodiment, the anti-TIGIT scFv comprises HCVR and LCVR, wherein the HCVR has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 66, and the LCVR has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 67.

[0117] In another embodiment, the anti-TIGIT scFv comprises: HCVR, which comprises a combination of (1) and (2) as follows: (1) HCDR1 of SEQ ID NO: 23, HCDR2 of SEQ ID NO: 24, and HCDR3 of SEQ ID NO: 25 and (2) HFR1 having at least 80%, 85%, or 90% identity with the amino acid sequence of SEQ ID NO: 260, HFR2 having at least 80%, 85%, or 90% identity with the amino acid sequence of SEQ ID NO: 247, HFR3 having at least 80%, 85%, or 90% identity with the amino acid sequence of SEQ ID NO: 261, and HFR4 having at least 80%, 85%, or 90% identity with the amino acid sequence of SEQ ID NO: 236; and further comprises immunoglobulin LCVR, which comprises a combination of (1) and (2) as follows: (1) LCDR1 of SEQ ID NO: 45, LCDR2 of SEQ ID NO: 46, and HCDR3 of SEQ ID NO: 260. LCDR3 of NO: 47, (2) LFR1 having at least 80%, 85% or 90% identity with the amino acid sequence of SEQ ID NO: 220, LFR2 having at least 80%, 85% or 90% identity with the amino acid sequence of SEQ ID NO: 228, LFR3 having at least 80%, 85% or 90% identity with the amino acid sequence of SEQ ID NO: 234, and LFR4 having at least 80%, 85% or 90% identity with the amino acid sequence of SEQ ID NO: 262.

[0118] In another embodiment, the first targeting domain comprises one or more variable regions from the anti-PD-1 antibody, and the second targeting domain comprises the anti-TIGIT scFv as described above.

[0119] In one embodiment, the anti-PD-1 targeting domain comprises one or more heavy chain CDRs selected from SEQ ID NO: 68-81 and / or one or more light chain CDRs selected from SEQ ID NO: 82-95.

[0120] In another embodiment, the anti-PD-1 targeting domain comprises: an HCVR having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with an amino acid sequence selected from SEQ ID NO: 96, 98, 100, 102, 104, and 106; an LCVR having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with an LCVR having an amino acid sequence selected from SEQ ID NO: 97, 99, 101, 103, 105, and 107; or both.

[0121] In another embodiment, the anti-PD-1 targeting domain comprises: HCVR, which comprises a combination of the following (1) and (2): (1) HCDR1 of SEQ ID NO: 79, HCDR2 of SEQ ID NO: 80 and HCDR3 of SEQ ID NO: 81, (2) HFR1 having at least 80%, 85% or 90% identity with the amino acid sequence of SEQ ID NO: 283, HFR2 having at least 80%, 85% or 90% identity with the amino acid sequence of SEQ ID NO: 277, HFR3 having at least 80%, 85% or 90% identity with the amino acid sequence of SEQ ID NO: 288, and HFR4 having at least 80%, 85% or 90% identity with the amino acid sequence of SEQ ID NO: 274; immunoglobulin LCVR, which comprises a combination of the following (1) and (2): (1) LCDR1 of SEQ ID NO: 93, LCDR2 of SEQ ID NO: 94 and LCDR3 of SEQ ID NO: 95, (2) HFR1 having at least 80%, 85% or 90% identity with the amino acid sequence of SEQ ID NO: 283, HFR2 having at least 80%, 85% or 90% identity with the amino acid sequence of SEQ ID NO: 288, and HFR4 having at least 80%, 85% or 90% identity with the amino acid sequence of SEQ ID NO: 274; and immunoglobulin LCVR, which comprises a combination of the following (1) and (2): (1) LCDR1 of SEQ ID NO: 93, LCDR2 of SEQ ID NO: 94 and LCDR3 of SEQ ID NO: 95, (2) HFR1 having at least 80%, 85% or 90% identity with the amino acid sequence of SEQ ID NO: 283, HFR3 having at least LFR1 having at least 80%, 85%, or 90% identity with the amino acid sequence of SEQ ID NO: 289, LFR2 having at least 80%, 85%, or 90% identity with the amino acid sequence of SEQ ID NO: 290, LFR3 having at least 80%, 85%, or 90% identity with the amino acid sequence of SEQ ID NO: 291, and LFR4 having at least 80%, 85%, or 90% identity with the amino acid sequence of SEQ ID NO: 292; or both.

[0122] In another embodiment, the anti-PD-1 targeting domain comprises HCVR having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 106; LCVR having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 107; or both.

[0123] In one particular embodiment, the bispecific anti-PD-1 / anti-TIGIT antagonist comprises: a first targeting domain comprising HCVR having the amino acid sequence of SEQ ID NO: 106 and / or LCVR having the amino acid sequence of SEQ ID NO: 107; and an anti-TIGIT scFv comprising HCVR having the amino acid sequence of SEQ ID NO: 66 and LCVR having the amino acid sequence of SEQ ID NO: 67.

[0124] In a more specific embodiment, the scFv in the bispecific anti-PD-1 / anti-TIGIT antagonist includes a linker that connects the heavy chain variable region to the light chain variable region in the second targeting domain, wherein the linker includes the amino acid sequence of SEQ ID NO: 191.

[0125] In another embodiment, the bispecific anti-PD-1 / anti-TIGIT antagonist comprises: an immunoglobulin heavy chain containing the amino acid sequence of SEQ ID NO: 160; an immunoglobulin light chain containing the amino acid sequence of SEQ ID NO: 161; or both.

[0126] In another embodiment, the bispecific anti-PD-1 / anti-TIGIT antagonist comprises: an immunoglobulin heavy chain containing the amino acid sequence of SEQ ID NO: 162; an immunoglobulin light chain containing the amino acid sequence of SEQ ID NO: 161; or both.

[0127] In another embodiment, the bispecific anti-PD-1 / anti-TIGIT antagonist comprises: an immunoglobulin heavy chain containing the amino acid sequence of SEQ ID NO: 160 and an immunoglobulin light chain containing the amino acid sequence of SEQ ID NO: 161.

[0128] In another embodiment, the first targeting domain comprises one or more variable regions from the anti-PD-L1 antibody and a second targeting domain comprising the anti-TIGIT scFv as described above.

[0129] In one embodiment, the anti-PD-L1 targeting domain comprises one or more heavy chain CDRs selected from SEQ ID NO: 108-122 and / or one or more light chain CDRs selected from SEQ ID NO: 123-138.

[0130] In another embodiment, the anti-PD-L1 targeting domain comprises an HCVR having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with an amino acid sequence selected from SEQ ID NO: 139, 141, 143, 145, 147, 149, 151, and 153 and / or an LCVR having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with an amino acid sequence selected from SEQ ID NO: 140, 142, 144, 146, 148, 152, and 154.

[0131] In another embodiment, the anti-PD-L1 targeting domain comprises an HCVR having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 153 and / or an LCVR having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 154.

[0132] In another embodiment, the anti-PD-L1 targeting domain comprises: immunoglobulin HCVR, which comprises a combination of the following (1) and (2): (1) HCDR1 of SEQ ID NO: 111, HCDR2 of SEQ ID NO: 114 and HCDR3 of SEQ ID NO: 115, (2) HFR1 having at least 80%, 85% or 90% identity with the amino acid sequence of SEQ ID NO: 283, HFR2 having at least 80%, 85% or 90% identity with the amino acid sequence of SEQ ID NO: 277, HFR3 having at least 80%, 85% or 90% identity with the amino acid sequence of SEQ ID NO: 300, and HFR4 having at least 80%, 85% or 90% identity with the amino acid sequence of SEQ ID NO: 274; immunoglobulin LCVR, which comprises a combination of the following (1) and (2): (1) LCDR1 of SEQ ID NO: 123, LCDR2 of SEQ ID NO: 124 and SEQ ID NO: 115. LCDR 3 of NO: 125, (2) LFR1 having at least 80%, 85% or 90% identity with the amino acid sequence of SEQ ID NO: 294, LFR2 having at least 80%, 85% or 90% identity with the amino acid sequence of SEQ ID NO: 295, LFR3 having at least 80%, 85% or 90% identity with the amino acid sequence of SEQ ID NO: 296, and LFR4 having at least 80%, 85% or 90% identity with the amino acid sequence of SEQ ID NO: 276; or both.

[0133] In a particular embodiment, the bispecific anti-PD-L1 / anti-TIGIT antagonist comprises a combination of a first targeting domain and a second targeting domain, the first targeting domain comprising: an HCVR having the amino acid sequence of SEQ ID NO: 153 and / or an LCVR having the amino acid sequence of SEQ ID NO: 154, and the second targeting domain being in the form of an anti-TIGIT scFv comprising an HCVR having the amino acid sequence of SEQ ID NO: 66 and an LCVR having the amino acid sequence of SEQ ID NO: 67. In a more specific embodiment, the scFv in the bispecific anti-PD-L1 / anti-TIGIT antagonist comprises a linker in the second targeting domain that links the anti-TIGIT HCVR to the anti-TIGIT LCVR, wherein the linker comprises the amino acid sequence of SEQ ID NO: 191.

[0134] In another embodiment, the first targeting domain comprises one or more variable regions from the anti-LAG-3 antibody, and the second targeting domain comprises the anti-TIGIT scFv as described above.

[0135] In one embodiment, the anti-LAG-3 targeting domain comprises one or more immunoglobulin heavy chain CDRs selected from SEQ ID NO: 163-171 and / or one or more immunoglobulin light chain CDRs selected from SEQ ID NO: 172-178.

[0136] In another embodiment, the anti-LAG-3 targeting domain comprises an HCVR having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with an amino acid sequence selected from SEQ ID NO: 180, 182, 184, and 186, and / or an LCVR having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with an amino acid sequence selected from SEQ ID NO: 181, 183, 185, and 187.

[0137] In another embodiment, the anti-LAG-3 targeting domain comprises: immunoglobulin HCVR, comprising a combination of the following (1) and (2): (1) HCDR1 of SEQ ID NO: 163, HCDR2 of SEQ ID NO: 164 and HCDR3 of SEQ ID NO: 165, (2) HFR1 having at least 80%, 85% or 90% identity with the amino acid sequence of SEQ ID NO: 316, HFR2 having at least 80%, 85% or 90% identity with the amino acid sequence of SEQ ID NO: 317, HFR3 having at least 80%, 85% or 90% identity with the amino acid sequence of SEQ ID NO: 318, and HFR4 having at least 80%, 85% or 90% identity with the amino acid sequence of SEQ ID NO: 319; immunoglobulin LCVR, comprising a combination of the following (1) and (2): (1) LCDR1 of SEQ ID NO: 172, LCDR2 of SEQ ID NO: 173 and SEQ ID NO: 165. LCDR3 of NO: 174, (2) LFR1 having at least 80%, 85% or 90% identity with the amino acid sequence of SEQ ID NO: 320, LFR2 having at least 80%, 85% or 90% identity with the amino acid sequence of SEQ ID NO: 321, LFR3 having at least 80%, 85% or 90% identity with the amino acid sequence of SEQ ID NO: 322, and LFR4 having at least 80%, 85% or 90% identity with the amino acid sequence of SEQ ID NO: 323; or both.

[0138] In another embodiment, the anti-LAG-3 targeting domain comprises immunoglobulin HCVR and / or immunoglobulin LCVR, wherein the immunoglobulin HCVR has an amino acid sequence having 90%, 95%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 180, and the immunoglobulin LCVR has an amino acid sequence having 90%, 95%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 181.

[0139] In a particular embodiment, the bispecific anti-LAG-3 / anti-TIGIT antagonist comprises a combination of a first targeting domain and an anti-TIGIT scFv, wherein the first targeting domain comprises an HCVR having the amino acid sequence of SEQ ID NO: 180 and / or an LCVR having the amino acid sequence of SEQ ID NO: 181, and the anti-TIGIT scFv comprises an HCVR having the amino acid sequence of SEQ ID NO: 66 and an LCVR having the sequence of SEQ ID NO: 67. In a more specific embodiment, the scFv in the bispecific anti-LAG-3 / anti-TIGIT antagonist comprises a linker in a second targeting domain that links the anti-TIGIT HCVR to the anti-TIGIT LCVR, wherein the linker comprises the amino acid sequence of SEQ ID NO: 189 or SEQ ID NO: 191.

[0140] In one embodiment, the bispecific anti-LAG-3 / anti-TI GIT antagonist comprises an immunoglobulin heavy chain, an immunoglobulin light chain, or both, wherein the immunoglobulin heavy chain comprises the amino acid sequence of SEQ ID NO: 192 or 193; and the immunoglobulin light chain comprises the amino acid sequence of SEQ ID NO: 181.

[0141] Anti-LAG-3 antibody and its antigen-binding fragment

[0142] In another aspect, this application provides an antibody that specifically binds to LAG-3, including its antigen-binding portion. Figure 19A The heavy chain CDR1, CD2, and CDR3 sequences corresponding to anti-LAG-3mAb 2L2A.1, 2L2A.6, 2L27B, and 3L1A are shown. Figure 19B The light chain CDR1, CD2, and CDR3 sequences corresponding to anti-LAG-3mAb 2L2A.1, 2L2A.6, 2L27B, and 3L1A are shown. Figure 20 The VH and VL sequences of anti-LAG-3mAb 2L2A.1, 2L2A.6, 2L27B and 3L1A are shown.

[0143] In one embodiment, the anti-LAG-3 antibody or its antigen-binding portion comprises: an immunoglobulin heavy chain CDR1 (HCDR1) having at least 80%, at least 85%, or at least 90% sequence identity with the amino acid sequences of HCDR1 selected from SEQ ID NO: 163, 166, and 169; an immunoglobulin heavy chain CDR2 (HCDR2) sequence having at least 80%, at least 85%, or at least 90% sequence identity with the amino acid sequences of HCDR2 selected from SEQ ID NO: 164, 167, and 170; an immunoglobulin heavy chain CDR3 (HCDR3) having at least 80%, at least 85%, or at least 90% sequence identity with the amino acid sequences of HCDR3 selected from SEQ ID NO: 165, 168, and 171; and an immunoglobulin light chain CDR1 (LCDR1) having at least 80%, at least 85%, or at least 90% sequence identity with the amino acid sequences of LCDR1 selected from SEQ ID NO: 172, 175, and 177. Immunoglobulin light chain CDR2 (LCDR2), having at least 80%, at least 85%, or at least 90% sequence identity with the amino acid sequence of LCDR2 selected from SEQ ID NO: 173 and 178; Immunoglobulin light chain CDR3 (LCDR3), having at least 80%, at least 85%, or at least 90% sequence identity with the amino acid sequence of LCDR3 selected from SEQ ID NO: 174, 176, and 179.

[0144] In another embodiment, the anti-LAG-3 antibody or its antigen-binding portion comprises: an immunoglobulin HCDR1 amino acid sequence selected from SEQ ID NO: 163, 166, and 169; an immunoglobulin HCDR2 amino acid sequence selected from SEQ ID NO: 164, 167, and 170; an immunoglobulin HCDR3 amino acid sequence selected from SEQ ID NO: 165, 168, and 171; an immunoglobulin LCDR1 amino acid sequence selected from SEQ ID NO: 172, 175, and 177; an immunoglobulin LCDR2 amino acid sequence selected from SEQ ID NO: 173 and 178; and an immunoglobulin LCDR3 amino acid sequence selected from SEQ ID NO: 174, 176, and 179.

[0145] In another embodiment, the anti-LAG-3 antibody or its antigen-binding portion comprises: an immunoglobulin HCVR having at least 80%, 85%, 90%, 95%, or 99% identity with an amino acid sequence selected from SEQ ID NO: 181, 182, 184, and 186; an immunoglobulin LCVR having at least 80%, 85%, 90%, 95%, or 99% identity with an amino acid sequence selected from SEQ ID NO: 181, 183, 185, and 1871; or both.

[0146] In another embodiment, the anti-LAG-3 antibody or its antigen-binding portion comprises: immunoglobulin HCVR having an amino acid sequence selected from SEQ ID NO: 180, 182, 184 and 186; immunoglobulin LCVR having an amino acid sequence selected from SEQ ID NO: 181, 183, 185 and 187; or both.

[0147] In another embodiment, the anti-LAG-3 antibody or its antigen-binding portion comprises: an immunoglobulin heavy chain sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 180; an immunoglobulin light chain sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 181; or both.

[0148] In a more specific embodiment, the anti-LAG-3 antibody or its antigen-binding portion comprises: the immunoglobulin heavy chain sequence of SEQ ID NO: 180; and the immunoglobulin light chain sequence of SEQ ID NO: 181; or both.

[0149] In another aspect, this application provides one or more nucleic acids encoding any anti-LAG-3 antibody described herein or any antigen-binding portion thereof.

[0150] In another aspect, this application provides one or more expression vectors comprising one or more nucleic acids encoding any anti-LAG-3 antibody as described herein or any antigen-binding portion thereof.

[0151] In another aspect, this application provides host cells transformed with one or more nucleic acids or one or more expression vectors encoding any of the anti-LAG-3 antibodies or any antigen-binding portions described herein.

[0152] In another aspect, this application provides a bispecific antitumor antagonist comprising a first targeting domain that specifically binds to LAG-3; and a second targeting domain that specifically binds to PD-1, PD-L1, or TIGIT, wherein the first targeting domain comprises any of the aforementioned LAG-3 binding portions. Preferably, the anti-LAG-3 bispecific antitumor antagonist comprises an immunoglobulin scaffold containing one or more IgG constant regions such as CH1, CH2, CH3, and / or CL.

[0153] In some embodiments, the first targeting domain is located at the N-terminus and the second targeting domain is located at the C-terminus. In other embodiments, the first targeting domain is located at the C-terminus and the second targeting domain is located at the N-terminus. In still other embodiments, the second targeting domain is inserted into a loop region, for example, within a CH3 domain.

[0154] In one embodiment, the bispecific antitumor antagonist comprises a first targeting domain that specifically binds to LAG-3 and a second targeting domain that specifically binds to PD-1, wherein the first targeting domain comprises any anti-LAG-3 binding fragment as described above, and wherein the second targeting domain comprises any PD-1 binding fragment as described below. For example, in one embodiment, the first targeting domain comprises a combination of the HCVR amino acid sequence of SEQ ID NO: 180 and the LCVR having the amino acid sequence of SEQ ID NO: 181, and the second targeting domain comprises a combination of the HCVR having the amino acid sequence of SEQ ID NO: 106 and the LCVR having the amino acid sequence of SEQ ID NO: 107. Alternatively, the second domain may be constructed in the form of a PD-1ECD.

[0155] In another embodiment, the bispecific antitumor antagonist comprises a first targeting domain that specifically binds to LAG-3 and a second targeting domain that specifically binds to PD-L1, wherein the first targeting domain comprises any of the aforementioned anti-LAG-3 binding fragments, and wherein the second targeting domain comprises any of the PD-L1 binding fragments described below. For example, in one embodiment, the first targeting domain comprises a combination of HCVR having the amino acid sequence of SEQ ID NO: 180 and LCVR having the amino acid sequence of SEQ ID NO: 181, and the second targeting domain comprises a combination of HCVR having the amino acid sequence of SEQ ID NO: 153 and LCVR having the amino acid sequence of SEQ ID NO: 154.

[0156] In another embodiment, the bispecific antitumor antagonist comprises a first targeting domain that specifically binds to LAG-3 and a second targeting domain that specifically binds to TIGIT, wherein the first targeting domain comprises any of the aforementioned anti-LAG-3 binding fragments, and wherein the second targeting domain comprises any of the aforementioned TIGIT binding fragments. For example, in one embodiment, the first targeting domain comprises a combination of HCVR having the amino acid sequence of SEQ ID NO: 180 and LCVR having the amino acid sequence of SEQ ID NO: 181, and the second targeting domain comprises a combination of HCVR having the amino acid sequence of SEQ ID NO: 66 and LCVR having the amino acid sequence of SEQ ID NO: 67. Alternatively, the second domain may be constructed in the form of TIGITECD.

[0157] Exemplary immunoglobulin scaffolds include, for example, complete CH1-CH2-CH3 fragments as shown in SEQ ID NO: 155-157 and 205-215, or Fc (hinge-CH2-CH3) containing the sequence amino acid sequence shown in any of SEQ ID NO: 195-202.

[0158] Anti-TIGIT antibody and anti-TIGIT antibody fragment

[0159] In some implementations, checkpoint regulator antagonists include anti-TIGIT antibodies or antigen-binding fragments thereof. Figure 1 The CDR sequence for anti-TIGIT mAb is shown. Figure 2A-2B Several embodiments of the anti-TIGIT antibody variable domain sequence used in this application are shown.

[0160] In one embodiment, the anti-TIGIT antibody or its antigen-binding fragment comprises: (1) an immunoglobulin HCVR containing three complementarity-determining regions (HCDRs): HCDR1, HCDR2, and HCDR3, wherein HCDR1 has an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with an amino acid sequence selected from SEQ ID NO: 1, 6, 11, 15, 17, 20, and 23; wherein HCDR2 has an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with an amino acid sequence selected from SEQ ID NO: 2, 4, 7, 9, 12, 13, 16, 18, 21, and 24; and wherein HCDR3 ... (1) The amino acid sequences of SEQ ID NOs 3, 5, 8, 10, 14, 19, 22, and 25 have at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity; and (2) Immunoglobulin LCVR comprising the sequences LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of LCDR1 has at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with the amino acid sequences selected from SEQ ID NOs 26, 29, 31, 33, 35, 39, 42, and 45, wherein the amino acid sequence of LCDR2 has at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with the amino acid sequences selected from SEQ ID NOs 27, 30, 36, 37, 40, 43, and 46, and the amino acid sequence of LCDR3 ...6, 29, 31, 33, 35, 39, 42, and 45, wherein the amino acid sequence of LCDR2 has at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or The amino acid sequences of NO: 28, 32, 34, 38, 41, 44 and 47 have at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity; wherein the antibody or its antigen-binding moiety specifically binds to human TIGIT.

[0161] In another embodiment, the anti-TIGIT antibody or its antigen-binding fragment comprises: (1) immunoglobulin HCVR, the amino acid sequence of which has at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with an amino acid sequence selected from SEQ ID NO: 48, 50, 52, 54, 56, 58, 60, 62, 64, and 66; and (2) immunoglobulin LCVR, the amino acid sequence of which has at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with an amino acid sequence selected from SEQ ID NO: 49, 51, 53, 55, 57, 59, 61, 63, 65, and 67; wherein the antibody or its antigen-binding fragment specifically binds to human TIGIT.

[0162] Anti-PD-1 antibodies and their antigen-binding fragments

[0163] In some implementations, checkpoint regulator antagonists include anti-PD-1 antibodies or antigen-binding fragments thereof. Figure 3 The CDR sequence for anti-PD-1mAb is shown. Figure 4A -4C illustrates several embodiments of the anti-PD-1 antibody variable domain sequence used in this application.

[0164] In one embodiment, the anti-PD-1 antibody or its antigen-binding fragment comprises: (1) an immunoglobulin HCVR containing HCDR1, HCDR2, and HCDR3 sequences, wherein the amino acid sequence of HCDR1 has at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with amino acid sequences selected from SEQ ID NO: 68, 71, 74, 76, and 79; wherein the amino acid sequence of HCDR2 has at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with amino acid sequences selected from SEQ ID NO: 69, 72, 77, and 80; and wherein the amino acid sequence of HCDR3 has at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with amino acid sequences selected from SEQ ID NO: 68, 71, 74, 76, and 79. (1) The amino acid sequences of NO: 70, 73, 75, 78 and 81 have at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity; (2) An immunoglobulin LCVR comprising the sequences LCDR1, LCDR2 and LCDR3, wherein the amino acid sequence of LCDR1 has at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with the amino acid sequences selected from SEQ ID NO: 82, 85, 88, 89, 90 and 93, wherein the amino acid sequence of LCDR2 has at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with the amino acid sequences selected from SEQ ID NO: 83, 86, 91 and 94, and wherein the amino acid sequence of LCDR3 ... The amino acid sequences of IDNO: 84, 87, 92, and 95 have at least 80%, 85%, 90%, 95%, 99%, or 100% identity, wherein the antibody or its antigen-binding moiety specifically binds to human PD-1.

[0165] In some embodiments, the anti-PD-1 antibody or its antigen-binding fragment comprises: (1) immunoglobulin HCVR, the amino acid sequence of which has at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with an amino acid sequence selected from SEQ ID NO: 96, 98, 100, 102, 104 and 106; and (2) immunoglobulin LCVR, the amino acid sequence of which has at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with an amino acid sequence selected from SEQ ID NO: 97, 99, 101, 103, 105 and 107, wherein the antibody or its antigen-binding fragment specifically binds to human PD-1.

[0166] Anti-PD-L1 antibody and its antigen-binding fragment

[0167] In some implementations, checkpoint regulator antagonists include anti-PD-L1 antibodies or antigen-binding fragments thereof. Figure 5 The CDR sequence for PD-L1 mAb is shown. Figures 6A-6C Several embodiments of the anti-PD-L1 antibody variable domain sequence used in this application are shown.

[0168] In one embodiment, the PD-L1 antibody or its antigen-binding fragment comprises: (1) an immunoglobulin HCVR containing HCDR1, HCDR2, and HCDR3 sequences, wherein the amino acid sequence of HCDR1 is selected from the amino acid sequences of SEQ ID NO: 108, 111, 117, and 120 and has at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity, wherein the amino acid sequence of HCDR2 has at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity, and wherein the amino acid sequence of HCDR3 is selected from the amino acid sequences of SEQ ID NO: 109, 112, 114, 116, 118, and 121 and has at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity, and wherein the amino acid sequence of HCDR3 is selected from the amino acid sequences of SEQ ID NO: 108, 111, 117, and 120 .... (1) The amino acid sequences of NO: 110, 113, 115, 119 and 122 have at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity; (2) Immunoglobulin LCVR, wherein the light chain variable region comprises three complementarity-determining regions (LCDRs): LCDR1, LCDR2 and LCDR3, wherein the amino acid sequence of LCDR1 has at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with the amino acid sequences selected from SEQ ID NO: 123, 126, 130, 133 and 136, wherein the amino acid sequence of LCDR2 has at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with the amino acid sequences selected from SEQ ID NO: 124, 127, 131, 134 and 137, and wherein the amino acid sequence of LCDR3 ... The amino acid sequences of NO: 125, 128, 129, 132, 135 and 138 have at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity, wherein the antibody or its antigen-binding moiety specifically binds to human PD-L1.

[0169] In some embodiments, the PD-L1 antibody or its antigen-binding fragment comprises: (1) immunoglobulin HCVR, the amino acid sequence of which has at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with an amino acid sequence selected from SEQ ID NO: 139, 141, 143, 145, 147, or 149, 151, and 153; and (2) immunoglobulin LCVR, the amino acid sequence of which has at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with an amino acid sequence selected from SEQ ID NO: 140, 142, 144, 146, 148, 150, 152, and 154, wherein the antibody or its antigen-binding fragment specifically binds to human PD-L1.

[0170] II. Other Implementation Plans

[0171] The HCVR and LCVR described herein can be linked to immunoglobulin scaffolds. In some embodiments, the immunological scaffold is constructed as IgG1, IgG2, or IgG4. The immunoglobulin scaffold may contain a CH1-CH2-CH3 region, or may contain a naturally occurring Fc region or a non-naturally occurring or mutated Fc region, such as an effectorless or predominantly effector Fc (e.g., human IgG2 or IgG4), or, alternatively, have an Fc that enhances binding to one or more activated Fc receptors (FcγRI, FcγRIIa, or FcγRIIIa), thereby enhancing T in the tumor setting. reg Consumption. Therefore, in some embodiments, the anti-TI GIT, anti-PD-1, anti-PD-L1, anti-LAG-3, HCVR, and LCVR described herein can typically be linked to an Fc containing one or more modifications to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cytotoxicity.

[0172] In one embodiment, the immunoglobulin scaffold used in this application comprises a CH1-CH2-CH3 region having the amino acid sequences shown in SEQ ID NO: 155-157 and 205-215. In another embodiment, the immunoglobulin scaffold comprises or is substantially composed of Fc receptors, such as Fc receptors having the amino acid sequences shown in any one of SEQ ID NO: 195-202.

[0173] Furthermore, the antibodies described herein may be chemically modified (e.g., one or more chemical moieties may be linked to the antibody) or may be modified to alter their glycosylation, thereby changing one or more functional properties of the antibody. More specifically, in some embodiments, the antibodies of this application may include modifications in the Fc region to produce antibodies with (a) increased or decreased antibody-dependent cell-mediated cytotoxicity (ADCC), (b) increased or decreased complement-mediated cytotoxicity (CDC), (c) increased or decreased affinity for Clq, and / or (d) increased or decreased affinity for the Fc receptor (relative to the parent Fc). Such Fc region variants will typically contain at least one amino acid modification in the Fc region. Combined amino acid modifications are considered particularly desirable. For example, the variant Fc region may include two, three, four, five, etc., substitutions therein (e.g., at specific Fc region positions identified herein).

[0174] For applications where effector function is completely avoided, such as when antigen binding alone is sufficient to produce the desired therapeutic benefit and effector function only results in undesirable side effects (or increases the risk of side effects), IgG4 antibodies can be used. Alternatively, antibodies or fragments thereof lacking the Fc region or a substantial segment can be devised, or the Fc can be mutated to completely eliminate glycosylation (e.g., N297A). Alternatively, hybrid constructs of human IgG2 (CH1 domain and hinge region) and human IgG4 (CH2 and CH3 domains) can be generated, lacking effector function and the ability to bind FcyR (as in IgG2) and activate complement (as in IgG4). When using the constant IgG4 domain, it is generally preferred to include a substitution of S228P, which mimics the hinge sequence in IgG1 and thereby stabilizes the IgG4 molecule, reducing Fab arm exchange between the therapeutic antibody and endogenous IgG4 in the treated patient.

[0175] In a preferred embodiment, the first and second targeting domains are present in the humanized immunoglobulin scaffold. Additionally, the IgG scaffold may have N297A or K447A amino acid substitutions.

[0176] In some embodiments, anti-TIGIT, anti-PD-1, anti-PD-L1, anti-LAG-3, or fragments thereof may be modified to increase their biological half-life. Various methods can be employed, including, for example, increasing the binding affinity of the Fc region to FcRn. In one embodiment, as described in U.S. Patent Nos. 5,869,046 and 6,121,022, the antibody is modified in the CH1 or CL region to include a salvage receptor-binding epitope truncated from two loops of the CH2 domain of the Fc region of IgG. The residue numbers in the Fc region are EU index numbers. Sequence variants disclosed herein are provided with reference to the residue numbers, the amino acid following the residue number being the substituted native amino acid, optionally a previously native residue at that position. Where multiple amino acids may be present at a given position, for example, if sequences differ between native isotypes, or if multiple mutations may be present at that position, they are separated by slashes (e.g., “X / Y / Z”).

[0177] Exemplary Fc variants that increase binding to FcRn and / or improve pharmacokinetic properties include substitutions at positions 259, 308, and 434, including, for example, 259I, 308F, 428L, 428M, 434S, 434H, 434F, 434Y, and 434M. Other variants that increase the binding of Fc to FcRn include: 250E, 250Q, 428L, 428F, 250Q / 428L (Hinton et al., 2004, J. Biol. Chem. 279(8):6213-6216, Hinton et al. 2006 Journal of Immunology 176:346-356), 256A, 272A, 305A, 307A, 31A, 312A, 378Q, 380A, 382A, 434A (Shields et al.). al. (2001) J. Biol. Chem., 276(9): 6591-6604), 252F, 252Y, 252W, 254T, 256Q, 256E, 256D, 433R, 434F, 434Y, 252Y / 254T / 256E, 433K / 434F / 436H (Dall'Acqua et al. (2002) J. Immunol., 169: 5171-5180, Dall'Acqua et al. (2006) J. Biol. Chem., 281: 23514-23524 and U.S. Patent No. 8,367,805.

[0178] Modification of certain conserved residues in IgG Fc (1253, H310, Q311, H433, N434), such as the N434A variant (Yeung et al. (2009) J. Immunol. 182:7663), has been proposed as a method to increase FcRn affinity and thus prolong the antibody's half-life in circulation (WO 98 / 023289). Combined Fc variants containing M428L and N434S have been shown to increase FcRn binding and prolong serum half-life by up to five times (Zalevsky et al. (2010) Nat. Biotechnol. 28:157). Combined Fc variants containing modifications of T307A, E380A, and N434A have also prolonged the half-life of IgG1 antibodies (Petkova et al. (2006) Int. Immunol. 18:1759). Furthermore, it has been shown that combined Fc variants containing M252Y-M428L, M428L-N434H, M428L-N434F, M428L-N434Y, M428L-N434A, M428L-N434M, and M428L-N434S can extend the half-life (US2006 / 173170). Additionally, it has been reported that combined Fc variants containing M252Y, S254T, and T256E increase the half-life by nearly four times (Dall'Acqua et al. (2006) J.Biol.Chem.281:23514).

[0179] The bispecific antitumor antagonists of this application can be constructed using an IgG backbone. More specifically, any bispecific antagonist of this application can be constructed using an IgG1 or IgG4 backbone. The use of an IgG1 backbone is preferred for cancer therapy, where the target is present on antigen-presenting cells that can mediate antibody-dependent cell-mediated cytotoxicity (ADCC). The use of an IgG4 backbone allows for targeting of antigens, where antigen binding alone is sufficient to produce the desired therapeutic benefit. IgG4-based antagonists exclude undesirable effector functions associated with, for example, IgG1 antibodies, including FcγR binding and complement activation.

[0180] Homopolymers and heterodimers

[0181] One of the challenges in effectively generating bispecific antibody formulations when co-expressing chains with different binding specificities involves mismatches between heavy and light chains. Table 1 lists several amino acid alternatives for overcoming mismatches between heavy chains with different binding specificities, which "enhance" or preferentially promote the correct binding between the desired heavy chains. Any method for preventing or reducing mismatches between heavy chains can be used to prepare the bispecific antitumor antagonist according to the present invention.

[0182] The "KiH" method relies on modification of the interface between two CH3 domains where most interactions occur. Typically, a large residue is introduced into the CH3 domain of the antibody heavy chain, acting like a key. In the other heavy chain, a "pore" is formed that accommodates the large residue, similar to a lock. The resulting heterodimer Fc moiety can be further stabilized with artificial disulfide bonds.

[0183] An alternative approach is based on charged residues with ionic interactions or spatial complementarity. This involves altering the charge polarity at the CH3 interface so that co-expression of electrostatically matched Fc domains supports favorable attractive interactions and heterodimer formation, while retaining the hydrophobic core and unfavorably repelling charge interactions to inhibit homodimerization. See Table 1. The amino acid numbering in Table 1 follows the Kabat numbering scheme and can be applied to the heavy chain amino acid sequences of the antibodies described herein.

[0184] In some embodiments, the immune scaffold can be replaced by another dimer structure containing, for example, a leucine zipper (LZ) domain. Leucine zippers are common three-dimensional structural motifs in proteins, often part of the DNA-binding domain in various transcription factors. A single LZ typically contains 4-5 leucine residues spaced approximately 7 residues apart, forming an amphiphilic α-helix with its hydrophobic region extending along one side. In a particular embodiment, the heterodimeric protein scaffold comprises LZs from the c-jun transcription factor and LZs from the c-fos transcription factor. Although c-jun is known to form jun-jun homodimers while c-fos is not, the formation of jun-fos heterodimers is more advantageous than that of jun-jun homodimers.

[0185] The leucine zipper domain can replace the CH2-CH3 sequence in the protein scaffold, or it can be placed at the carboxyl terminus of the two heavy chains in a bispecific antitumor antagonist. In the latter case, a furin protease cleavage site can be introduced between the carboxyl terminus of CH3 and the amino terminus of the leucine zipper. When the heavy and light chains of the bispecific antitumor antagonist are co-expressed in a suitable mammalian cell expression system, furin protease-mediated cleavage of the leucine zipper can be promoted after the heterodimerization step (see Wranik et al., J. Biol. Chem., 287(5):43331-43339, 2012).

[0186] Table 1

[0187]

[0188] The amino acid numbers in Table 1 follow the Kabat numbering scheme and can be applied to the heavy chain amino acid sequences of the antibodies described herein. The mutations described in Table 1 can be applied to sequences of any immunoglobulin IgG1 heavy chain and other immunoglobulin classes and subclasses (or isotypes) therein (as published or otherwise).

[0189] When heavy and light chains of monospecific and bispecific antibodies are co-expressed, the light chain with one binding specificity may mismatch with the heavy chain with the other binding specificity. Therefore, in some embodiments, portions of the heavy chain, light chain, or both may be modified relative to the “wild-type” antibody chain from which they are derived to prevent or reduce mismatches between the two heavy chain constant regions and between the light chain constant region and its heavy chain counterpart.

[0190] Light chain mismatch problems can be addressed in several ways. In some embodiments, spatially complementary mutations and / or disulfide bonds can be introduced into both VL / VH interfaces. In other embodiments, mutations can be introduced based on ionic or electrostatic interactions. In some embodiments, light chain mismatches can be prevented or reduced by using a first arm having an S183E mutation in the CH1 domain of the heavy chain and an S176K mutation in the CL domain of the light chain. A second arm may contain the S183K mutation in the CH1 domain of the heavy chain and the S176E mutation in the CL domain of the light chain. In other embodiments, a “CrossMab” approach is employed, in which one arm of a bispecific antitumor antagonist (e.g., Fab) remains unchanged, but in the other arm containing additional binding specificity, one or more domains in the light chain are swapped with one or more domains in the heavy chain at the heavy chain:light chain interface.

[0191] The methods and immunoglobulin domain sequences, including specific mutations as described above for preventing heavy and light chain mismatches, are further described in U.S. Patent Application Publication Nos. 2014 / 0243505 and 2013 / 0022601.

[0192] Coupled

[0193] In some embodiments, the antitumor antagonist of this application is chemically coupled to one or more peptides and / or small molecule drugs. The peptides or small molecule drugs may be the same or different. The peptides or small molecule drugs may, for example, be linked to reduced SH groups and / or carbohydrate side chains. Methods for preparing covalent or non-covalent conjugates of peptides or small molecule drugs with antibodies are known in the art, and any such known methods may be used.

[0194] In some embodiments, the peptide or small molecule drug is attached to the hinge region of the reduced antibody component via a disulfide bond. Alternatively, a heterobifunctional crosslinking agent, such as N-succinoyl 3-(2-pyridyl dithio)propionate (SPDP), can be used to link such reagents. Common techniques for this conjugation are well known in the art. In some embodiments, the peptide or small molecule drug is conjugated via a carbohydrate moiety in the Fc region of the antibody. The carbohydrate group can be used to increase the loading of the same reagent that binds to the thiol group, or the carbohydrate group can be used to bind different therapeutic or diagnostic agents. Methods of conjugating peptide inhibitors or small molecule drugs to antibodies via the antibody carbohydrate moiety are well known to those skilled in the art. For example, in one embodiment, the method includes reacting an antibody component having an oxidized carbohydrate moiety with a carrier polymer having at least one free amine function. This reaction produces an initial Schiff base (imine) bond, which can be stabilized by reduction to a secondary amine to form the final conjugate. Exemplary methods for conjugating small molecule drugs and peptides to antibodies are described in U.S. Patent Publication No. 2014 / 0356385.

[0195] Preferably, the antitumor antagonists of this disclosure retain certain desired characteristics and pharmacokinetic properties of antibodies, including desired in vitro and in vivo stability (e.g., individual half-life and half-life stability), desired efficient delivery to target cells, increased affinity for binding chaperone molecules, desired antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity, and reduced renal clearance or excretion. Therefore, consideration can be given to size and the need for specific constant-region effector functions in the design of antitumor antagonists.

[0196] Anti-TIGIT, anti-PD-1, and anti-PD-L1 inhibitors, including their monospecific and bispecific antitumor antagonists, can range in size from 50kD to 300kD, 50kD to 250kD, 60kD to 250kD, 80kD to 250kD, 100kD to 250kD, 125kD to 250kD, 150kD to 250kD, 60kD to 225kD, 75kD to 225kD, 100kD to 225kD, 125kD to 225kD, and 150kD. Any range covered by any combination of all the numbers listed above, or any range specified by any combination of all the values ​​between any ranges listed above.

[0197] Reagent test kit

[0198] This application further provides a kit comprising any one or more of the checkpoint modulator antagonists or antitumor antagonists of this application. In some embodiments, the kit further comprises other components, including syringes and needles for administration, and reagents, including a secondary antibody for detection, and other antibodies described herein for combination therapy. Kits typically include labels and / or instructions indicating the intended use of the kit contents. Labels or instructions may include any written or documented material provided on or with the kit or accompanying the kit.

[0199] III. Methods of using antitumor antagonists

[0200] The antitumor antagonist of this application has many in vitro and in vivo effects, including, for example, enhancing immune responses and treating cancer, infectious diseases, or autoimmune diseases.

[0201] In some embodiments, this application provides a method for treating a proliferative disease; a method for reducing or depleting regulatory T cells in a tumor; a method for treating a microbial infection; or a method for treating an immune disease, wherein the method comprises administering an effective amount of the antitumor antagonist according to this application to a subject in need.

[0202] In some embodiments, the antitumor antagonists of this application are administered in vitro or ex vivo to cultured cells, or in vivo to human subjects, to enhance immunity in a variety of diseases. Therefore, this document provides methods for altering immune responses in subjects, comprising administering to the subject the antibodies described herein or antigen-binding fragments thereof to enhance, stimulate, or upregulate the subject's immune response. Preferred subjects include human patients requiring enhanced immune responses. This method is particularly suitable for treating human patients with diseases that can be treated by enhancing immune responses (e.g., T-cell-mediated immune responses). The method is particularly suitable for in vivo treatment of cancer or chronic infections. For example, an anti-TIGIT, anti-PD-1, anti-PD-L1, or anti-LAG-3 composition may be administered together with an antigen of interest, or the antigen may already be present in the subject to be treated (e.g., a subject with a tumor or carrying a virus) to enhance antigen-specific immunity. When an anti-TIGIT antibody is administered together with another agent, the two components may be administered separately or simultaneously.

[0203] In some embodiments, the checkpoint regulator antagonist used in the above methods is anti-TIGIT, anti-PD-1, anti-PD-L1 antibody, anti-LAG-3 antibody, fragments thereof, or combinations thereof. In some embodiments, the checkpoint regulator antagonist is a monospecific or bispecific antibody.

[0204] In some embodiments, the checkpoint regulator antagonist or antitumor antagonist is in the form of an antibody or antibody fragment. In some embodiments, the antibody described herein is a human or humanized antibody.

[0205] It also includes a method for detecting and / or measuring the presence of human TIGIT, human PD-1, human PD-L1, or human LAG3 in a sample, the method comprising contacting a sample and a control sample with a human monoclonal antibody or an antigen-binding fragment thereof, the human monoclonal antibody or antigen-binding fragment thereof specifically binding to the human TIGIT, human PD-1, or human PD-L1 under conditions allowing the formation of a complex between the antibody or fragment thereof and the human TIGIT, human PD-1, or human PD-L1. The formation of the complex is then detected, wherein a difference in complex formation between the sample and the control sample indicates the presence of human TIGIT antigen in the sample.

[0206] Given the ability of anti-TIGIT, anti-PD-1, anti-PD-L1, and anti-LAG-3 antibodies to block the inhibition or co-inhibition of T cell responses (e.g., antigen-specific T cell responses), this document provides in vitro and in vivo methods for stimulating, enhancing, or upregulating antigen-specific T cell responses, such as anti-tumor T cell responses, using the antibodies described herein. In some embodiments, CD3 stimulation (e.g., by co-incubation with cells expressing cell membrane CD3) is also provided, said stimulation may be provided concurrently with, before, or after treatment with anti-TIGIT, anti-PD-1, anti-PD-L1, or anti-LAG-3 antibodies. For example, this application provides a method for enhancing antigen-specific T cell responses comprising contacting T cells with the anti-TIGIT, anti-PD-1, anti-PD-L1, or anti-LAG-3 antibodies described herein, and optionally with CD3, thereby enhancing the antigen-specific T cell response, for example, by removing the inhibition mediated by TIGIT, PD-1, PD-L1, or LAG-3. Any suitable indicator of antigen-specific T cell response may be used to measure the antigen-specific T cell response. Non-limiting examples of such suitable indicators include increased T cell proliferation in the presence of an antibody and / or increased cytokine production in the presence of an antibody. In a preferred embodiment, the production of interleukin-2 and / or interferon-γ by antigen-specific T cells is enhanced.

[0207] Further comprising a method for enhancing an immune response (e.g., antigen-specific T-cell response) in a subject, the method comprising administering to the subject an anti-TIGIT antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-LAG-3 antibody, or a bispecific antitumor antagonist as described herein, thereby enhancing an immune response (e.g., antigen-specific T-cell response) in the subject. In a preferred embodiment, the subject is a subject with a tumor, and the antitumor immune response is enhanced. The tumor can be a solid tumor or a liquid tumor, such as a hematologic malignancy. In some embodiments, the tumor is an immunogenic tumor. In other embodiments, the tumor is non-immunogenic. In some embodiments, the tumor is PD-L1 positive. In other embodiments, the tumor is PD-L1 negative. The subject may also be a subject carrying a virus, and the administration of an anti-TIGIT antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-LAG-3 antibody, a monospecific antitumor antagonist as described herein, or a bispecific antitumor antagonist results in an enhanced antiviral immune response.

[0208] In one embodiment, a method for inhibiting tumor cell growth in a subject includes administering an anti-TIGIT antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-LAG-3 antibody, or a bispecific antitumor antagonist described herein to the subject, thereby inhibiting tumor growth in the subject. A method for treating chronic viral infection in a subject is also provided, comprising administering an anti-TIGIT antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-LAG-3 antibody, or a bispecific antitumor antagonist described herein to the subject, thereby treating chronic viral infection in the subject.

[0209] This article also includes the consumption of T from the tumor microenvironment of subjects with tumors, such as cancerous tumors. reg A cellular approach comprising administering to a subject a therapeutically effective amount of an anti-TIGIT antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-LAG-3 antibody, or a bispecific antitumor antagonist described herein, wherein the bispecific antitumor antagonist comprises substances that stimulate T cells in the tumor microenvironment. reg Fc consumed by the cell. Fc can be, for example, an effector with or enhanced effector function, such as an Fc that binds to or enhances binding to one or more activated Fc receptors.

[0210] In a preferred embodiment, T occurs in the tumor microenvironment. reg Consumes without significantly consuming or inhibiting T eff And it did not significantly deplete or inhibit T cells outside the tumor microenvironment. eff and T reg Cells. In some implementations, such as in the tumor microenvironment, the subject in T cells.reg TIGIT levels on cells are higher than in T cells. eff The level of TIGIT in the tumor. In some implementations, anti-TIGIT antibodies or antagonists can deplete TIGIT levels in the tumor. regs and / or T cells in tumor-infiltrating lymphocytes (TILs) regs For example, in the CT26 tumor model, anti-mouse TIGIT antibodies that form mouse IgG2a (exhibiting effector function) partially deplete T2. reg and CD8 + T cells, but do not consume CD4 + T cells. The anti-TIGIT antibody that forms an effectless response to mouse IgG1 D265A does not deplete T cells.

[0211] When considering whether to use anti-TIGIT antibodies with or without Fc effector function, appropriate consideration must be given to the potential for T cells to enhance the anti-tumor immune response. regs The consumption and elimination of some of the CD8 cells needed to actually kill tumor cells + The balance between T cell depletion and other factors. Although T cell depletion is expected. regs To enhance anti-tumor activity, but recent studies have shown that TIGIT... + T regs The ligation of TIGIT on the surface promotes T reg Cell-mediated T eff Cell proliferation inhibition (Joller et al. (2014) Immunity 40:569) indicates that blocking TIGIT signaling (e.g., using the antagonist anti-TIGIT antibody of the present invention) can also enhance antitumor activity. Therefore, the use of antagonistic anti-TIGIT antibodies lacking effector function may be most effective, having the following functions: i) blocking T regs (ii) By blocking the inhibitory effect of TIGIT on TIGIT signaling, thereby reducing their immunosuppressive activity; + T cells, while avoiding their depletion mediated by effector function; iii) enhancing DNAM-mediated activation by allowing DNAM to bind to PVR (CD155, TIGIT ligand) that would otherwise bind to TIGIT (and reducing direct TIGIT-DNAM interaction) (Johnston et al. (2014) CancerCell 26:923). This also applies to the use of anti-PD-1 antibodies, anti-PD-L1 antibodies, or bispecific antitumor antagonists.

[0212] In some embodiments, anti-TIGIT antibodies, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-LAG-3 antibodies, or bispecific antitumor antagonists described herein are administered to subjects as adjuvant therapy. Treatment of cancer patients with anti-TIGIT antibodies, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-LAG-3 antibodies, or bispecific antitumor antagonists according to this application may result in a long-term sustained response relative to current standards of care; long-term survival of at least 1, 2, 3, 4, 5, 10 years or longer, and recurrence-free survival of at least 1, 2, 3, 4, 5, or 10 years or longer. In some embodiments, treatment of cancer patients with anti-TIGIT antibodies, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-LAG-3 antibodies, or bispecific antitumor antagonists may prevent or delay cancer recurrence for, for example, 1, 2, 3, 4, 5, or 10 years or longer. Anti-TIGIT, anti-PD-1, anti-PD-L1, and / or anti-LAG-3 therapy can be used as primary or secondary treatment options.

[0213] In some preferred embodiments, the subject has a proliferative disease or cancer. Anti-TIGIT antibodies enhance the immune response against the patient's cancer cells by blocking PVR / Nectin-2 signaling via TIGIT. Similarly, the blocking described herein is a method for treating a subject with cancer, comprising administering to the subject an anti-TIGIT, anti-PD-1, anti-PD-L1, anti-LAG-3, or a bispecific antitumor antagonist thereof as described herein. This treats the subject, for example, by inhibiting or reducing the growth of cancerous tumors and / or causing tumor regression. Anti-TIGIT, anti-PD-1, anti-PD-L1, anti-LAG-3, or a bispecific antitumor antagonist thereof as described herein can be used alone to inhibit the growth of cancerous tumors. Alternatively, any of these antitumor antagonists can be used in combination with another agent, such as other anticancer targets, immunogenic agents, standard cancer treatments, or other antibodies, as described below.

[0214] Therefore, this article provides a method for treating cancer by, for example, inhibiting tumor cell growth in a subject, the method comprising administering to the subject a therapeutically effective amount of an anti-TI GIT, anti-PD-1, anti-PD-L1, or anti-LAG-3 antagonist, or a bispecific antitumor antagonist as described herein. Preferably, the antibody is a human anti-TIGIT, anti-PD-1, anti-PD-L1, or anti-LAG-3 HCVR and LCVR comprising the anti-TIGIT, anti-PD-1, anti-PD-L1, or anti-LAG-3 antibodies described herein, or may be a chimeric, humanized, or non-human anti-hu TIGIT, anti-hu PD-1, anti-PD-L1, or anti-LAG-3 antibody, such as a chimeric, humanized, or non-human anti-TIGIT, anti-PD-1, anti-PD-L1, or anti-LAG-3 antibody that competitively binds to the same epitope as at least one of the anti-TIGIT, anti-PD-1, anti-PD-L1, or anti-LAG-3 antibodies described herein, or binds to the same epitope as at least one of the anti-TIGIT, anti-PD-1, anti-PD-L1, or anti-LAG-3 antibodies described herein.

[0215] Cancers whose growth can be inhibited using the antibodies of this invention include cancers that typically respond to immunotherapy. Non-limiting examples of cancers for treatment include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, squamous non-small cell lung cancer (NSCLC), non-NSCLC, glioma, gastrointestinal cancer, renal cancer (e.g., cell carcinoma), ovarian cancer, liver cancer, colorectal cancer, endometrial cancer, kidney cancer (e.g., renal cell carcinoma (RCC)), prostate cancer (e.g., hormone-refractory prostate adenocarcinoma), thyroid cancer, neuroblastoma, pancreatic cancer, glioblastoma (glioblastoma multiforme), cervical cancer, gastric cancer, bladder cancer, liver cancer, breast cancer, colon cancer and head and neck cancer (or cancers), gastric cancer, germ cell tumors, pediatric sarcoma, sinus natural killer cells. Killer,), melanoma (e.g., metastatic malignant melanoma, such as cutaneous or ocular malignant melanoma), bone cancer, skin cancer, uterine cancer, anal region cancer, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, esophageal cancer, small intestine cancer, endocrine system cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, childhood solid tumors, ureteral cancer, renal pelvis cancer, central nervous system tumors (CNS), primary CNS lymphoma, tumor angiogenesis, spinal cord axis tumors, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers (including those induced by asbestos), virus-related cancers (e.g., tumors related to human papillomavirus (HPV)), and cancers derived from one of the two main blood cell lines, namely the myeloid cell line (which produces granulocytes, erythrocytes, platelets, macrophages). Hematologic malignancies of the blood system, such as all types of leukemia, lymphoma, and myeloma, including acute, chronic, lymphocytic, and / or myeloid leukemias, such as acute leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL) and chronic myeloid leukemia (CML), undifferentiated AML (MO), myeloblastic leukemia (M1), myeloblastic leukemia (M2; as cells mature), promyelocytic leukemia (M3 or M3 variant [M3V]), myelomonocytic leukemia (M4 or M4 with eosinophilia [M4E]), monocytic leukemia (M5), erythroleukemia (M6), megakaryocytic small cell leukemia (M7), solitary granulocytic sarcoma, and chloroma.Lymphomas such as Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NEIL), B-cell lymphoma, T-cell lymphoma, lymphoplasmacytic lymphoma, mononuclear B-cell lymphoma, mucosa-associated lymphoid tissue (MALT) lymphoma, anaplastic (e.g., Ki 1+) large cell lymphoma, adult T-cell lymphoma / leukemia, mantle cell lymphoma, angioimmunoblastic T-cell lymphoma, angiocentric lymphoma, intestinal T-cell lymphoma, primary mediastinal B-cell lymphoma, precursor T-lymphoblastic lymphoma, T-lymphocytic lymphoma / leukemia (T-Lbly / T-ALL), peripheral T-cell lymphoma, lymphoblastic lymphoma, post-transplant lymphoproliferative disorders, true histiocytic lymphoma, primary central nervous system lymphoma. Lymphoma, primary exudative lymphoma, lymphoblastic lymphoma (LBL), hematopoietic lymphoid neoplasms, acute lymphoblastic leukemia, diffuse large B-cell lymphoma, Burkitt lymphoma, follicular lymphoma, diffuse histiocytic lymphoma (DHL), immunogenic large cell lymphoma, precursor B-cell lymphoblastic lymphoma, cutaneous T-cell lymphoma (CTLC) (also known as mycosis or Sezary syndrome), and lymphoma with Waldenstrom macroglobulin. Lymphoplasmacytic lymphoma (LPL) of leukemia; myeloma, such as IgG myeloma, light chain myeloma, non-secreting myeloma, smoldering myeloma (also known as indolent myeloma), solitary plasmacytoma and multiple myeloma, chronic lymphocytic leukemia (CLL), pilocellular lymphoma; myeloid hematopoietic tumors, stromal tumors, including fibrosarcoma and rhabdomyosarcoma; seminoma, teratoma, central and peripheral nerve tumors, including astrocytoma, schwannoma Mesenchymal tumors, including fibrosarcoma, rhabdomyosarcoma, and osteosarcoma; and other tumors, including melanoma, xeroderma pigmentosum, keratinocyte acanthoma, seminoma, thyroid follicular carcinoma, and teratoma; lymphoid hematopoietic tumors, such as T-cell and B-cell tumors, including but not limited to T-cell diseases such as T-cell prolymphocytic leukemia (T-PLL), including small cell and brain-like cell types; large granular lymphocytic leukemia (LGL), preferably T-cell type; a / d T-NHL hepatosplenic lymphoma; peripheral / postthymic T-cell lymphoma (pleomorphic and immunoblastic subtypes); angiogenic (nasal) T-cell lymphoma; head and neck cancer, renal cell carcinoma, rectal cancer, thyroid cancer; acute myeloid lymphoma; and any combination of the above cancers. The methods described herein can also be used to treat metastatic cancers, refractory cancers (e.g., cancers refractory to previous immunotherapy, such as with blocking CTLA-4 or PD-1 antibodies), and relapsed cancers.

[0216] Anti-TIGIT, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-LAG-3 antibodies, or bispecific antitumor antagonists can be administered alone, in combination with another antitumor antagonist, or concurrently with another antitumor antagonist. Anti-TIGIT, anti-PD-1 antibodies, anti-LAG-3 antibodies, or bispecific antitumor antagonists can also be administered concurrently with immunogenic agents (e.g., cancer cells, tumor vaccines, purified tumor antigens (including recombinant proteins, peptides, and carbohydrate molecules), cells transfected with genes encoding immunostimulatory cytokines in cancer vaccine strategies (He et al. (2004) J. Immunol. 173:4919-28), or oncolytic viruses.

[0217] Numerous experimental strategies for tumor vaccination have been designed. Among these strategies, vaccines are prepared using autologous or allogeneic tumor cells. Some of these cell vaccines have proven most effective when tumor cells are transduced to express GM-CSF. GM-CSF has been shown to be an effective antigen-presenting activator for tumor vaccination (Dranoff et al. (1993) Proc. Natl. Acad. Sci. USA 90:3539-43). In preclinical models, cancer vaccines have been shown to enhance the infiltration of effector T cells into tumors. The main types of cancer vaccines include peptide vaccines, vector-based antigen-specific vaccines, whole-cell vaccines, and dendritic cell vaccines. All vaccine-based therapies are designed to deliver single or multiple antigenic epitopes or antigens from whole cells to the patient and induce tumor-specific effector T cells. Therefore, vaccine-based therapies may be the most effective method for inducing T cell infiltration into tumors.

[0218] Studies of gene expression and large-scale gene expression patterns in various tumors have led to the definition of so-called tumor-specific antigens (Rosenberg, SA (1999) Immunity 10:281-7). In many cases, these tumor-specific antigens are differentiation antigens expressed in tumors and tumor-originating cells, such as melanocyte antigen gp100, MAGE antigen, and Trp-2. More importantly, many of these antigens can be shown to be targets of tumor-specific T cells found in the host.

[0219] Inhibition of TIGIT, PD-1, PD-L1, and / or LAG-3 can be used in conjunction with a series of recombinant proteins and / or peptides expressed in tumors to generate an immune response against these proteins. These proteins can be recognized as self-antigens by the immune system and thus tolerated. Tumor antigens may include telomerases, proteins essential for telomere synthesis in chromosomes, expressed in over 85% of human cancers and only in a limited number of somatic tissues (Kim et al. (1994) Science 266:2011-2013). Tumor antigens can also be “neoantigens” in cancer cells because somatic mutations can alter protein sequences or form fusion proteins between two unrelated sequences (i.e., bcr-abl in the Philadelphia chromosome), or from idiotypes specific to B-cell tumors.

[0220] Non-limiting examples of tumor vaccines include sipuleucel-T An FDA-approved tumor vaccine for metastatic prostate cancer; tumor cells transfected with the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF), such as whole-cell GM-CSF secretory irradiated allogeneic pancreatic cancer vaccine (GVAX; Johns Hopkins); a peptide vaccine consisting of immunogenic peptides derived from breast cancer antigens, neu, legumain, and β-catenin, which, when used in combination with an anti-PD-1 antibody, prolongs progression-free survival in vaccine-induced tumor-bearing mice (Karyampudi L. et al. (2014) Cancer Res 74: 2974-2985); peptides of melanoma antigens, such as gp100, MAGE antigen, Trp-2, MARTI, and / or peptides of tyrosinase, or other tumor vaccines, including viral proteins associated with human cancer, such as human papillomavirus (HPV) (e.g. and Hepatitis B virus (e.g., Engerix-B and Recombivax HB); Hepatitis C virus (HCV); Kaposi's sarcoma-associated herpessarcoma virus (KSHV). Another form of tumor-specific antigen that can be used in conjunction with TIGIT inhibition is purified heat shock proteins (HSPs) isolated from tumor tissue itself. These heat shock proteins contain protein fragments derived from tumor cells, and these HSPs are highly effective in delivering them to antigen-presenting cells to elicit tumor immunity. Talimogenelaherparepvec (T-VEC or ) is an FDA-approved oncolytic virus used to treat some patients with unresectable metastatic melanoma.

[0221] Dendritic cells (DCs) are effective antigen-presenting cells that can be used to elicit antigen-specific responses. DCs can be generated in vitro and loaded with various protein and peptide antigens, as well as tumor cell extracts (Nestle et al. (1998) Nature Medicine 4:328-332). DCs can also be genetically transduced to express these tumor antigens. For immunization purposes, DCs have also been directly fused to tumor cells (Kugler et al. (2000) Nature Medicine 6:332-336). As a vaccination method, DC immunization can be effectively combined with TIGIT blockade to activate (release) a more effective anti-tumor response.

[0222] TIGIT, PD-1, PD-L1, and / or LAG-3 inhibition can also be used in combination with standard cancer treatments such as surgery, radiotherapy, and chemotherapy. In particular, TIGIT, PD-1, PD-L1, and / or LAG-3 inhibition can be effectively combined with chemotherapy regimens. In these cases, it is possible to reduce the dose of the administered chemotherapy agent (Mokyr et al. (1998) Cancer Research 58:5301-5304). An example of such a combination is an antitumor antagonist in combination with decarbazine for the treatment of melanoma. Another example of such a combination is a checkpoint regulator antagonist or antitumor antagonist in combination with interleukin-2 (IL-2) for the treatment of melanoma. For example, the scientific basis for using TIGIT, PD-1, PD-L1, and / or LAG-3 inhibition in combination with chemotherapy to promote cell death is the cytotoxic effects of most chemotherapy compounds, which should lead to elevated levels of tumor antigens in the antigen presentation pathway. Other combination therapies that synergize with TIGIT, PD-1, PD-L1, and / or LAG-3 inhibition through cell death include radiation, surgery, and hormone deprivation. Each of these regimens generates a source of tumor antigens in the host. Angiogenesis inhibitors can also be combined with TIGIT, PD-1, PD-L1, and / or LAG-3 inhibition. Inhibition of angiogenesis leads to tumor cell death, which may allow tumor antigens to enter the host antigen presentation pathway.

[0223] Anti-TIGIT antibodies, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-LAG-3 antibodies, and the bispecific antitumor antagonists described herein can also be used in combination with bispecific antibodies targeting Fcα or Fcγ receptor-expressing effector cells against tumor cells (see, for example, U.S. Patent Nos. 5,922,845 and 5,837,243). Bispecific antibodies can be used to target two separate antigens. For example, anti-Fc receptor / antitumor antigen (e.g., Her-2 / neu) bispecific antibodies have been used to target macrophages to tumor sites. This targeting can more effectively activate tumor-specific responses. The T-cell arms of these responses will be enhanced by inhibiting TIGIT, PD-1, PD-L1, and / or LAG-3. Alternatively, antigens can be delivered directly to dendritic cells (DCs) using bispecific antibodies that bind to tumor antigens and dendritic cell-specific cell surface markers.

[0224] Tumors evade host immune surveillance through a variety of mechanisms. Many of these mechanisms can be overcome by inactivating immunosuppressive proteins expressed by the tumor. These include TGF-β, IL-10, and Fas ligand. Antibodies against each of these entities can be used in combination with the antitumor antagonists described herein to counteract the effects of immunosuppressants and promote the host's tumor immune response.

[0225] Other antibodies that activate the host immune response can be used in combination with the antitumor antagonists described herein. These include molecules on the surface of dendritic cells that activate DC function and antigen presentation. Anti-CD40 antibodies can effectively replace the helper activity of T cells (Ridge et al. (1998) Nature 393:474-478) and can be used in conjunction with anti-TIGIT antibodies. Activating antibodies against T cell co-stimulatory molecules, such as OX-40 (Weinberg et al. (2000) Immunol 164:2160-2169), CD137 / 4-1BB (Melero et al. (1997) Nature Medicine 3:682-685 (1997), and ICOS (Hutloff et al. (1999) Nature 397:262-266), can also be used to increase T cell activation levels. Additionally, as further described below, inhibitors of other immune checkpoint modulators can also be used in combination with the other antitumor antagonists described herein.

[0226] Bone marrow transplantation is currently used to treat various hematopoietic tumors. Although graft-versus-host disease is a consequence of this treatment, TIGIT inhibition can be used to enhance the effectiveness of donor-transplanted tumor-specific T cells by reducing graft-versus-tumor responses.

[0227] In vitro activation and expansion of antigen-specific T cells, as well as adoptive transfer of these cells to recipients to stimulate antigen-specific T cells against cancer or viral infections in the presence of anti-TIGIT antibodies, can increase the frequency and activity of adoptive T cell transfer.

[0228] Several experimental treatment options exist, including in vitro activation and expansion of antigen-specific T cells, and adoptive transfer of these cells to recipients to stimulate anti-tumor antigen-specific T cells (Greenberg & Riddell (1999) Science 285:546-51). These methods can also be used to activate T cell responses to infectious agents such as CMV. In the presence of anti-TIGIT antibodies, in vitro activation can increase the frequency and activity of adoptive T cell transfers.

[0229] In some implementations, the antitumor antagonists described herein can be administered to subjects suffering from infectious diseases, particularly chronic ones. In this context, similar to their application in cancer, antibody-mediated TIGIT, PD-1, PD-L1, and / or LAG-3 inhibition can be used alone or as adjuvants in combination with vaccines to enhance immune responsiveness to pathogens, toxins, and self-antigens. Exemplary pathogens to which this treatment may be applied include, but are not limited to, HIV, hepatitis (A, B, and C), influenza, herpes, Giardia lamblia, malaria, Leishmaniasis, Staphylococcus aureus, and Pseudomonas aeruginosa. TIGIT, PD-1, PD-L1, and / or LAG-3 inhibition is particularly suitable for combating infections caused by pathogens such as HIV that are known to occur during infection by presenting new or altered antigens. Administration of anti-TIGIT antibodies, anti-PD-1 antibodies, anti-PD-L1 antibodies, or bispecific antitumor antagonists can allow these antigens to be recognized as foreign, thereby evoking an appropriate T-cell response.

[0230] Other pathogenic viruses that can cause infection and can be treated by the methods described herein include HIV, hepatitis (A, B, and C), herpesvirus infections (e.g., VZV, HSV-1, HAV-6, HSV-II, and CMV, Epstein-Barr Virus), and infections caused by adenovirus, influenza virus, flavivirus, echovirus, rhinovirus, Coxsackievirus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, human papillomavirus, mollusc virus, poliovirus, rabies virus, JC virus, arboviral encephalitis virus, or combinations thereof.

[0231] Exemplary pathogens or diseases caused by the methods described herein that can be treated include Chlamydia, Rickettsia, Mycobacteria, Staphylococci, Streptococci, Pneumococci, Meningococci, and Gonococci, Klebsiella, and Proteus. Serratia, Pseudomonas, Legionella, Diphtheria, Salmonella, Bacilli, Cholera, Leptospirosis, Botulinum toxin, Anthrax, Plague, and Lyme disease.

[0232] Exemplary pathogenic fungi causing infection that can be treated by the methods described herein include Candida (e.g., Candida albicans, Krusei, Glabrata, Tropicalis, etc.), Cryptococcus neoformans, Aspergillus (e.g., Fumigatus niger, etc.), Mucorales (e.g., Mucor, Absidia, Rhizopus), Sporothrix schenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis, and Histoplasma capsulatum.

[0233] Exemplary pathogenic parasites causing infection that can be treated by the methods described herein include *Entamoeba histolytica*, *Balantidium coli*, *Naegleria fowleri*, *Acanthamoeba sp.*, *Giardia Zambia*, *Cryptosporidium sp.*, *Pneumocystis carinii*, *Plasmodium vivax*, *Babesia microti*, *Trypanosoma brucei*, *Trypanosoma cruzi*, *Leishmania donovani*, *Toxoplasma gondii*, and *Nippostrongylus brasiliensis*.

[0234] In all of the above methods, TIGIT, PD-1, PD-L1 and / or LAG-3 inhibition can be combined with other forms of immunotherapy (e.g., cytokine therapy (e.g., interferon, GM-CSF, G-CSF, IL-2)) or bispecific antibody therapy using two different binding specificities to enhance the presentation of tumor antigens.

[0235] Anti-TIGIT antibodies, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-LAG-3 antibodies, and the bispecific antitumor antagonists described herein can enhance antigen-specific immune responses by co-administering one or more of these antibodies with an antigen of interest (e.g., a vaccine). Therefore, this document provides a method for enhancing an immune response to an antigen in a subject, the method comprising administering to the subject: (i) an antigen; and (ii) an anti-TIGIT antibody, anti-PD-1 antibody, anti-PD-L1 antibody, anti-LAG-3 antibody, or a bispecific antitumor antagonist, or a combination thereof, to enhance an immune response to the antigen in the subject. The antigen may be, for example, a tumor antigen, a viral antigen, a bacterial antigen, or an antigen derived from a pathogen. Non-limiting examples of such antigens include those discussed in the foregoing sections, such as the tumor antigens (or tumor vaccines) discussed above, or antigens derived from the aforementioned viruses, bacteria, or other pathogens.

[0236] In some implementations, peptides or fusion proteins containing epitopes that bind to anti-TIGIT antibodies, anti-PD-1 antibodies, anti-PD-L1 antibodies, LAG-3 antibodies, or bispecific antitumor antagonists can be used as vaccines or additional vaccines in place of antitumor antagonists.

[0237] Suitable routes of administration, both in vivo and in vitro, of the antibody compositions described herein (e.g., human monoclonal antibodies, multispecific antibodies, or antagonists and immunoconjugates) are well known in the art and can be chosen by a person skilled in the art. For example, the antibody composition may be administered by injection (e.g., intravenous or subcutaneous). The appropriate dose of the molecules used will depend on the age and weight of the subject, as well as the concentration and / or dosage form of the antibody composition.

[0238] Combination therapy

[0239] In another aspect, this application provides a combination therapy for enhancing antigen-specific T-cell responses in subjects. In one embodiment, the method includes contacting T cells with an anti-TIGIT antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-LAG-3 antibody, an antibody fragment thereof, or a bispecific antitumor antagonist, and combining this with a second antibody, antibody fragment, antagonist, or drug, thereby enhancing antigen-specific T-cell responses or apoptosis pathways. For example, in some embodiments, the first antibody or antibody fragment specifically binds to TIGIT, while the second antibody or antibody fragment specifically binds to PD-1, PD-L1, or LAG-3.

[0240] In related aspects, methods for reducing or depleting regulatory T cells in the tumors of subjects in need include administering an effective amount of an antibody or antibody fragment, in combination with a second antibody, antibody fragment, antagonist, or drug, thereby reducing the number of regulatory T cells in the subject.

[0241] In some implementations, the subject suffers from the proliferative disorders or cancers described herein.

[0242] In other implementations, the subject has a chronic viral infection, inflammatory disease, or autoimmune disease as described herein.

[0243] Providing two distinct signals to T cells is a widely accepted model of lymphocyte activation by antigen-presenting cells (APCs) in response to quiescent T lymphocytes. This model further provides a distinction between self and non-self, as well as immune tolerance. The primary signal, or antigen-specific signal, is transduced via the T cell receptor (TCR) and then recognizes foreign antigenic peptides present in the context of the major histocompatibility complex (MHC). The second, or co-stimulatory signal, is delivered to T cells via co-stimulatory molecules expressed on antigen-presenting cells (APCs). This induces T cells to promote clonal expansion, cytokine secretion, and effector function. In the absence of co-stimulation, T cells may become refractory to antigen stimulation, leading to a tolerant response to either exogenous or endogenous antigens.

[0244] In the two-signal model, T cells simultaneously receive positive co-stimulatory signals and negative co-inhibitory signals. The regulation of these positive and negative signals is crucial for maximizing the host's protective immune response while maintaining immune tolerance and preventing autoimmunity. Negative signaling appears to be necessary for inducing T cell tolerance, while positive signaling promotes T cell activation. Both co-stimulatory and co-inhibitory signals are provided to T cells exposed to antigens, and the interaction between them is critical for controlling the magnitude of the immune response. Furthermore, the signals provided to T cells change with the clearance, deterioration, or persistence of infection or immune provocation, and these changes powerfully influence responding T cells and remodel the immune response.

[0245] Co-stimulatory mechanisms have therapeutic implications because manipulation of co-stimulatory signals has been shown to provide means to enhance or terminate cell-based immune responses. Recently, it has been found that T cell dysfunction or unresponsiveness can occur concurrently with the induction and sustained expression of immune checkpoint regulators such as programmed death-1 peptide (PD-1) and its ligands PD-L1 and PD-L2. PD-L1 is overexpressed in many cancers and is generally associated with poor prognosis (Thompson RH et al., Cancer Res 2006, 66(7):3381). Furthermore, unlike T lymphocytes in normal tissues and peripheral blood, most tumor-infiltrating T lymphocytes predominantly express PD-1, suggesting that upregulation of PD-1 on tumor-responsive T cells can lead to impaired antitumor immune responses (Blood 2009 114(8):1537). This may be due to the use of PD-L1 signal transduction resulting from the interaction between PD-L1-expressing tumor cells and PD-L1-expressing T cells, leading to reduced T cell activation and evasion of immune surveillance. Inhibition of PD-L1 / PD-1 interaction provides a means to enhance T-cell immunity, including CD8. + T cell-mediated killing of cancer cells and tumors. Similar enhancements to T cell immunity have been observed by inhibiting the binding of PD-L1 to its chaperone molecule B7-1. Therefore, therapeutic targeting of PD-1 and other immune checkpoint modulators is an area of ​​widespread interest.

[0246] Inhibition of TIGIT, PD-1, PD-L1, and / or LAG-3 signaling pathways, combined with other dysregulated signaling pathways in tumor cells, can provide a means to enhance therapeutic efficacy. In recent years, numerous immune checkpoint modulators in the form of receptors and their ligands have been identified. An important family of membrane-bound ligands that bind to co-stimulatory or co-inhibitory receptors is the B7 family, which includes CTLA-4 and its ligands B7-1 and B7-2; PD-1 and its ligands PD-L1 (B7-H1) and PD-L2 (B7-DC); B7-H2 (ICOS-L), B7-H3, B7-H4, B7-H5 (VISTA), and B7-H6. Other immune checkpoint antagonists include, but are not limited to, TIM-3 and its ligand galactoglobulin-9; LAG-3 and its ligands, including hepatic sinusoidal endothelial cell lectin (LSECtin) and galactoglobulin-3; CD122 and its CD122R ligand; CD70, B7H3, B and T lymphocyte attenuators (BTLA) and VISTA (Le Mercier et al. (2015) Front. Immunol., (6), Article 418). In addition, numerous checkpoint regulator antagonists have been identified and tested in various clinical and preclinical models, and / or have received FDA approval (Kyi et al., FEBS Letters, 588:368-376 (2014)). The concept of inhibitory receptor blockers (also known as immune checkpoint blockers) has been validated by, for example, FDA-approved PD-1 inhibitors nivolumab and pembrolizumab, and the anti-CTLA-4 antibody ipilimumab (for metastatic melanoma).

[0247] Immune checkpoint antagonists modulate or interfere with the activity of immune checkpoint modulators, thereby blocking or inhibiting signaling through checkpoint modulator receptors by binding to the checkpoint modulators or their ligands. By inhibiting this signaling, immunosuppression can be reversed, thereby restoring or enhancing T-cell immunity against cancer cells. Conversely, immune checkpoint agonists (e.g., co-stimulatory molecules) stimulate the activity of immune checkpoint modulators, thereby stimulating signaling through checkpoint modulator receptors by binding to the checkpoint modulators or their ligands. By stimulating this signaling, T-cell immunity against cancer cells can be restored or enhanced.

[0248] Therefore, in one embodiment, a method for stimulating an immune response in a subject includes administering to the subject in combination an anti-TIGIT antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-LAG-3 antibody, an antibody fragment thereof (e.g., anti-TIGITHCVR and / or anti-LCVR), or a bispecific antitumor antagonist described herein with another immune checkpoint modulator described above, thereby stimulating an immune response in the subject, such as inhibiting tumor growth or stimulating an antiviral response.

[0249] In one embodiment, an anti-TIGIT antibody, anti-PD-1 antibody, anti-PD-L1 antibody, anti-LAG-3 antibody, antibody fragment thereof, or a bispecific antitumor antagonist according to the present application is administered in combination with another immune checkpoint modulator as a single antibody or comprising a multispecific antibody having binding specificity to multiple products. Typically, the anti-TIGIT antibody, anti-PD-1 antibody, anti-PD-L1 antibody, anti-LAG-3 antibody, or bispecific antitumor antagonist described herein may be combined with the following substances to stimulate an immune response: (i) antagonists of IgSF family proteins, B7 family proteins, or TNF family proteins that inhibit T cell activation, or cytokine antagonists that inhibit T cell activation (e.g., IL-6, IL-10, TGF-β, VEGF, or other immunosuppressive cytokines) and / or (ii) agonists of IgSF family, B7 family, or TNF family stimulating receptors or cytokine agonists that stimulate T cell activation for stimulating an immune response.

[0250] In one embodiment, a subject is given a combination of an anti-TIGIT antibody or its HCVR and / or LCVR fragment and an anti-PD-1 antibody or PD-1 antagonist. In another embodiment, a subject is given a combination of an anti-TIGIT antibody or its HCVR and / or LCVR fragment and an anti-PD-L1 antibody or PD-L1 antagonist. In yet another embodiment, a subject is given a combination of an anti-TIGIT antibody or its HCVR and / or LCVR fragment and an anti-CTLA-4 antibody or CTLA-4 antagonist.

[0251] In some embodiments, only subjects with cancer exhibiting high expression of ligands against immune checkpoint modulators are selected for treatment with anti-TIGIT, anti-PD-1, anti-PD-L1, and / or anti-LAG-3 antibodies, fragments thereof, or any bispecific antagonist of this application. For example, in one embodiment, subjects with cancer exhibiting high expression of PVR (CD155) and / or adhesion protein-2 (CD112) and / or low expression of PD-L1 may be selected for monotherapy with an anti-TIGIT antibody, fragment thereof, or a TIGIT antagonist of this application, or in combination therapy with a PD-1 antagonist or other immune checkpoint modulators.

[0252] Anti-TIGIT antibodies, anti-PD-1 antibodies, and anti-PD-L1 antibodies can be administered separately from a second antibody, antibody fragment, or antagonist, or multispecific antibodies or antagonists can be administered, including at least one binding specificity against TIGIT and a second binding specificity against other target products. Furthermore, anti-TIGIT, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-LAG-3 antibodies, or bispecific antagonists according to this application can be co-administered with one or more other agents, such as antibodies, antagonists, or drugs, in amounts that can effectively stimulate the subject's immune response and / or apoptosis to further enhance, stimulate, or upregulate the subject's immune response and / or apoptosis.

[0253] In some embodiments, anti-TIGIT, anti-PD-1, anti-PD-L1, or anti-LAG-3 antibodies or fragments thereof are administered after treatment with different antitumor antagonists. For example, in one embodiment, anti-TIGIT, anti-PD-1, anti-PD-L1, or anti-LAG-3 antibodies may be administered only after failure of treatment with PD-1 / PD-L1 antagonists, incomplete treatment response, or tumor recurrence or relapse (or "PD-1 failure"). In some embodiments, cancers exhibiting such failure may be screened for based on the expression of PVR and / or cohesin-2, and only those with high levels of expression may be treated with the anti-TIGIT, anti-PD-1, anti-PD-L1, or anti-LAG-3 antibody, fragment, or antagonist of this application.

[0254] In one embodiment, an anti-TIGIT, anti-PD-1, anti-PD-L1, or anti-LAG-3 antibody or a fragment thereof is administered in combination with a PD-1, PD-L1, PD-L2, TIGIT, or LAG-3 antagonist.

[0255] Other anti-PD-1 antibodies include, but are not limited to, nivolumab (BMS-936558, MDX-1106, OPDIVO). TM Humanized immunoglobulin G4 (IgG4) mAb (Bristol-Myers Squibb); pembrolizumab (MK-3475, lambolizumab, KEYTRUDA™) (Merck); pidilizumab (CT-0111) (Medivation); and AMP-224 (Merck). Anti-PD-1 antibodies are commercially available, for example from ABCAM (AB137132), BIOLEGEND. TM (EH12.2H7, RMP1-14) and AFFYMETRIX EBIOSCIENCE (J105, Jl 16, MIH4).

[0256] Other anti-PD-L1 antibodies include atezolizumab (MPDL3280A, RG7446), a fully human IgG4 mAb (Genentech / Roche); BMS-936559 (MDX-1105), a fully humanized IgG4 mAb (Bristol-Myers Squibb); MEDI4736, a humanized IgG antibody (Medimmune / AstraZeneca); and MSB0010718C, a fully humanized IgG4 monoclonal antibody (Merck, EMD Serono).

[0257] Exemplary anti-CTLA-4 antibodies used according to this method include ipilimumab, trevilizumab, and tremelimumab.

[0258] In some embodiments, the antitumor antagonist is a dominant-negative protein of an immune checkpoint regulator. In some specific embodiments, the dominant-negative protein comprises an extracellular domain derived from the group consisting of members selected from: PD-L1, PD-L2, PD-1, B7-1, B7-2, B7H3, CTLA-4, LAG-3, TIM-3, TIGIT, BTLA, VISTA, CD70, and combinations thereof. In some specific embodiments, these extracellular domains are fused to an immunoglobulin constant region or Fc receptor in the antibody described herein. Such mutants can bind to endogenous receptors, thereby forming a signal transduction-deficient complex. In some embodiments, the extracellular domains are fused to monomers in an immunoglobulin constant region or Fc fragment or oligomeric protein complex.

[0259] In some embodiments, the dominant-negative PD-L1 antagonist comprises an extracellular domain of PD-1. An exemplary dominant-negative protein is AMP-224 (co-developed by Glaxo Smith Kline and Amplimmune), a recombinant fusion protein comprising an extracellular domain of PD-L2 and the Fc region of human IgG. In another embodiment, the dominant-negative PD-L1 antagonist comprises one or more mutations in PD-1 that prevent its ability to bind to PD-L1.

[0260] Exemplary immune checkpoint regulator agonists include, but are not limited to, members of the tumor necrosis factor (TNF) receptor superfamily, such as CD27, CD40, OX40, GITR, and 4-1BB (CD137) and their ligands, or members of the B7-CD28 superfamily, including CD28 and ICOS (CD278). Other checkpoint regulator agonists include CD2, CDS, ICAM-1, LFA-1 (CD11a / CD18), CD30, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, and CD83 ligands. Immune checkpoint agonists may include antibodies or soluble fusion protein agonists comprising one or more co-stimulatory domains. Agonist antibodies include, but are not limited to, anti-CD40 mAbs, such as CP-870, 893, lucatumumab, and dacetuzumab. Anti-CD137 mAbs, such as BMS-663513urelumab and PF-05082566; anti-OX40 monoclonal antibodies; anti-GITR mAbs, such as TRX518; anti-CD27 mAbs, such as CDX-1127; and anti-ICOS mAbs.

[0261] Exemplary GITR agonists include, for example, GITR fusion proteins and anti-GITR antibodies (e.g., bivalent anti-GITR antibodies), such as U.S. Patent Nos. 6,111,090 and 8,586,023; European Patent No. 090505B1; and U.S. Patent Publication Nos. WO2010 / 003118 and 2011 / 090754. Anti-GITR antibodies are described, for example, in U.S. Patent Nos. 7,025,962, 7,618,632, 7,812,135, 8,388,967, and 8,591,886; European Patent Nos. 1947183B1 and 1866339; and PCT Publications WO 2011 / 028683, WO2013 / 039954, WO2005 / 007190, WO 2007 / 133822, WO2005 / 055808, WO 99 / 40196, WO 2001 / 03720, WO99 / 20758, WO2006 / 083289, WO 2005 / 115451, and WO2011 / 051726. An exemplary anti-GITR antibody is TRX518.

[0262] Another family of membrane-bound ligands that bind to co-stimulatory or co-inhibitory receptors is the TNF family of molecules that bind to members of the homologous TNF receptor family, including CD40 and CD40L, OX-40, OX-40L, CD70, CD27L, CD30, CD30L, 4-1BBL, CD137 / 4-1BB, TRAIL / Apo2-L, TRAILR1 / DR4, TRAILR2 / DR5, TRAILR3, TRAILR4, OPG, RANK, RANKL, TWEAKR / Fnl4, TWEAK, BAFFR, EDAR, XEDAR, TACI, APRIL, BCMA, LTβR, LIGHT, DcR3, HVEM, VEGI / TL1A, and TRAMP / D. R3, EDAR, EDA1, XEDAR, EDA2, TNFR1, Lymphotoxin α / TNFγ, TNFR2, TNFα, LTβR, Lymphotoxin α1 (32, FAS, FASL, RELT, DR6, TROY, NGFR (see, for example, Tansey, M. Get et al. (2009) Drug Discovery Today, 14(23-24):1082-1088).

[0263] Immune checkpoint agonists or co-stimulatory molecules include cell surface molecules other than antigen receptors or their ligands required for an effective immune response, including but not limited to MHC class I molecules, MHC class II molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signal transduction lymphocyte activating molecules (SLAM proteins), activated NK cell receptors, BTLA, Toll ligand receptors, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD11a / CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS (C D278), GITR, BAFFR, LIGHT, HVEM(LIGHTR), KIRDS2, SLAMF7, NKp80(KLRF1), NKp44, NKp30, NKp46, CD19, CD4 , CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 1d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB 7. NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM 1. CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, and ligands that specifically bind to CD83.

[0264] In one aspect, the T cell response can be stimulated by combining the anti-TIGIT, anti-PD-1, anti-PD-L1, or anti-LAG-3 mAb of the present invention with one or more of the following: (i) antagonists of proteins that inhibit T cell activation (e.g., immune checkpoint inhibitors), such as CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, TIM-3, galactoglobulin 9 (CEACAM-1), BTLA, CD69, galactoglobulin-1, CD113, GPR 56, VISTA, 2B4, CD48, GARP, PD-1H, LAIR1, TIM-1, CD96 and TIM-4, and (ii) agonists of proteins that stimulate T cell activation, such as B7-1, B7-2, CD28, 4-1BB (CD137), 4-1BBL, ICOS, CD40, ICOS-L, OX40, OX40L, GITR, GITRL, CD70, CD27, CD40, DR3 and CD28H.

[0265] Exemplary agents that regulate one of the above proteins and can be combined with the anti-TIGIT antibody, anti-PD-1 antibody, anti-PD-L1 antibody, and / or anti-LAG-3 antibody of this application to treat cancer include: YERVOY. TM / Pilimumab or tremelimumab (anti-CTLA-4), galiximab (anti-B7.1), OPDIVO TM / nivolumab / BMS-936558 (anti-PD-1), pidilizumab / CT-01111 (anti-PD-1), KEYTRUDA TM Pembrolizumab / MK-3475 (anti-PD-1), AMP224 (anti-B7-DC / PD-L2), BMS-936559 (anti-B7-H1), MPDL3280A (anti-B7-H1), MEDI-570 (anti-ICOS), AMG557 (anti-B7H2), MGA271 (anti-B7H3), IMP321 (anti-LAG-3), urelumab / BMS-6635l3 and PF-05082566 (anti-CD137 / PD-L2), PK135 / PD-L2 ... 4-1BB), CDX-1127 (anti-CD27), MEDI-6383 and MEDI-6469 (anti-OX40), RG-7888 (anti-OX40L), Atacicept (anti-TACI), CP-870893 (anti-CD40), lucatumumab (anti-CD40), dacetuzumab (anti-CD40), and muromonab-CD3 (anti-CD3).

[0266] Other molecules that can be combined with the antitumor antagonists described herein for the treatment of cancer include antagonists of inhibitory receptors on NK cells or agonists of activating receptors on NK cells. For example, antagonists such as anti-TIGIT, anti-PD-1 and / or anti-PD-L1 antibodies can be combined with antagonists of KIRs (e.g., lirilumab) and CSF-1R antagonists such as RG7155.

[0267] Tumors evade host immune surveillance through a variety of mechanisms. Many of these mechanisms can be overcome by inactivating tumor-expressed immunosuppressive proteins. These include TGF-β, IL-10, and Fas ligands. Antibodies against each of these entities can be used in combination with the antitumor antagonists described herein to counteract the effects of immunosuppressants and promote the host's tumor immune response.

[0268] Other antibodies that activate the host immune response can be used in combination with the antitumor antagonists described herein. These include molecules on the surface of dendritic cells that activate DC function and antigen presentation. Anti-CD40 antibodies can effectively replace T cell helper activity and can be used in combination with the antitumor antagonists described herein. Antibodies that activate T cell co-stimulatory molecules (e.g., OX-40, CD137 / 4-1BB, and ICOS) can also enhance T cell activation levels.

[0269] In some embodiments, the antitumor antagonists described herein may be co-administered with one or more other therapeutic agents, such as anticancer agents, radiotoxic agents, or immunosuppressants. Such co-administration can address problems arising from the development of drug resistance, antigenic changes in tumor cells (which will render them unresponsive to antibodies), and toxicity (from the administration of lower doses of one or more agents).

[0270] The antitumor antagonists described herein can be conjugated with a reagent (as an immune complex) or administered separately from the reagent. In the latter case (separate administration), the antibody can be administered before, after, or simultaneously with the reagent, or co-administered with other known therapies such as anticancer therapies such as radiation. The antitumor antagonists described herein can be co-administered with one or more anticancer agents to provide two anticancer agents that act synergistically via different mechanisms, thereby producing cytotoxic effects in human cancer cells.

[0271] The antitumor antagonists described herein can be combined with anticancer agents, such as alkylating agents; anthracycline antibiotics; antimetabolites; detoxifying agents; interferons; polyclonal or monoclonal antibodies; EGFR inhibitors; HER2 inhibitors; histone deacetylase inhibitors; hormones; mitotic inhibitors; phosphatidylinositol-3-kinase (PI3K) inhibitors; Akt inhibitors; mammalian targets of rapamycin (mTOR) inhibitors; proteasome inhibitors; poly(ADP-ribose) polymerase (PARP) inhibitors; Ras / MAPK pathway inhibitors; centrosome declustering agents; multikinase inhibitors; serine / threonine kinase inhibitors; tyrosine kinase inhibitors; VEGF / VEGFR inhibitors; taxanes or taxane derivatives; aromatase inhibitors; anthracyclines; microtubule-targeting drugs; topoisomerase poisoning drugs; inhibitors of molecular targets or enzymes (e.g., kinases or protein methyltransferases); cytidine analogs or combinations thereof.

[0272] Exemplary alkylating agents include, but are not limited to, cyclophosphamide (Cytoxan; Neosar); chlorambucil (Leukeran); melphalan (Alkeran); carmustine (BiCNU); busulfan (Busulfex); lomustine (CeeNU); dacarbazine (DTIC-Dome); oxatin; carmustine (Gliadel); ifosfamide (Ifex); methyl ethylamine (Mustargen); busulfan (Myleran); carboplatin (Paraplatin); cisplatin (CDDP; platinum alcohol); temozolomide (temoda); thiotepa (thioether); bendamustine (Tereanda); or zoxacin (Zanosar).

[0273] Exemplary anthracycline antibiotics include, but are not limited to, doxorubicin (Adriamycin); doxorubicin liposome (Doxil); Novantrone; Blenoxane; Cerubidine; DaunoXome; Cosmegen; Ellence; Idarubicin (Idamycin); Premycin (Thioflavin); Mitamycin; Pentostatin (Nipent); or Valrubicin (Valstar).

[0274] Exemplary antimetabolites include, but are not limited to, fluorouracil (Adrucil); capecitabine (Hydrea); hydroxyurea (Hydrea); mercaptopurine (purinol); pemetrexed (Alimta); fludarabine (Fludara); nerabine (Arranon); and cladribine (Cladribine). Novaplus); Clolar; Cytosar-U; Decitabine; DepoCyt; Hydroxyurea; Pralatrexate (Folotyn); Fluorouracil (FUDR); Gemzactabine; Leustatin; Fludarabine (Oforta); Methotrexate (MTX; Rheumatrex); Methotrexate (Trexall); Tabloid; TS-1 or Tarabine PFS.

[0275] Exemplary antidotes include, but are not limited to, ethyol or mesna (Mesnex).

[0276] Exemplary interferons include, but are not limited to, interferon α-2b (Intron A) or interferon α-2a (Roferon-A).

[0277] Exemplary polyclonal or monoclonal antibodies include, but are not limited to, trastuzumab (Herceptin); ofatumumab (Arzerra); bevacizumab (Avastin); rituximab (Rituxan); cetuximab (Erbitux); panitumumab (Vectibix); tositumomab / iodine-131 tosimomab (Bexxar); alemtuzumab (Campath); ibritumomab (Zevalin; In-111; Y-90Zevalin); gemutumab (Mylotarg); and ordenosumab (Soliris).

[0278] Exemplary EGFR inhibitors include, but are not limited to, gefitinib (Iressa); lapatinib (Tykerb); cetuximab (Erbitux); erlotinib (Tarceva); panitumumab (Vectibix); PKI-166; cannitinib (0-1033); matuzumab (Emd7200); or EKB-569.

[0279] Exemplary HER2 inhibitors include, but are not limited to, trastuzumab (Herceptin); lapatinib (Tykerb); or AC-480.

[0280] Exemplary histone deacetylase inhibitors include, but are not limited to, vorinostat, valproic acid, romedixin, entecavir, abaxinostat, givitosta, and mocetinostat.

[0281] Exemplary hormones include, but are not limited to, tamoxifen (Soltamox; Nolvadex); raloxifene (Evista); progesterone (Megace); leuprorelin (Lupron; Lupron Depot; Eligar; Viadur); fulvestrant (Faslodex); letrozole (Femara); and triptorelin (Trelstar LA; Trelstar). Depot); exemestane (Aromasin); goserelin (Zoladex); bicalutamide (Casodex); anastrozole (Arimidex); flumethasone (Androxy; Halotesin); medroxyprogesterone (Provera; Depo-Provera); estrustetrastine (Emcyt); flutamide (Oral-X); toremifene (Fareston); degarelix (Firmagon); nilandron; abarelix (Plenaxis); or testosterone (Teslac).

[0282] Exemplary mitotic inhibitors include, but are not limited to, paclitaxel (Taxol; Onxol; Abraxane); docetaxel (paclitaxel); vincristine (Oncovin; Vincasar PFS); vinblastine (Velban); etoposide (Toposar; Etopophos; VePesid); teniposide (Vumon); ixabepilone (Ixempra); nocodazole; epothilone; vinorelbine (Navelbine); camptothecin (CPT); irinotecan (Camptosar); topotecan (Hycamtin); amclophene or lamellarin D (LAM-D).

[0283] Exemplary phosphatidylinositol 3-kinase (PI3K) inhibitors include wortmannin (an irreversible PI3K inhibitor), demethoxyviridin (a derivative of wortmannin), LY294002 (a reversible PI3K inhibitor); BKM120 (Buparlisib); Idelalisib (a PI3K Delta inhibitor); duvelisib (IPI-145, a PI3Kδ and γ inhibitor); alpelisib (BYL719) (an α-specific PI3K inhibitor); TGR 1202 (formerly known as RP5264, an oral PI3Kδ inhibitor); and copanlisib (BAY 80-6946, primarily an inhibitor of the RI3Kα,δ subtype).

[0284] Exemplary Akt inhibitors include, but are not limited to, Miltefosine, AZD5363, GDC-0068, MK2206, Perifosine, RX-0201, PBI-05204, GSK2141795, and SR13668.

[0285] Exemplary mTOR inhibitors include, but are not limited to, everolimus (Afinitor) or tesirobolimus (Torisel); rapamune, ridaforolimus; deforolimus (AP23573), AZD8055 (AstraZeneca), OSI-027 (OSI), INK-128, BEZ235, PI-103, Torinl, PP242, PP30, Ku-0063794, WAY-600, WYE-687, WYE-354, and CC-223.

[0286] Exemplary proteasome inhibitors include, but are not limited to, bortezomib (PS-341), ezomib (MLN2238), MLN 9708, dilanzomib (CEP-18770), carfilzomib (PR-171), YU101, opozomib (ONX-0912), marizomib (NPI-0052), and desuferam.

[0287] Exemplary PARP inhibitors include, but are not limited to, olaparib, iniparib, velaparib, BMN-673, BSI-201, AG014699, ABT-888, GPI21016, MK4827, INO-1001, CEP-9722, PJ-34, Tiq-A, Phen, PF-01367338, and combinations thereof.

[0288] Exemplary Ras / MAPK pathway inhibitors include, but are not limited to, trametinib, selumetinib, cobimetinib, 0-1040, PD0325901, AS703026, R04987655, R05068760, AZD6244, GSK1120212, TAK-733, U0126, MEK162, and GDC-0973.

[0289] Exemplary centrosome declusors include, but are not limited to, griseofulvin; noscapine, noscapine derivatives such as brominated noscapine (e.g., 9-bromonoscapine), reduced bromonoscapine (RBN), N-(3-bromobenzyl)noscapine, aminonoscapine and their water-soluble derivatives; CW069; phenanthrene-derived poly(ADP-ribose) polymerase inhibitor PJ-34; N2-(3-pyridylmethyl)-5-nitro-2-furanamide, N2-(2-thienylmethyl)-5-nitro-2-furanamide and N2-benzyl-5-nitro-2-furanamide.

[0290] Exemplary multi-kinase inhibitors include, but are not limited to, regorafenib; sorafenib (Nexavar); sunitinib (Sutent); BIBW 2992; E7080; Zd6474; PKC-412; moticanib or AP24534.

[0291] Exemplary serine / threonine kinase inhibitors include, but are not limited to, ruboxistaurin; eril / esdil hydrochloride; flavonoid piperidine; sebcicbb (CYC202; Roscovitrine); SNS-032 (BMS-387032); Pkc4l2; bromostatin; KAI-9803; SF1126; VX-680; Azdl l52; Arry-142886 (AZD-6244); SCIO-469; GW681323; CC-401; CEP-1347 or PD 332991.

[0292] Exemplary tyrosine kinase inhibitors include, but are not limited to, erlotinib (Tarceva); gefitinib (Iressa); imatinib (Gleevec); sorafenib (Nexavar); sunitinib (Sutent); trastuzumab (Herceptin); bevacizumab (Avastin); rituximab (Rituxan); lapatinib (Tykerb); cetuximab (Erbitux); panitumumab (Vectibix); Everobmus (Afmitor); alemtuzumab (Campath); gemtuzumab (Mylotarg); temsirobmus (Torisel); pazopanib (Votrient); and dasatinib (Spry cel); Nilotinib (Tasigna); Vatalani (Ptk787; ZK222584); CEP-701; SU5614; MLN518; XL999; VX-322; Azd0530; BMS-354825; SKI-606CP-690; AG-490; WHI-P154; WHI-P131; AC-220; or AMG888.

[0293] Exemplary VEGF / VEGFR inhibitors include, but are not limited to, bevacizumab (Avastin); sorafenib (Nexavar); sunitinib (Sutent); ranitumab (pegapanib); or vandetinib (Vandetinib).

[0294] Exemplary microtubule-targeting drugs include, but are not limited to, paclitaxel, docetaxel, vincristine, vinblastine, nocodazole, epothilone, and navirbine.

[0295] Exemplary topoisomerase poisoning drugs include, but are not limited to, teniposide, etoposide, doxorubicin, camptothecin, daunorubicin, actinomycin, mitoxantrone, aminophylline, epirubicin, and idarubicin.

[0296] Exemplary taxanes or taxane derivatives include, but are not limited to, paclitaxel and docetaxel.

[0297] Exemplary general chemotherapeutic agents, antitumor agents, and antiproliferative agents include, but are not limited to, octreotide (Hexalen); isotretinoin (Accutane; Amnesteem; Claravis; Sotret); retinoic acid (Vesanoid); azacitidine (Vidaza); bortezomib (Velcade); asparaginase (Elspar); levamisole (Ergamisol); mitotane (Lysodren); promethazine (Matulane); pegaspargase (Oncaspar); and denileukin. diftitox (Ontak); porfimer (Photofrin); aldesleukin (Proleukin); lenalidomide (Revlimid); bexarotine (Targretin); thalidomide (Thalomid); temsirolimus (Torisel); arsenic trioxide (Trisenox); verteporfm (Visudyne); momexin (Leucenol); (1M tegafur-0.4M 5-chloro-2,4-dihydroxypyrimidine-1M potassium taurate) or lovastatin.

[0298] In some implementations, TIGIT, PD-1, PD-L1, and / or LAG-3 inhibition is combined with standard cancer treatments (e.g., surgery, radiation, and chemotherapy). TIGIT, PD-1, PD-L1, and / or LAG-3 inhibition can be effectively combined with chemotherapy regimens. In these cases, it is possible to reduce the dosage of the chemotherapy agents used. An example of such a combination is the combination of anti-TIGIT, anti-PD-1, anti-PD-L1, or anti-LAG-3 antibodies with decarbazine for the treatment of melanoma. Another example of this combination is the combination of anti-TIGIT, anti-PD-1, anti-PD-L1, or anti-LAG-3 antibodies with interleukin-2 (IL-2) for the treatment of melanoma. It is believed that the combined use of TIGIT, PD-1, PD-L1, and / or LAG-3 inhibition with chemotherapy can enhance apoptosis and increase tumor antigen presentation against cytotoxic immunity. When used in combination with radiation, surgery, or hormone deprivation, other synergistic combination therapies include TIGIT, PD-1, PD-1L1, and / or LAG-3 through the inhibition of cell death. Each of these regimens generates a source of tumor antigens in the host.

[0299] In some embodiments, the checkpoint regulator antagonists described herein can be used as multispecific antagonists or in combination with bispecific antibodies targeting tumor cells that express Fcα or Fcγ receptors (see, for example, U.S. Patent Nos. 5,922,845 and 5,837,243). Bispecific antibodies can be used to target two separate antigens. For example, anti-Fc receptor / anti-tumor antigen (e.g., Her-2 / neu) bispecific antibodies have been used to target macrophages to cancer cells or tumors. This targeting can more effectively activate tumor-specific responses. The T-cell arms of these responses will be enhanced by inhibiting TIGIT, PD-1, PD-L1, and / or LAG-3. Alternatively, antigens can be delivered directly to dendritic cells (DCs) using bispecific antibodies that bind to both tumor antigens and dendritic cell-specific cell surface markers.

[0300] IV. Nucleic acids and host cells used to express checkpoint regulators

[0301] In another aspect, this application provides nucleic acids encoding the antitumor antagonists (including heavy and light chains) of this application, and expression vectors containing such nucleic acids. Specifically, the nucleic acids encode one or more HCDR, LCDR, HCVR, and / or LCVR corresponding to any antibody, antagonist, or fragment described herein.

[0302] Therefore, in one aspect, this application provides one or more nucleic acids that encode any of the antitumor antagonists, antibodies or their antigen-binding portions described herein.

[0303] In another aspect, this application provides one or more expression vectors comprising one or more nucleic acids encoding any of the antitumor antagonists, antibodies, or antigen-binding portions thereof as described herein.

[0304] In another aspect, this application provides host cells transformed using one or more expression vectors, said expression vectors comprising one or more nucleic acids encoding any of the antitumor antagonists, antibodies or their antigen-binding portions described herein.

[0305] DNA encoding antigen-binding sites can be isolated and sequenced from monoclonal antibodies produced by hybridoma cells using conventional methods (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of monoclonal antibodies). Alternatively, the amino acid sequence from the immunoglobulin of interest can be determined by direct protein sequencing, and suitable coding nucleotide sequences can be designed based on a universal codon table. In other cases, the nucleotide and amino acid sequences of the antigen-binding sites or other immunoglobulin sequences, including constant regions, hinge regions, etc., can be obtained from publicly available sources known in the art.

[0306] Expression vectors encoding specific monospecific or bispecific antitumor antagonists can be used to synthesize the antitumor antagonists of this disclosure in vitro in cultured cells, or they can be administered directly to patients to express the antitumor antagonists in vivo or in vitro. As used herein, "expression vector" refers to a viral or non-viral vector containing polynucleotides encoding one or more polypeptide chains corresponding to the monospecific or bispecific antitumor antagonists of this invention in a form suitable for expression from host cells for antibody preparation or direct administration as a therapeutic agent.

[0307] When the former has a functional relationship with the latter, a nucleic acid sequence is "operably linked" to another nucleic acid sequence. For example, if the DNA of a sequence or signal peptide is expressed as a preprotein involved in polypeptide secretion, then the DNA of that sequence or signal peptide can be operably linked to the DNA of the polypeptide. If a promoter or enhancer affects the transcription of a coding sequence, then that promoter or enhancer can be operably linked to that sequence; or a ribosome binding site is operably linked to the coding sequence if said ribosome binding site is positioned to facilitate translation. Generally, "operably linked" means that the linked DNA sequences are contiguous, and in the case of a signal peptide, contiguous and in the reading phase. However, enhancers do not need to be contiguous. Ligation is accomplished by linking to a convenient restriction site. If such a site is not available, synthetic oligonucleotide adaptors or linkers can be used according to conventional practice.

[0308] Nucleic acid sequences used to express antitumor antagonists typically include an N-terminal signal peptide sequence, which is removed from the mature protein. Because the signal peptide sequence can affect expression levels, polynucleotides can encode any of a variety of different N-terminal signal peptide sequences. Those skilled in the art will understand that the design of expression vectors can depend on factors such as the choice of host cells to be transformed and the desired protein expression level.

[0309] The term "regulatory sequence" refers to a DNA sequence necessary for the expression of a coding sequence that is effectively linked in one or more host organisms. The term "regulatory sequence" is intended to include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). Regulatory sequences include those that direct constitutive expression of nucleotide sequences in many types of host cells or those that direct nucleotide sequence expression only in certain host cells (e.g., tissue-specific regulatory sequences). Expression vectors typically contain a sequence for transcription termination and may additionally contain one or more elements that positively influence mRNA stability.

[0310] Expression vectors contain one or more transcriptional regulatory elements, including promoters and / or enhancers for directing the expression of antitumor antagonists. The promoter contains a DNA sequence that functions to initiate transcription from a relatively fixed position relative to the transcription start site. The promoter contains the core elements required for the fundamental interactions between RNA polymerases and transcription factors and can be used in conjunction with other upstream and response elements.

[0311] As used herein, the term "promoter" should be used in its broadest context and includes transcriptional regulatory elements (TREs) derived from or containing chimeric TREs of genomic genes, including TATA boxes or promoter elements for precise transcription initiation, with or without additional TREs (i.e., upstream activating sequences, transcription factor binding sites, enhancers, and silencers), which regulate the activation or repression of genes operatively linked to them in response to developmental and / or external stimuli, as well as transactivation of regulatory proteins or nucleic acids. Promoters may contain genomic segments or may contain chimeras of one or more TREs linked together.

[0312] Preferred promoters are those capable of directing high-level expression in target cells. Promoters can include constitutive promoters (e.g., HCMV, SV40, elongation factor-1α (EF-1α)) or those that exhibit preferential expression in specific cell types of interest. Enhancers are generally DNA sequences that function outside the transcription start site and can be located at the 5' or 3' end of the transcription unit. Furthermore, enhancers can be within introns and within coding sequences. They are typically between 10 and 300 bp in length and function in cis. Enhancers act to increase and / or regulate transcription from nearby promoters. Preferred enhancers are those that direct high-level expression in antibody-producing cells. Cell- or tissue-specific transcriptional regulatory elements (TREs) can be introduced into expression vectors to restrict expression to the desired cell type. PolIII promoters (H1 or U6) are particularly suitable for expressing shRNAs from which they express siRNAs. Expression vectors can be engineered to promote the expression of antitumor antagonists in one or more cell types.

[0313] In some implementations, one or more expression vectors can be modified to express one or more siRNAs that target the Tie2 pathway, VEGF pathway, or immune checkpoint regulators.

[0314] siRNA is a double-stranded RNA that can be engineered to induce sequence-specific post-transcriptional gene silencing of mRNA. Synthesized siRNAs structurally mimic the type of siRNA typically processed in cells using the Dicer enzyme. When expressed from an expression vector, the vector is engineered to transcribe short, double-stranded hairpin-like RNA (shRNA), which is then processed into intracellular targeting siRNAs. Synthesized siRNAs and shRNAs can be designed using well-known algorithms and synthesized using conventional DNA / RNA synthesizers.

[0315] To co-express the individual chains of an antitumor antagonist, suitable splice donor and acceptor sequences can be introduced to express two products. Alternatively, an internal ribosome-binding sequence (IRES) or a 2A peptide sequence can be used to express multiple products from a single promoter. An IRES provides a structure that the ribosome can bind to, which does not need to be at the 5' end of the mRNA. Therefore, it can direct the ribosome to initiate translation at the second start codon within the mRNA, allowing the production of more than one polypeptide from a single mRNA. A 2A peptide contains a short, self-cleaving sequence mediating the co-translation of peptides upstream and downstream of the 2A site, allowing the production of equimolar amounts of two different proteins from a single transcript. CHYSEL is a non-restrictive example of a 2A peptide that causes the translating eukaryotic ribosome to release a synthesizing, growing polypeptide chain without dissociating from the mRNA. The ribosome continues translation, resulting in a second polypeptide.

[0316] Expression vectors can be viral or non-viral. Viral vectors can be derived from adeno-associated virus (AAV), adenovirus, herpesvirus, vaccinia virus, poliovirus, poxvirus, retrovirus (including lentiviruses such as HIV-1 and HIV-2), Sindbis virus and other RNA viruses, alpha viruses, astroviruses, coronaviruses, orthomyxoviruses, papillomaviruses, paramyxoviruses, parvoviruses, dermatoviruses, encapsulated viruses, etc. Non-viral vectors are simply "naked" expression vectors, not packaged with viral-derived components (such as capsids and / or envelopes).

[0317] In some cases, vectors can be engineered to target specific diseases or cell populations by utilizing their inherent targeting properties or by engineering these vectors into viral vectors. Specific cells can be “targeted” for both the delivery and expression of polynucleotides. Thus, in this context, the term “targeting” can be based on the use of capsid, envelope proteins, endogenous or heterologous binding agents in the form of antibodies for delivery to specific cells, the use of tissue-specific regulatory elements to restrict expression to specific cell subgroups, or both.

[0318] In some embodiments, antibody chain expression is controlled by regulatory elements such as tissue-specific or ubiquitous promoters. In some embodiments, ubiquitous promoters, such as the CMV promoter, the CMV-chicken β-actin hybrid (CAG) promoter, tissue-specific or tumor-specific promoters control the expression of a specific antibody heavy chain or light chain, or a single-chain derivative thereof.

[0319] Nonviral expression vectors can be used for nonviral gene transfer, either by direct injection of naked DNA or by encapsulating polynucleotides encoding antitumor antagonists in liposomes, microparticles, microcapsules, virus-like particles, or red blood cells. Such compositions can be further linked to targeting domains via chemical conjugation to facilitate targeted delivery of nucleic acids and / or entry into desired target cells. Additionally, plasmid vectors can be incubated with synthetic gene transfer molecules such as polymerized DNA-binding cations like polylysine, protamine, and albumin, and linked to ligands targeting cells such as asialoside, insulin, galactose, lactose, or transferrin.

[0320] Alternatively, naked DNA can be used. The uptake efficiency of naked DNA can be improved by compaction or by using biodegradable latex beads. This delivery can be further improved by treating the beads to increase hydrophobicity and thereby promote the disruption of endosomes and the release of DNA into the cytoplasm.

[0321] V. Methods for generating monospecific or multispecific antibodies

[0322] In another aspect, this application provides host cells transformed with anti-TIGIT, anti-PD-1, anti-PD-L1 and / or anti-LAG-3HCVR and / or LCVR, encoding nucleic acids or expression vectors, or nucleic acid / expression vectors encoding the monospecific or bispecific antitumor antagonist of this application. The host cells can be any bacterial or eukaryotic cell capable of expressing the encoding nucleic acid or expression vector for anti-TIGIT, anti-PD-1, anti-PD-L1 and / or anti-LAG-3HCVR and / or LCVR, or any other co-administered antibody or antagonist described herein.

[0323] In another aspect, methods for producing antitumor antagonists include culturing host cells transformed with one or more encoding nucleic acids or expression vectors of anti-TIGIT, anti-PD-1, anti-PD-L1 and / or anti-LAG-3HCVR and / or LCVR under conditions that allow for the production of antibodies or fragments, and purifying the antibodies from the cells.

[0324] In another aspect, this application provides a method for generating antibodies, the method comprising culturing cells to transiently or stably express one or more constructs encoding one or more polypeptide chains in an antibody; and purifying the antibody from the cultured cells. Any cell capable of generating a functional antibody can be used. In some preferred embodiments, the cells expressing the antibody are of eukaryotic or mammalian origin, preferably human cells. Cells from various tissue cell types can be used to express antibodies. In some other embodiments, the cells are yeast cells, insect cells, or bacterial cells. Preferably, the antibody-generating cells are stably transformed with a vector expressing the antibody.

[0325] One or more expression vectors encoding the heavy or light chain of an antibody can be introduced into cells using any conventional method, such as naked DNA technology, cationic lipid-mediated transfection, polymer-mediated transfection, peptide-mediated transfection, virus-mediated infection, physical or chemical reagents or treatments, electroporation, etc. Additionally, cells can be transfected with one or more expression vectors to express antibodies along with selective markers that promote secretion by stable transformed clones expressing the antibody. Antibodies produced by such cells can be collected and / or purified according to techniques known in the art, such as centrifugation, chromatography, etc.

[0326] Examples of selection markers suitable for mammalian cells include dihydrofolate reductase (DHFR), thymidine kinase, neomycin, the neomycin analog G418, aquamycin, and puromycin. When such selection markers are successfully transferred to mammalian host cells, the transformed mammalian host cells can survive if subjected to selective pressure. There are two widely used different classes of selection schemes. The first class is based on cell metabolism and the use of mutant cell lines that lack the ability to grow independently of supplemental culture medium. Two examples are CHO DHFR cells and mouse LTK cells. - These cells lack the ability to grow without the addition of nutrients such as thymidine or hypoxanthine. Because these cells lack certain genes essential for the complete nucleotide synthesis pathway, they will not survive unless the missing nucleotides are provided in supplemental medium. Another method of supplemental medium is to alter the growth requirements by introducing the complete DHFR or TK gene into cells lacking the corresponding gene. Individual cells not transformed with the DHFR or TK gene will not survive in unsupplemented medium.

[0327] The second type is dominant selection, which refers to selection protocols used in any cell type and without the need for mutant cell lines. These protocols typically use drugs to inhibit the growth of host cells. Those cells with novel genes will express proteins that transmit resistance and survive the selection process. Examples of this dominant selection use the drugs neomycin, mycophenolic acid, or hygromycin. These three examples employ bacterial genes under eukaryotic control to transmit resistance to the appropriate drugs G418 or neomycin (geneticin), xgpt (mycophenolic acid), or hygromycin, respectively. Others include neomycin analogues G418 and puromycin.

[0328] Exemplary antibody-expressing cells include human Jurkat, human embryonic kidney (HEK) 293, Chinese hamster ovary (CHO) cells, mouse WEHI fibrosarcoma cells, and single-celled protozoan species such as Leishmania tarentolae. Alternatively, primary cells immortalized by c-myc or other immortalizing agents can be used to generate stable, transformed antibody-producing cell lines.

[0329] In one implementation, the cell line comprises a stably transformed Leishmania cell line, such as *Leishmania tarentolae*. Leishmania are known to provide robust, rapidly growing single-cell hosts for high-level expression of eukaryotic proteins exhibiting mammalian-type glycosylation patterns. Leishmania eukaryotic expression kits are commercially available (Jena Bioscience GmbH, Jena, Germany).

[0330] In some implementations, the cell line expresses at least 1 mg, at least 2 mg, at least 5 mg, at least 10 mg, at least 20 mg, at least 50 mg, at least 100 mg, at least 200 mg, at least 300 mg, at least 400 mg, or at least 500 mg of antibody per liter of culture.

[0331] In any suitable culture medium (e.g., RPMI, DMEM, and AIM) After being cultured and maintained in the culture medium, the antibodies described in this application can be isolated from the cells expressing the antibodies. Antibodies can be purified using conventional protein purification methods (e.g., affinity purification, chromatography, etc.), including purification using Protein-A or Protein-G immunoaffinity purification. In some embodiments, the antibody is engineered to be secreted into the culture supernatant for separation.

[0332] VI. Pharmaceutical Compositions and Treatment Methods

[0333] Another aspect of this application relates to pharmaceutical compositions and methods for treating proliferative disorders such as cancer, chronic infections, or immunologically impaired disease states. In one embodiment, the pharmaceutical composition comprises one or more of the antitumor antagonists of this application. In some embodiments, the antitumor antagonist comprises one or more checkpoint regulator antagonists, such as anti-T cell Ig and ITIM domain (TIGIT) inhibitors, PD-1 inhibitors, PD-L1 inhibitors, and LAG-3 inhibitors. The antagonist is formulated with a pharmaceutically acceptable carrier. The pharmaceutical compositions of this application may comprise one or more different antibodies, one or more multispecific antibodies, one or more immunoconjugates, or combinations thereof, as described herein.

[0334] As described above, the method of using the pharmaceutical composition described herein includes administering an effective amount of the pharmaceutical composition according to the invention to a subject in need.

[0335] Antibodies or antagonists can be administered to patients in therapeutic or preventative doses via any suitable route or method of administration. Exemplary routes or methods of administration include parenteral (e.g., intravenous, intra-arterial, intramuscular, subcutaneous, intratumoral), oral, topical (nasal, transdermal, intradermal, or intraocular), mucosal (e.g., nasal, sublingual, buccal, rectal, vaginal), inhalation, intralymphatic, intraspinal, intracranial, intraperitoneal, intratracheal, intrabladder, intrathecal, intraintestinal, intrapulmonary, intralymphatic, intracavitary, intraorbital, intracystic, and intraurethral delivery, as well as local delivery via catheters or stents.

[0336] Pharmaceutical compositions containing antibodies or antagonists according to this disclosure can be formulated in any pharmaceutically acceptable carrier or excipient. As used herein, the term "pharmaceutically acceptable carrier" includes any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delay agents, etc. Pharmaceutical compositions may contain suitable solid-phase or gel-phase carriers or excipients. Exemplary carriers or excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers (e.g., polyethylene glycol). Exemplary pharmaceutically acceptable carriers include one or more of water, saline, phosphate-buffered saline, dextran, glycerol, ethanol, etc., and combinations thereof. In many cases, isotonic agents, such as sugars, polyols like mannitol, sorbitol, or sodium chloride, are preferably included in the composition. Pharmaceutically acceptable carriers may further contain small amounts of excipients, such as wetting agents or emulsifiers, preservatives, or buffers, which may prolong the shelf life or effectiveness of the therapeutic agent.

[0337] Antitumor antagonists can be incorporated into pharmaceutical compositions suitable for parenteral administration. Suitable buffers include, but are not limited to, sodium succinate, sodium citrate, sodium phosphate, or potassium phosphate. Sodium chloride can be used at concentrations of 0-300 mM (optimally 150 mM for liquid formulations) to modify the toxicity of the solution. For lyophilized formulations, cryoprotectants can be included, primarily 0-10% sucrose (preferably 0.5-1.0%). Other suitable cryoprotectants include trehalose and lactose. For lyophilized formulations, fillers can be included, primarily 1-10% mannitol (preferably 2-4%). ​​Stabilizers can be used in both liquid and lyophilized formulations, primarily 1-50 mM (preferably 5-10 mM) L-methionine. Other suitable fillers include glycine and arginine, which can be included as 0-0.05% (preferably 0.005-0.01%) polysorbate-80. Additional surfactants include, but are not limited to, polysorbate 20 and BRIJ surfactants.

[0338] Therapeutic antitumor antagonist formulations can be lyophilized and stored in sterile powder form, preferably under vacuum, and then reconstituted in antibacterial water (e.g., containing benzyl alcohol preservative) or sterile water prior to injection. Pharmaceutical compositions can be formulated for parenteral administration by injection, for example, by large-dose injection or continuous infusion.

[0339] Therapeutic agents in pharmaceutical compositions can be formulated at either a "therapeutic effective amount" or a "prophylactic effective amount." A "therapeutic effective amount" refers to the amount that effectively achieves the desired therapeutic outcome within the necessary dose and time period. The therapeutic effective amount of a recombinant vector can be varied based on factors such as: the disease to be treated, the severity and duration of the disease, the route of administration, whether the antibody or drug is administered for preventative or therapeutic purposes, the bioavailability of the specific drug, the ability of the antitumor antagonist to elicit the desired response in the individual, prior treatment, the patient's age, weight, and sex, the patient's clinical history and response to the antibody, the type of antitumor antagonist used, and the judgment of the attending physician. A therapeutic effective amount is also an amount in which any toxic or harmful effects of the recombinant vector are less important than the beneficial therapeutic effects. A "prophylactic effective amount" refers to the amount that effectively achieves the desired preventative outcome within the necessary dose and time period.

[0340] Preferably, the polypeptide domains in the antitumor antagonist are derived from the same host in which they are to be administered, in order to reduce the inflammatory response to the administered therapeutic agent.

[0341] Antitumor antagonists are appropriately administered to the patient in a single treatment or as part of a series of treatments, and can be administered at any time from the onset of diagnosis. Antitumor antagonists can be administered as a single treatment or in combination with other drugs or therapies available for treating the condition.

[0342] As a general recommendation, the dosage of an antitumor antagonist in a therapeutic or prophylactic effective dose is from about 1 ng / kg body weight / day to about 100 mg / kg body weight / day, whether administered once or multiple times.In one specific implementation, each antitumor antagonist is administered in the following dosage ranges: approximately 1 ng / kg body weight / day to approximately 10 mg / kg body weight / day, approximately 1 ng / kg body weight / day to approximately 1 mg / kg body weight / day, approximately 1 ng / kg body weight / day to approximately 100 μg / kg body weight / day, approximately 1 ng / kg body weight / day to approximately 10 μg / kg body weight / day, approximately 1 ng / kg body weight / day to approximately 1 μg / kg body weight / day, approximately 1 ng / kg body weight / day to approximately 100 ng / kg body weight / day, approximately 10 ng / kg body weight / day to approximately 100 mg ... / kg body weight / day to approximately 10mg / kg body weight / day, approximately 10ng / kg body weight / day to approximately 1mg / kg body weight / day, approximately 10ng / kg body weight / day to approximately 100μg / kg body weight / day, approximately 10ng / kg body weight / day to approximately 10μg / kg body weight / day, approximately 10ng / kg body weight / day to approximately 1μg / kg body weight / day, 10ng / kg body weight / day to approximately 100ng / kg body weight / day, approximately 100ng / kg body weight / day to approximately 100mg / kg body weight / day, approximately 100ng / kg body weight / day to approximately 10mg / kg body weight / day, approximately 100ng / kg body weight / day to approximately 1mg / kg body weight / day, approximately 100n g / kg body weight / day to about 100 μg / kg body weight / day, about 100 ng / kg body weight / day to about 10 μg / kg body weight / day, about 100 ng / kg body weight / day to about 1 μg / kg body weight / day, about 1 μg / kg body weight / day to about 100 mg / kg body weight / day, about 1 μg / kg body weight / day to about 10 mg / kg body weight / day, about 1 μg / kg body weight / day to about 100 μg / kg body weight / day, about 1 μg / kg body weight / day to about 10 μg / kg body weight / day, about 10 μg / kg body weight / day to about 100 mg / kg body weight / day, about 10 μg / kg body weight / day to about 100 mg / kg body weight / day, about 10 μg / kg body weight / day to about 100 mg / kg body weight / day, about 10 μg / kg body weight / day g body weight / day to about 10 mg / kg body weight / day, about 10 μg / kg body weight / day to about 1 mg / kg body weight / day, about 10 μg / kg body weight / day to about 100 μg / kg body weight / day, about 100 μg / kg body weight / day to about 100 mg / kg body weight / day, about 100 μg / kg body weight / day to about 10 mg / kg body weight / day, about 100 μg / kg body weight / day to about 1 mg / kg body weight / day, about 1 mg / kg body weight / day to about 100 mg / kg body weight / day, about 1 mg / kg body weight / day to about 10 mg / kg body weight / day, about 10 mg / kg body weight / day to about 100 mg / kg body weight / day.

[0343] In other implementations, the antitumor antagonist is administered at a dose of 500 μg to 20 g every three days or at a dose of 25 mg / kg body weight every three days.

[0344] In other embodiments, the dosage range of each antitumor antagonist is approximately 10 ng to 100 ng per dose, approximately 10 ng to 1 μg per dose, approximately 10 ng to 10 μg per dose, approximately 10 ng to 100 μg per dose, approximately 10 ng to 1 mg per dose, approximately 10 ng to 10 mg per dose, approximately 10 ng to 100 mg per dose, approximately 10 ng to 1000 mg per injection, approximately 10 ng to 10,000 mg per dose, approximately 100 ng to 1 μg per dose, and approximately 100 ng to 1 μg per dose. From g to about 10 μg, each time administered alone, about 100 ng to about 100 μg, each time administered alone, about 100 ng to about 1 mg, each time administered alone, about 100 ng to about 10 mg, each time administered alone, about 100 ng to about 100 mg, each time administered alone, about 100 ng to about 1000 mg, each time administered alone, about 100 ng to about 10,000 mg, each time administered alone, about 1 μg to about 10 μg, each time administered alone, about 1 μg to about 100 μg, each time administered alone, about 1 μg to about 1 mg, each time administered alone, about 1 μg to about 10 mg, each time administered alone, about 1 μg to about 100 mg, each time administered alone, about 1 μg Up to 1000 mg, administered alone at approximately 1 μg to 10,000 mg, administered alone at approximately 10 μg to 100 μg, administered alone at approximately 10 μg to 1 mg, administered alone at approximately 10 μg to 10 mg, administered alone at approximately 10 μg to 100 mg, administered by injection at approximately 10 μg to 1000 mg, administered alone at approximately 10 μg to 10,000 mg, administered alone at approximately 100 μg to 1 mg, administered alone at approximately 100 μg to 10 mg, administered alone at approximately 100 μg to 100 mg, administered by injection at approximately 100 μg to 1000 mg. Each dose can be administered alone at a dose of approximately 100 μg to approximately 10,000 mg; each dose can be administered alone at a dose of approximately 1 mg to approximately 10 mg; each dose can be administered alone at a dose of approximately 1 mg to approximately 100 mg; each injection can be administered alone at a dose of approximately 1 mg to approximately 10,000 mg; each dose can be administered alone at a dose of approximately 10 mg to approximately 100 mg; each injection can be administered alone at a dose of approximately 10 mg to approximately 10,000 mg; each injection can be administered alone at a dose of approximately 100 mg to approximately 10,000 mg; each dose can be administered alone at a dose of approximately 100 mg to approximately 10,000 mg. Antitumor antagonists can be administered daily, every 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, or 4 weeks.

[0345] In other specific embodiments, the amount of the antitumor antagonist may be administered at the following doses: approximately 0.0006 mg / day, 0.001 mg / day, 0.003 mg / day, 0.006 mg / day, 0.01 mg / day, 0.03 mg / day, 0.06 mg / day, 0.1 mg / day, 0.3 mg / day, 0.6 mg / day, 1 mg / day, 3 mg / day, 6 mg / day, 10 mg / day, 30 mg / day, 60 mg / day, 100 mg / day, 300 mg / day, 600 mg / day, 1000 mg / day, 2000 mg / day, 5000 mg / day, or 10,000 mg / day. As anticipated, the dosage will depend on the patient's disease, size, age, and condition.

[0346] In some implementations, the coding sequence of the antitumor antagonist is introduced into a suitable expression vector (e.g., a viral or non-viral vector) to express an effective amount of the antitumor antagonist in patients with proliferative disorders.

[0347] In some embodiments, including the administration of, for example, one or more recombinant AAV (rAAV) viruses, the pharmaceutical composition may contain rAAV in an amount of at least 10. 10 At least 10 11 At least 10 12 At least 10 13 Or at least 10 14 One genome copy (GC) or recombinant viral particle / kg, or any range thereof. In some embodiments, the pharmaceutical composition comprises an effective amount of recombinant virus, such as rAAV, comprising at least 10 10 At least 10 11 At least 10 12 At least 10 13 At least 10 14 At least 10 15 One genome copy or a recombinant viral particle genome copy, or any range thereof.

[0348] The dose can be tested in several well-established animal models applicable to any specific cell proliferative disease.

[0349] Delivery methods may also include the use of polycationic condensed DNA linked to or not linked to a virus-killing agent, ligand-linked DNA, liposomes, eukaryotic cell delivery vector cells, deposition of photopolymer hydrogel materials, use of handheld gene transfer particle guns, ionizing radiation, nucleic acid neutralization or fusion with cell membranes, particle-mediated gene transfer, etc.

[0350] The invention is further illustrated by the following embodiments, which should not be construed as limiting. All references cited throughout this application, the contents of patents and published patent applications, as well as the accompanying drawings and tables, are incorporated herein by reference.

[0351] example

[0352] Example 1: Production of monoclonal antibodies

[0353] The monoclonal antibodies (mAbs) of this application were generated and screened using techniques well known in the art, see, for example, Harlow and Lane (1988).

[0354] Antibodies, Laboratory Manual, Cold Spring Harbor Publications, New York. Antigen-specific hybridoma mAbs are cloned, sequenced, and engineered using techniques well-known in the art; see, for example, Lo. BKC Methods in Molecular Biology™. Volume 248, 2004. Antibody Engineering.

[0355] Figure 1 The CDR sequence for anti-TIGIT mAb is shown. Figure 2A-2B Several implementation schemes of the variable domain sequence of the anti-TIGIT antibody are shown. Figure 3 The CDR sequence for anti-PD-1mAb is shown. Figure 4A -4C shows several implementation schemes of the variable domain sequence of the anti-PD-1 antibody. Figure 5 The CDR sequence for PD-L1 mAb is shown. Figures 6A-6C Several implementation schemes of the variable domain sequence of the anti-PD-L1 antibody are shown.

[0356] Example 2: Design of a bispecific anti-PD-1 antibody with anti-TIGIT scFv

[0357] Figures 7A-7C Three exemplary bispecific antitumor antagonists were shown, including Bi-TPM-93 ( Figure 7A Bi-TPM-94A Figure 7B ) and Bi-TPM-94B ( Figure 7C These antagonists contain an anti-PD-1 (PD-01) antibody backbone ( Figure 7A ) or anti-PD-1 (PD-06 / 2P17) antibody backbone ( Figure 7A ,7B) and anti-TIGIT scFv, via 3xG4S connector ( Figure 7A 7B) or 6xG4S connector ( Figure 7C It can differentiate between heavy and light chains from anti-TIGIT mAb T-10 / B21.

[0358] Figure 8 shows that... Figures 7A-7C The functional domain sequences present in the bispecific antibody are shown in the figure.

[0359] Figures 9A to 9B It shows the corresponding Figures 7A-7C The exemplary heavy chain (HC) and light chain (LC) sequences of the bispecific antibody are shown in the figure.

[0360] Example 3: Expression and functional characterization of bispecific anti-PD-1 / anti-TIGIT antibodies

[0361] To evaluate the PD-1 blocking ability of Bi-TPM-93 and Bi-TPM-94A, a PD-1 IC50 analysis was performed. Serial dilutions of the bispecific mAbs were incubated with human PD-1-transfected CHOK1 cells and 7 μg / ml FITC-labeled human PD-L1-Fc protein at 4°C for 30 min. Cells were then washed and fixed, and analysis was performed using the iQue intelligent system. The assay... Figure 10 The results showed that Bi-TPM-94 (IC50 = 0.15 nM) containing VH and VL sequences from PD-06 / 2P17 blocked the interaction between PD-1 and its ligand PD-L1 better than Bi-TPM-93 (IC50 = 0.83 nM) containing VH and VL sequences from PD-01.

[0362] Figure 11 Non-reducing PAGE analysis was shown to demonstrate robust transient expression of Bi-TPM-94A and Bi-TPM-94B in human embryonic kidney (HEK) 293 cells.

[0363] To evaluate the homogeneity of the antagonist species corresponding to Bi-TPM93, Bi-TPM-94A, and Bi-TPM-94B, samples were purified and analyzed by size exclusion ultra-high performance liquid chromatography (SE-UHPLC). Sample purification was performed as follows: First, the harvested cell culture medium (HCCF) was filtered through a 0.2 μm filter, followed by affinity purification using Hitrap Protein A HP chromatography (GE Healthcare) at a rate of 1 mL / min. After affinity purification, the samples were... The material was subjected to cation exchange (CEX) chromatography using an SCX-NP5 column with a gradient elution rate of 0.8 mL / min. The amounts of aggregates (high molecular weight, HMW), dimers, and low molecular weight (LMW) fragments were determined by SE-UPLC using a Tosoh TSKgel UP-G3000SWXL column.

[0364] The results of this analysis ( Figure 12Unexpectedly, it was revealed that the level of species heterogeneity in Bi-TPM 93 and Bi-TP M-94A was eliminated by the joint modification in Bi-TPM-94B (i.e., increasing the joint length from 3xG4S to 6xG4S).

[0365] To assess the binding affinity and binding kinetics of His-labeled human PD-1 using Bi-TPM-94A, Bi-TPM-94B, and parental anti-PD-1 benchmark (BM) mAbs, biolayer interferometry was performed using the Octet RED96 system (ForteBio). Briefly, 20 nM of the bispecific antagonist was loaded onto the anti-human IgG capture biosensor. Association of the analyte (His-labeled human PD-1 protein or His-labeled human TIGIT protein) was observed by placing the biosensor in wells containing serially diluted His-labeled PD-1 or His-labeled TIGIT for 5 minutes. Dissociation was measured after transferring the biosensor individually to kinetic buffer and monitoring the interferometer signal for 10 minutes. The observed on-off rates (K0.05) were fitted using a 1:1 binding global fitting model including at least five test concentrations. a and K d Then calculate the equilibrium binding constant K. D .

[0366] Figure 13A The analysis results showed that Bi-TPM-94A and Bi-TPM-94B had a stronger binding affinity for PD-1 than the baseline anti-PD-1 antibody. Similarly, Figure 13B The results showed that Bi-TPM-94A and Bi-TPM-94A had a stronger binding affinity for TIGIT than the baseline anti-TIGIT antibody.

[0367] Blocking assays were performed using CHO cells stably expressing TIGIT and 1 μg / ml biotinylated PVR-muFc to compare the ability of Bi-TPM-94A and Bi-TPM-94B to block the binding of TIGIT to its PVR ligand, TIGIT. Briefly, cells were incubated with biotinylated PVR-Fc and Bi-TPM molecules, washed, and the binding of PVR-muFc was detected using PE streptavidin in the iQue Intellicyt system. Figure 14A The results showed that both molecules similarly blocked the binding of TIGIT and PVR. Similarly, both molecules could block the binding of PD-1 to its PD-L1 ligand. Figure 14B These results further indicate that Bi-TPM-94A and Bi-TPM-94B exhibited slightly better IC50 values ​​than their respective anti-TIGIT and anti-PD-1 baseline (BM) antibodies.

[0368] To determine whether Bi-TPM-94A and Bi-TP M-94B can simultaneously bind PD-1 and TIGIT, 96-well ELISA plates coated with huPD-1-Fc (5 μg / ml) were blocked with 1% BSA in PBS and incubated for 2 hours with serially diluted anti-PD-1 / anti-TIGIT bispecific antibodies. His-tagged huTIGIT protein was then added and incubated for another 2 hours. After washing, HRP-conjugated anti-Elis tag Ab and TMB substrate were added as detection reagents, and quantification was performed using a Perkin Elmer multimode plate reader. Figure 15 The results showed that Bi-TPM-94A and Bi-TPM-94B simultaneously bind PD-1 and TIGIT.

[0369] To evaluate the ability of Bi-TPM-94B to induce IFN-γ production, 250,000 individual PBMCs from donors were screened for CMV antigen reactivity; specifically, donor 287 cells were stimulated with 0.1 μg / ml CMV-infected cell lysate (lanes 2-7). Figure 16A ) and donor 401 ( Figure 16B CMV-reactive T cells were stimulated with CMV-infected cell lysates or without CMV-infected cell lysates (lane 1). Shp-77 cells were co-cultured with PBMCs to provide an immunosuppressive environment and further incubated with human IgG (lane 3), parental anti-TIGIT mAb B21-35 (lane 4), parental anti-PD-1 mAb 2P17 (lane 5), a combination of parental anti-TIGIT mAb B21-35 and parental anti-PD-1 mAb 2P17 (lane 6), or Bi-TPM-94B (lane 7). After 5 days, IFN-γ production in the cell culture supernatant was detected by ELISA.

[0370] Figures 16A-16B The analysis results showed that, compared with monospecific parental mAb or the combination of monospecific parental anti-PD-1 and TIGIT antibodies and negative controls, human PBMCs (donor 287, ...) using Bi-TPM-94B... Figure 16A Donor 401 Figure 16B Increased IFN-γ secretion.

[0371] To assess the ability of Bi-TPM-94B to induce T cell proliferation, cells derived from donor 287 cells were stimulated with 0.1 μg / ml CMV-infected cell lysate. Figure 17A ) and donor 401 ( Figure 17BTwo hundred and fifty thousand human PBMCs were used for two days to stimulate CMV-responsive T cells, and then labeled with carboxyfluorescein succinimide (CFSE). The CFSE-labeled PBMCs were then co-cultured with Shp-77 cells to provide an immunosuppressive environment and further incubated with human IgG (lane 1), parental anti-TIGIT mAb B21-35 (lane 2), parental anti-PD-1 mAb2P17 (lane 3), a combination of parental anti-TIGIT mAb B21-35 and parental anti-PD-1 mAb2P17 (lane 4), or Bi-TPM-94B (lane 5). After five days, CSFE signaling on CD3+ T cells was analyzed using the iQue intelligence system, and the proliferation index was calculated based on the loss of CFSE signaling.

[0372] Figures 17A-17B The analysis results showed that Bi-TPM-94B significantly enhanced the levels of donor 287PBMCs compared to parental anti-PD-1 and anti-TIGIT alone, or in combination thereof, and the negative control. Figure 17A ) and donor 401PBMC ( Figure 17B The proliferation of primary human T cells.

[0373] To evaluate the pharmacokinetic properties of Bi-TPM-94A and Bi-TPM-94B in vivo, pharmacokinetic curves were generated. Briefly, 10 mg / kg of Bi-TPM-94A or Bi-TPM-94B was intravenously injected into the tail vein of 6-10 week old female CD1 mice (n = 2 mice). Serum was harvested at 3 minutes, 3 hours, 1 day, 3 days, 7 days, and 10 days post-injection. To detect antibodies in the serum, 96-well ELISA plates were coated with 5 μg / ml goat anti-human IgG (Fc-specific) F(ab')2 fragment (Sigma, #SAB370l274) and then blocked with 5% milk in PBS. After the blocking step, mouse serum and purified Bi-TPM-94A or Bi-TPM-94B molecules (as standards) were serially diluted in 5% milk, added to the plates, and incubated for 2 hours. After incubation, the wells were washed and then incubated with peroxidase AffmiPure mouse anti-human IgG Fcγ fragment specific antibody (Jackson ImmunoResearch #209-035-098) and TMB-ELISA substrate solution (Thermo Scientific #34029). Quantification was performed using a Perkin Elmer multimode plate reader based on the OD650 signal.

[0374] Figure 18The analysis results showed that the half-lives (T0) of the bispecific antagonists Bi-TPM-94A and Bi-TMP-94B were... 1 / 2 The reaction time is 7-10 days. Therefore, compared with Bi-TPM-93, Bi-TPM-94A and Bi-TPM-94B molecules have higher affinity binding properties, and compared with Bi-TPM-93 and Bi-TPM-94A, Bi-TPM-94B with extended G4S joints has further improved homogeneity in resistance to TIGIT scfv.

[0375] Example 4: Identification and functional characterization of anti-LAG-3 monoclonal antibodies

[0376] In another aspect, this application relates to the screening and characterization of monoclonal antibodies or antigen-binding moieties that specifically bind to the human immune checkpoint regulator LAG-3. Figure 19A The heavy chain CDR1, CD2, and CDR3 sequences corresponding to the isolated anti-LAG-3mAb 2L2A.1, 2L2A.6, 2L27B, and 3L1A are shown. Figure 19B The light chain CDR1, CD2, and CDR3 sequences corresponding to anti-LAG-3mAb2L2A.1, 2L2A.6, 2L27B, and 3L1A are shown. Figure 20 The VH and VL sequences of anti-LAG-3mAb 2L2A.1, 2L2A.6, 2L27B and 3L1A are shown.

[0377] The blocking assays performed showed that monoclonal antibodies 2L2A and 2L27B ( Figure 21A ) and 2L37A and 3L1A ( Figure 21B This blocked the interaction between human LAG-3 and its major ligand, and the major histocompatibility complex II (MHC II) antigen expressed on Raji cells. In short, mAbs 2L2A and 2L27B were prepared. Figure 21A ) and 2L37A and 3L1A ( Figure 21B The serial dilutions were prepared by incubating the serial dilutions and human LAG-3-huFc at room temperature for 30 minutes, then adding them to Raji cells and incubating for another 30 minutes on ice. Raji cells were then washed, and the binding of LAG-3-huFc on Raji cells was detected using anti-human IgG PE. Cells were fixed and then analyzed using the iQue Intellicyt system. Figures 21A-21B The analysis results confirm that mouse anti-LAG-3 mAb has the ability to block the interaction between LAG-3 and its major ligand, namely the major histocompatibility complex II (MHCII) antigen, to a degree comparable to that of anti-LAG-3 baseline (BM) mAb.

[0378] The aforementioned blocking assays were also used to calculate IC50 values, which reflect the inhibitory effect of anti-LAG-3 antibodies, particularly humanized anti-LAG-3 variant 2L2A.1, anti-LAG-3 baseline antibody (BM) (BMS-986016, Bristol-Myers Squibb), and chimeric 2L2A antibodies containing mouse 2L2A CDR in human Ig, on the binding of LAG-3 to MHC II.

[0379] Figure 22 These assays show that the humanized anti-LAG-3mAb2L2A.1 is a better blocker than BM mAb and chimeric 2L2A antibodies containing mouse 2L2A CDRs in human Ig, as reflected by the lower IC50 values ​​obtained.

[0380] To evaluate the affinity and kinetics of anti-LAG-3 mAb 2L2A.1 for His-labeled human LAG-3, biolayer interferometry was performed using the Octet RED96 system (ForteBio), essentially as described in Example 3 (refer to Example 3). Figures 13A-13B As determined by surface plasmon resonance (SPR) and the corresponding binding affinity constants are shown in the figure. Figure 23A The binding affinity of the humanized 2L2A.1 antibody to His-labeled human LAG-3 was demonstrated. Figure 23B Similarly, the binding affinity constant of the humanized 2L2A.1 antibody to human LAG-3 fused into mouse IgG2a (huLAG-3-mIgG2a) was shown.

[0381] To further evaluate the ability of 2L2A.1 to bind to human LAG-3, serial dilutions of either 2L2A.1 or LAG-3 baseline (BM) antibodies were added to CHO-K1 cells expressing human LAG-3 (20,000 cells / well). The mixture was incubated at 4°C for 20 min, washed three times, and stained with the secondary antibody, PE-labeled F(ab')2-goat anti-human IgG Fc (Thermo Scientific #H10104), by incubation at 4°C for 20 min. Cells were washed, resuspended in 7AAD solution, fixed in 10% neutral formalin buffer for 15 min, and then analyzed using the iQue Intellicyt system. The corresponding EC was also determined. 50 The value of the EC 50 The value reflects the maximum effective concentration (EC) relative to human LAG-3 that produces half the response between baseline and maximum response. 50 ).like Figure 24 As shown, this result indicates that 2L2A.1 has a higher binding affinity for human LAG-3 than BM antibody.

[0382] Figure 25 Non-denaturing polyacrylamide gel (PAGE) analysis demonstrates robust expression of the humanized anti-LAG-3 mAb variant 2L2A.1 in transiently expressed human embryonic kidney (HEK) 293 cells, compared to the control antibody (ctrl).

[0383] To assess whether LAG-3 and PD-1 are co-expressed in activated human CD3+ T cells, FACS analysis was performed on donor PBMCs activated with anti-human CD3 / CD28. Figure 26 The analysis results confirmed that human PBMCs activated with staphylococcal enterotoxin B (SEB) co-expressed LAG-3 and PD-1.

[0384] To evaluate the ability of 2L2A.1 to induce IFN-γ production, samples from two donors (i.e., donor 0105) were used. Figure 27A ) and donor 0817 ( Figure 27B 100,000 individual PBMCs were plated in 96-well ELISA plates. PBMCs were either unstimulated (lane 1) or stimulated with 0.5 μg / ml staphylococcal enterotoxin B (SEB; lanes 2-4). Standard (BM) anti-LAG-3 mAb (lane 3), 2L2A.1 mAb (lane 4), or isotype-matched control antibody (lane 2) were added to the stimulated cells, and the plates were incubated at 37°C for 5 days. IFN-γ production in the cell culture supernatant was then examined by ELISA. Figures 27A-27B The results showed that, compared to anti-LAG-3BM, both human donor PBMCs with 2L2A.1 had increased IFN-γ secretion.

[0385] A similar analysis was performed, but with three additional donors, namely donor 223. Figure 28A ), donor 224 ( Figure 28B ) and donor 225 ( Figure 28C PBMCs are used to assess PBMC proliferation. Figures 28A-28B Further confirmation indicated that IFN-γ secretion was increased in human donor PBMCs with 2L2A.1 compared to anti-LAG-3BM. To assess proliferation, three donor PBMCs were labeled with CSFE and stimulated with 100 ng / ml SEB and Benchmark (BM) anti-LAG-3 mAb (lane 3), 2L2A.1 mAb (lane 4), or an isotype-matched control antibody (lane 2). The PBMC mixture was incubated at 37°C for 5 days, and the loss of CFSE signaling on CD4 T cells was quantified by FACS to generate a proliferation index. Figure 29A and 29BThe results showed that 2L2A.1 induced more primary T cell proliferation than the baseline (BM) anti-LAG-3 mAb.

[0386] Example 5: Design and functional characterization of bispecific anti-LAG-3 / anti-TIGIT scFv antagonists

[0387] Based on the design and characterization of the bispecific anti-PD-1 / anti-TIGIT scFv described in Example 1, it is of interest to evaluate whether the manufacturability and functionality of this design can be extended to similar bispecific anti-LAG-3 / anti-TIGIT scFv designs. Figures 30A-30B Two exemplary bispecific antitumor antagonists are shown, namely Bi-LT-1 (with an extended scfv linker 4xG4S). Figure 30A )) and Bi-LT-3 (with extended SCFV connector, 6xG4S ( Figure 30B )). Figure 31 Summary Figures 30A-30B The functional domains in the bispecific antagonist shown in the figure. Figure 32 Displayed corresponding to Figures 30A-30B The heavy chain (HC) and light chain (LC) amino acid sequences of the bispecific antagonist are shown in the figure.

[0388] To evaluate the ability of the bispecific antitumor antagonists Bi-LT-1 and Bi-LT-3 to block the interaction between TIGIT and its ligand human PVR (CD 155), and to further block the interaction between LAG-3 and its major ligand, major histocompatibility complex II (MHC II) antigen, cell-based blocking assays were performed.

[0389] In summary, to demonstrate that Bi-LT-1 and Bi-LT-3 can block the interaction between TIGIT and its ligand human PVR (CD 155), a cell-based blocking assay was performed in which serial dilutions of Bi-LT-1, Bi-LT-3, or Bi-TPM-94B (as described in Example 1 above) were incubated on ice for 30 min with human TIGIT-transfected CHOK1 cells and CD155 / PVR-mouse IgG2a. Binding of CD155 / PVR-mouse IgG2a to CHOK1 cells was detected using anti-mouse IgG PE. Cells were fixed and then analyzed using the iQue Smart System. Results from this analysis showed... Figure 33A The study revealed that Bi-LT-1 and Bi-LT-3 with extended scfv connectors retained their bioactivity toward TIGIT.

[0390] To demonstrate that the bispecific antitumor antagonists Bi-LT-1 and Bi-LT-3 can also block the interaction between LAG-3 and its major ligand, the major histocompatibility complex II (MHC II) antigen, a cell-based blocking assay was performed. Serial dilutions of Bi-LT-1, Bi-LT-3, or anti-LAG-3 baseline antibody (BM) were incubated with LAG-3-mouse IgG2a at room temperature for 30 minutes, added to Raji cells, and further incubated on ice for 30 minutes. Raji cells were then washed, and the binding of LAG-3-mouse IgG2a to Raji cells was detected using anti-mouse IgG PE. Cells were fixed and then analyzed using the iQue intelligent system. The results of this assay are shown in… Figure 33B The results indicate that, similar to the baseline anti-LAG3 antibody, Bi-LT-1 and Bi-LT-1 retain their bioactivity against LAG-3. This assay data was also used to calculate the IC50 value (nm) shown, which is comparable to the IC50 value of the baseline (BM) anti-LAG-3 antibody. In summary, Figure 33A and 33B The results confirmed that Bi-LT-1 and Bi-LT-3 retained the bioactivity of both TIGIT and LAG-3.

[0391] To examine whether Bi-LT-1 and Bi-LT-3 could simultaneously bind to LAG-3 and TIGIT, 5 μg / ml LAG-3-mIgG was coated onto an ELISA plate overnight at 4°C, followed by blocking with serial dilutions of LT-1, LT-3, or parental LAG-3 mAb. After incubation at room temperature for 1 hour, the plate was washed and 500 ng / ml His-labeled HuTIGIT was added, followed by incubation at room temperature for 1 hour. The binding of His-labeled HuTIGIT to the plate was then detected using anti-His-tag HRP and TMB substrates. Figure 34 The results of this study demonstrate that Bi-LT-1 and Bi-LT-3 can simultaneously bind to LAG-3 and TIGIT.

[0392] Figures 35A-35D The corresponding parental anti-LAG-3mAb was shown. Figure 35A ), anti-LAG-3 benchmark mAb( Figure 35B ), Bi-LT-1 ( Figure 35C ) or Bi-LT-3( Figure 35D Pharmacokinetic profiles and in vivo half-life (T1) following tail vein injection in 6-10 week old female CD1 mice 1 / 2Antibodies and bispecific antagonists were recovered from serum collected at different time points after injection and analyzed by ELISA. The results showed that Bi-LT-1 and Bi-LT-3 had similar pharmacokinetics compared to the anti-LAG3 parental antibody and the baseline antibody, with similar half-lives (T0, T0, T0). 1 / 2 It takes 5-6 days.

[0393] To evaluate the species homogeneity and stability of purified Bi-LT-1 and Bi-LT-3 of protein A, size exclusion chromatography (SEC) curves of Bi-LT-1 and Bi-LT-3 were generated as described above. Figure 36A The results of this analysis are consistent with the material formed by the extended SCFV connector, and show good stability after 7 days at 4°C. Figure 36B The size exclusion ultra-high performance liquid chromatography (SE-UHPLC) analysis performed as described above is illustrated. The results of this analysis are consistent with... Figure 36A The observed species homogeneity was consistent, as reflected in the following results: On day 0 and day 7, Bi-LT-1 was 1.3% and 1.5% for low-level high molecular weight (HMW) species, respectively; and Bi-LT-3 was 1.3% and 1.4% for HMW species, respectively. On day 0 and day 7, Bi-LT-1 was 0.2% and 0.2% for low molecular weight (LMW) species, respectively; and Bi-LT-3 was 0.3% and 0.5% for LMW species, respectively. In contrast, for dimers, Bi-LT-1 was 98.4% and 98.3% on day 0 and day 7, respectively; and Bi-LT-3 was 98.4% and 98.1% for LMW species, respectively.

[0394] To evaluate the ability of Bi-LT-1 and Bi-LT-3 to induce IFN-γ production, experiments were conducted in SHP-77 cells ( Figure 37A ) or H358 cells ( Figure 37B In the presence of SEB, human PBMCs were stimulated with 100 ng / ml SEB to provide an immunosuppressive signal. Then, human IgG control (lane 3), anti-TIGIT mAb alone (B21-35) (lane 4), anti-LAG-3 mAb alone (lane 5), a combination of anti-TIGIT mAb and anti-LAG-3 mAb (lane 6), Bi-LT-1 (lane 7), or Bi-LT-3 (lane 8) were added to rescue T cell function. Figure 37A (37B). Four days later, the cell culture supernatant was collected, and the IFN-γ level was examined by ELISA. Figure 37A and 37B These analyses show that LT-1 and LT-3 are more effective than the combination of parental TIGIT and LAG-3 antibodies.

[0395] Figure 38The ability of Bi-LT-1 and Bi-LT-3 to induce CD4 T cell proliferation was assessed. Human PMBCs were labeled with CSFE and then stimulated with 100 ng / ml SEB in the presence of SHP-77 cells to provide an immunosuppressive signal. T cell function was rescued using either a 128 nM human IgG control (lane 3), anti-TIGIT mAb B21-35 (lane 4), anti-LAG-3 mAb (lane 5), a combination of anti-TIGIT mAb and anti-LAG-3 mAb (lane 6), Bi-LT-1 (lane 7), or Bi-LT-3 (lane 8). After 5 days, the loss of CFSE signaling on CD4 T cells was quantified by FACS to determine the proliferation index. Similar to the increased IFNγ stimulation by the parental antibody and the combination of two parental antibodies, both Bi-LT-1 and Bi-LT-3 demonstrated the ability to enhance the stimulation of human CD4 T cell proliferation.

[0396] The above description is intended to teach those skilled in the art how to practice the invention, and not to detail all those modifications and variations that will become obvious to those skilled in the art upon reading this description. However, all such obvious modifications and variations should be included within the scope of the invention, which is defined by the appended claims. Unless the context clearly indicates otherwise, the claims are intended to cover the claimed components and steps in any order that effectively achieves the desired objective. sequence list <110> Jingshang Biopharmaceutical Company <120> Antitumor immune checkpoint modulators antagonists <130> 2022-003 PCT3 (130781) <150> US 62 / 691,658 <151> 2018-06-29 <150> US 62 / 823,989 <151> 2019-03-26 <160> 337 <170> PatentIn version 3.5 <210> 1 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 1 Ser Asp Tyr Ala Trp Asn 1 5 <210> 2 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 2 Tyr Ile Ser Tyr Ser Gly Ser Thr Gly Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 <210> 3 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 3 Arg Met Ile Gly Tyr Ala Met Asp Tyr 1 5 <210> 4 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 4 Tyr Ile Thr Tyr Ser Gly Gly Ser Thr Ser Tyr Asn Pro Ser Leu Lys 1 5 10 15 Ser <210> 5 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 5 Arg Gln Ile Gly Leu Gly Phe Thr Tyr 1 5 <210> 6 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 6 Asp His Thr Ile His 1 5 <210> 7 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 7 Tyr Phe Tyr Pro Arg Asp Gly Ser Thr Lys Tyr Asn Glu Lys Phe Lys 1 5 10 15 Gly <210> 8 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 8 Gly Met Leu Arg Trp Phe Ala Asp 1 5 <210> 9 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 9 Tyr Ile Tyr Pro Arg Asp Gly Ser Ser Lys Tyr Asn Val Lys Phe Lys 1 5 10 15 Gly <210> 10 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 10 Gly Met Leu Arg Trp Phe Ala Tyr 1 5 <210> 11 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 11 Asp Gln Ala Ile His 1 5 <210> 12 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 12 Tyr Ile Tyr Pro Arg Asp Gly Ser Thr Lys Tyr Asn Glu Thr Phe Lys 1 5 10 15 Gly <210> 13 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 13 Tyr Ile Thr Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 <210> 14 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 14 Arg Gln Val Gly Leu Gly Phe Ala Tyr 1 5 <210> 15 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 15 Ser Asp Ser Ala Trp Asn 1 5 <210> 16 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 16 Tyr Ile Thr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Arg Ser 1 5 10 15 <210> 17 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 17 Asn Tyr Gly Met Asn 1 5 <210> 18 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 18 Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe Lys 1 5 10 15 Gly <210> 19 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 19 Ala Pro Pro Tyr Gly Tyr Asp Val Arg Phe Ala Tyr 1 5 10 <210> 20 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 20 Thr Phe Ala Met Gly Val Gly 1 5 <210> twenty one <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> twenty one His Ile Trp Trp Asp Asp Asp Lys Tyr Tyr Asn Pro Ala Leu Lys Ser 1 5 10 15 <210> twenty two <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> twenty two Met Asp Tyr Ser Tyr Phe Ala Trp Phe Ala Tyr 1 5 10 <210> twenty three <211> 5 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> twenty three Ser Tyr Tyr Met His 1 5 <210> twenty four <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> twenty four Ile Asn Pro Ser Gly Gly Arg Thr Ser Tyr Ala Gln Met Phe Gln Gly 1 5 10 15 <210> 25 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 25 Asp Arg Glu Glu Gln Trp Pro Val Gly Gly Phe Asp Tyr 1 5 10 <210> 26 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 26 Lys Ala Ser Gln Asp Val Ser Thr Val Val Ala 1 5 10 <210> 27 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 27 Ser Ala Ser Tyr Arg Tyr Thr 1 5 <210> 28 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 28 Gln Gln His Tyr Ser Thr Pro Trp Thr 1 5 <210> 29 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 29 Lys Ala Ser Gln Asp Leu Ser Thr Ala Val Ala 1 5 10 <210> 30 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 30 Ser Ser Ser Tyr Arg Tyr Thr 1 5 <210> 31 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 31 Lys Ala Ser Gln Asp Val Ser Thr Thr Val Ala 1 5 10 <210> 32 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 32 Gln Gln His Tyr Ser Thr Pro Leu Thr 1 5 <210> 33 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 33 Lys Ala Ser Gln Asp Val Phe Thr Ala Val Ala 1 5 10 <210> 34 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 34 Gln Gln His Tyr Ser Ile Pro Leu Thr 1 5 <210> 35 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 35 Lys Ala Ser Gln Asp Val Ser Thr Ala Val Ala 1 5 10 <210> 36 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 36 Ser Ala Ser Tyr His Tyr Thr 1 5 <210> 37 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 37 Ser Ala Ser Tyr Arg Phe Thr 1 5 <210> 38 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 38 Gln His His Tyr Ser Thr Pro Trp Thr 1 5 <210> 39 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 39 Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr Leu Glu 1 5 10 15 <210> 40 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 40 Lys Val Ser Asp Arg Phe Ser 1 5 <210> 41 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 41 Phe Gln Gly Ser His Val Pro Trp Thr 1 5 <210> 42 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 42 Arg Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn 1 5 10 <210> 43 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 43 Gly Thr Asn Asn Arg Ala Pro 1 5 <210> 44 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 44 Ala Leu Trp Tyr Ser Asn His Trp Val 1 5 <210> 45 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 45 Arg Ala Ser Gln Ser Ile Arg Arg Tyr Leu Asn 1 5 10 <210> 46 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 46 Ser Ala Ser Asn Leu Gln Ser 1 5 <210> 47 <211> 9 <212> PRT <213> artificial sequence <220> <223> synthetic <400> 47 Gln Gln Ser Tyr Ile Ile Pro Pro Thr 1 5 <210> 48 <211> 118 <212> PRT <213> artificial sequence <220> <223> synthetic <400> 48 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 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 Ser Tyr Ser Gly Ser Thr Gly 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 Arg Met Ile Gly Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Ser Val Thr Val Ser Ser 115 <210> 49 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 49 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 Asp Val Ser Thr Val 20 25 30 Val Ala Trp His Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Tyr 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 Ser Tyr Ser Thr Pro Gln[[ID=?]] 85 90 95 Gln His Tyr Ser Thr Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu 100 105 11 Ile Lys Arg It seems there is a mistake in the original text where "100 105 11" should probably be "100 105 110" in line . The translation above has been done based on the given text with this noted potential error. 115 <210> 50 <211> 118 <212> PRT <213> artificial sequence <220> <223> synthetic <400> 50 Gln Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr 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 Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Thr Tyr Ser Gly Gly 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 Ser Cys 85 90 95 Ala Arg Arg Gln Ile Gly Leu Gly Phe Thr Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ala 115 <210> 51 <211> 114 <212> PRT <213> artificial sequence <220> <223> synthetic <400> 51 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 Pro Cys Lys Ala Ser Gln Asp Leu Ser Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ser Ser Tyr Arg Tyr 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 Ser Tyr Ser Thr Pro Gln 85 90 95 Gln His Tyr Ser Thr Pro Trp Thr Phe Gly Glu Gly Thr Lys Leu Glu 100 105 110 Island Light <210> 52 <211> 118 <212> PRT <213> artificial sequence <220> <223> synthetic <400> 52 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr Asp His 20 25 30 Thr Ile His Trp Val Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Tyr Phe Tyr Pro Arg Asp Gly Ser Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asp Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Gly Met Leu Arg Trp Phe Ala Asp Trp Gly Gln Gly Thr Leu 100 105 110 Ile Thr Val Ser Val Ala 115 <210> 53 <211> 107 <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 Asp Val Ser Thr Thr 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Tyr 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 Ser Thr Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 <210> 54 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 54 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr Asp His 20 25 30 Thr Ile His Trp Val Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Tyr Ile Tyr Pro Arg Asp Gly Ser Ser Lys Tyr Asn Val Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asp Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Gly Met Leu Arg Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 55 <211> 107 <212> PRT <213> artificial sequence <220> <223> synthetic <400> 55 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 Asp Val Phe Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln His Tyr Ser Ile Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 56 <211> 117 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 56 Glu Val Gln Leu Lys Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr Asp Gln 20 25 30 Ala Ile His Trp Val Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Tyr Ile Tyr Pro Arg Asp Gly Ser Thr Lys Tyr Asn Glu Thr Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asp Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Arg Gly Met Leu Arg Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 57 <211> 107 <212> PRT <213> artificial sequence <220> <223> synthetic <400> 57 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 Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Tyr 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 Ser Thr Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 <210> 58 <211> 118 <212> PRT <213> artificial sequence <220> <223> synthetic <400> 58 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Val Ser 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 Thr 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 Arg Gln Val Gly Leu Gly Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ala 115 <210> 59 <211> 107 <212> PRT <213> artificial sequence <220> <223> synthetic <400> 59 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 Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr His Tyr 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 Ser Thr Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 60 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 60 Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Asp 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Thr Ser Asp 20 25 30 Ser Ala Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Thr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Arg Ser Arg Val Thr Met Ser Val Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Val Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Arg Gln Val Gly Leu Gly Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ala 115 <210> 61 <211> 107 <212> PRT <213> artificial sequence <220> <223> synthetic <400> 61 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 Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Phe Thr Gly Ala 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 Gly Ile Tyr Tyr Cys Gln His His Tyr Ser Thr Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Phe Lys 100 105 <210> 62 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 62 Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe 50 55 60 Lys Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr 65 70 75 80 Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ala Pro Pro Tyr Gly Tyr Asp Val Arg Phe Ala Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 63 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 63 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Phe Gln Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg Val Leu Ile Tyr Lys Val Ser Asp Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Trp Thr Phe Gly Arg Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 64 <211> 121 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 64 Gln Val Thr Leu Lys Glu Ser Gly Pro Thr Leu Val Lys Pro Thr Gln 1 5 10 15 Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Phe 20 25 30 Ala Met Gly Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu 35 40 45 Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Tyr Tyr Asn Pro Ala 50 55 60 Leu Lys Ser Arg Leu Thr Ile Thr Lys Asp Thr Ser Lys Asn Gln Val 65 70 75 80 Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr 85 90 95 Cys Ala Arg Met Asp Tyr Ser Tyr Phe Ala Trp Phe Ala Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 65 <211> 109 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 65 Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30 Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Gln Leu Phe Arg Gly 35 40 45 Leu Ile Gly Gly Thr Asn Asn Arg Ala Pro Trp Val Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Ile Gly Asp Lys Ala Ala Leu Thr Leu Ser Gly Val 65 70 75 80 Gln Pro Glu Asp Glu Ala Glu Tyr Phe Cys Ala Leu Trp Tyr Ser Asn 85 90 95 His Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 66 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 66 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala [[ID=5​Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Ile Ile Asn Pro Ser Gly Gly Arg Thr Ser Tyr Ala Gln Met Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Arg Glu Glu Gln Trp Pro Val Gly Gly Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 67 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 67 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 Gln Ser Ile Arg Arg Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Asn Leu Gln 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 Tyr Ile Ile Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Arg 100 105 <210> 68 <211> 5 <212> PRT <213> artificial sequence <220> <223> synthetic <400> 68 Asn Phe Leu Met Ser 1 5 <210> 69 <211> 17 <212> PRT <213> artificial sequence <220> <223> synthetic <400> 69 Thr Ile Ser Gly Gly Gly Arg Asp Thr Tyr Tyr Val Asp Ser Val Lys 1 5 10 15 Gly <210> 70 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 70 Arg Thr Thr Tyr Ser Met Asp Tyr 1 5 <210> 71 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 71 Asn Ser Tyr Leu Tyr 1 5 <210> 72 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 72 Gly Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe Asn Glu Lys Phe Lys 1 5 10 15 Thr <210> 73 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 73 Arg Asp Tyr Asn Tyr Asp Gly Gly Phe Asp Ser 1 5 10 <210> 74 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 74 Asn Ser Tyr Ile Tyr 1 5 <210> 75 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 75 Arg Arg Asp Tyr Arg Tyr Asp Gly Gly Phe Asp Ser 1 5 10 <210> 76 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 76 Thr Tyr Tyr Ile Tyr 1 5 <210> 77 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 77 Gly Ile Asn Pro Gly Asn Gly Gly Thr Asn Phe Asn Glu Lys Phe Lys 1 5 10 15 Ile <210> 78 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 78 Arg Tyr His Gly Tyr Asp Gly Gly Leu Asp Tyr 1 5 10 <210> 79 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 79 Ser Tyr Tyr Ile His 1 5 <210> 80 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 80 Trp Ile Phe Pro Gly Ser Gly Asn Ser Lys Tyr Asn Glu Asn Phe Lys 1 5 10 15 Gly <210> 81 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 81 Ser Glu Thr Tyr Asp Tyr Gly Asp Tyr 1 5 <210> 82 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 82 Leu Ala Ser Gln Thr Ile Gly Thr Trp Leu Ala 1 5 10 <210> 83 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 83 Ala Ala Thr Ser Leu Ala Asp 1 5 <210> 84 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 84 Gln Gln Phe Tyr Ser Ile Pro Trp Thr 1 5 <210> 85 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 85 Arg Ala Ser Ser Thr Leu Tyr Ser Asn Tyr Leu His 1 5 10 <210> 86 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 86 Arg Ala Ser Phe Leu Ala Ser 1 5 <210> 87 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 87 Gln Gln Gly Ser Ser Ile Pro Leu Thr 1 5 <210> 88 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 88 Ser Ala Ser Ser Ser Leu Tyr Ser Ser Tyr Leu His 1 5 10 <210> 89 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 89 Arg Ala Ser Ser Ser Leu Tyr Ser Asn Tyr Leu His 1 5 10 <210> 90 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 90 Arg Ala Ser Lys Ser Val Ser Thr Ser Gly Phe Ser Tyr Ile His 1 5 10 15 <210> 91 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 91 Leu Ala Ser Asn Leu Glu Ser 1 5 <210> 92 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 92 Gln His Thr Trp Glu Leu Pro Asn Thr 1 5 <210> 93 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 93 Lys Ala Ser Gln Asn Val Gly Thr Asn Val Ala 1 5 10 <210> 94 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 94 Ser Ala Ser Tyr Arg Tyr Ser 1 5 <210> 95 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 95 Gln Gln Tyr Tyr Ser Tyr Pro Tyr Thr 1 5 <210> 96 <211> 117 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 96 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Phe 20 25 30 Leu Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Thr Ile Ser Gly Gly Gly Arg Asp Thr Tyr Tyr Val Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 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 Ala Lys Arg Thr Thr Tyr Ser Met Asp Tyr Trp Gly Gln Gly Thr Ser 100 105 110 Val Thr Val Ser Ser 115 <210> 97 <211> 107 <212> PRT <213> artificial sequence <220> <223> synthetic <400> 97 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Leu Ala Ser Gln Thr Ile Gly Thr Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Thr Ser Leu Ala Asp 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 Phe Tyr Ser Ile Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 98 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 98 Gln 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 Asp Tyr Thr Phe Thr Asn Ser 20 25 30 Tyr Leu Tyr Trp Leu Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe Asn Glu Lys Phe​​​​​​ Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Val Val Tyr Tyr Cys 85 90 95 Thr Arg Arg Asp Tyr Asn Tyr Asp Gly Gly Phe Asp Ser Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 99 <211> 108 <212> PRT <213> artificial sequence <220> <223> synthetic <400> 99 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Phe Thr Cys Arg Ala Ser Ser Thr Leu Tyr Ser Asn 20 25 30 Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Arg Ala Ser Phe Leu Ala Ser Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Ser Ser Ile Pro 85 90 95 Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 100 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 100 Gln 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 Asp Tyr Thr Phe Thr Asn Ser 20 25 30 Tyr Ile Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe Asn Glu Lys Phe 50 55 60 Lys Thr Arg Val Thr Ser 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 Val Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Asp Tyr Arg Tyr Asp Gly Gly Phe Asp Ser Trp Gly Gln 100 105 110 Gly Thr Thr Leu Thr Val Ser Ser 115 120 <210> 101 <211> 108 <212> PRT <213> artificial sequence <220> <223> synthetic <400> 101 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 Ser Ala Ser Ser Ser Leu Tyr Ser Ser 20 25 30 Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Arg Ala Ser Phe Leu Ala Ser Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Ser Ser Ile Pro 85 90 95 Leu Thr Phe Gly Ala Gly Thr Lys Leu Asp Leu Lys 100 105 <210> 102 <211> 120 <212> PRT <213> artificial sequence <220> <223> synthetic <400> 102 Gln 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 Asp Tyr Thr Phe Thr Asn Ser 20 25 30 Tyr Ile Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe Asn Glu Lys Phe 50 55 60 Lys Thr Arg Val Thr Ser 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 Val Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Asp Tyr Asn Tyr Asp Gly Gly Phe Asp Ser Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 103 <211> 108 <212> PRT <213> artificial sequence <220> <223> synthetic <400> 103 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Phe Thr Cys Arg Ala Ser Ser Ser Leu Tyr Ser Asn 20 25 30 Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Arg Ala Ser Phe Leu Ala Ser Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Ser Ser Ile Pro 85 90 95 Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 104 <211> 120 <212> PRT <213> artificial sequence <220> <223> synthetic <400> 104 Gln 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 Thr Tyr 20 25 30 Tyr Ile Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Asn Pro Gly Asn Gly Gly Thr Asn Phe Asn Glu Lys Phe 50 55 60 Lys Ile Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Tyr His Gly Tyr Asp Gly Gly Leu Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 105 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 105 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Pro Gly 1 5 10 15 Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Lys Ser Val Ser Thr Ser 20 25 30 Gly Phe Ser Tyr Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn 65 70 75 80 Pro Val Glu Ala Asn Asp Thr Ala Asn Tyr Tyr Cys Gln His Thr Trp 85 90 95 Glu Leu Pro Asn Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 106 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 106 Gln 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 Ser Tyr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Phe Pro Gly Ser Gly Asn Ser Lys Tyr Asn Glu Asn Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Ala Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Glu Thr Tyr Asp Tyr Gly Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 107 <211> 107 <212> PRT <213> artificial sequence <220> <223> synthetic <400> 107 Asp Ile Gln Met Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Asn 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Ala Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ser Tyr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 108 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 108 Asn Tyr Trp Met His 1 5 <210> 109 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 109 Met Ile His Pro Asn Thr Asn Asn Tyr Asn Tyr Asn Glu Lys Phe Lys 1 5 10 15 Ser <210> 110 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 110 Ser Asp Tyr Gly Ser Ser Pro Tyr Tyr Phe Asp Tyr 1 5 10 <210> 111 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 111 Ser Tyr Trp Met His 1 5 <210> 112 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 112 Met Ile His Pro Asn Val Gly Ser Thr Asn Tyr Asn Glu Lys Phe Lys 1 5 10 15 Ser <210> 113 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 113 Ser Arg Tyr Gly Ser Ser Pro Tyr Tyr Phe Asp Tyr 1 5 10 <210> 114 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 114 Met Ile His Pro Asn Ser Gly Gly Asn Asn Tyr Asn Glu Lys Phe Lys 1 5 10 15 Ser <210> 115 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 115 Ser Trp Tyr Gly Ser Ser Pro Tyr Tyr Phe Asp Tyr 1 5 10 <210> 116 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 116 Met Ile His Pro Thr Gly Val Ser Thr Asp Tyr Asn Glu Lys Phe Lys 1 5 10 15 Ser <210> 117 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 117 Ser Asp Tyr Ala Trp Asn 1 5 <210> 118 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 118 Tyr Ile Ser Asp Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 <210> 119 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 119 Ser Phe Leu Arg Leu Arg Ser Tyr Phe Asp His 1 5 10 <210> 120 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 120 Ser Tyr Gly Ile Asn 1 5 <210> 121 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 121 Cys Ile Tyr Ile Gly Asn Asp Tyr Thr Asn Tyr Asn Glu Lys Phe Lys 1 5 10 15 Gly <210> 122 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 122 Ala Tyr Tyr Gly Ser Arg Val Asp Tyr 1 5 <210> 123 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 123 Arg Ala Ser Gln Asp Ile Asp Asn Tyr Leu Asn 1 5 10 <210> 124 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 124 Tyr Thr Ser Arg Leu His Ser 1 5 <210> 125 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 125 Gln Gln Gly Tyr Thr Leu Pro Trp Thr 1 5 <210> 126 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 126 Arg Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn 1 5 10 <210> 127 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 127 Tyr Thr Ser Arg Leu Gln Ser 1 5 <210> 128 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 128 Gln Gln Gly Asn Thr Leu Pro Trp Thr 1 5 <210> 129 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 129 Gln Gln Gly Asp Thr Leu Pro Trp Thr 1 5 <210> 130 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 130 Lys Ala Ser Gln Asp Val Asn Val Ala Val Ala 1 5 10 <210> 131 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 131 Trp Ala Ser Thr Arg His Ile 1 5 <210> 132 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 132 Gln Gln His Tyr Ser Thr Pro Tyr Thr 1 5 <210> 133 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 133 Lys Ala Ser Gln Asp Ile Asn Lys Tyr Ile Ala 1 5 10 <210> 134 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 134 Tyr Thr Ser Thr Leu Gln Pro 1 5 <210> 135 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 135 Leu Gln Tyr Asp Asn Leu Tyr Thr 1 5 <210> 136 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 136 Gln Ser Ile Ser Asp Tyr Leu His 1 5 <210> 137 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 137 Cys Ala Ser Gln Ser Ile Ser Gly 1 5 <210> 138 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 138 Gln Asn Gly His Ser Phe Pro Tyr Thr 1 5 <210> 139 <211> 121 <212> PRT <213> artificial sequence <220> <223> synthetic <400> 139 Gln 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 Asn Tyr 20 25 30 Trp Met His Trp Met Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Met Ile His Pro Asn Thr Asn Asn Tyr Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Ser Arg Val Thr Ser 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 Val Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Asp Tyr Gly Ser Ser Pro Tyr Tyr Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 140 <211> 107 <212> PRT <213> artificial sequence <220> <223> Synthetic <400> 140 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 Ser Cys Arg Ala Ser Gln Asp Ile Asp Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Lys Tyr Thr Ser Arg Leu His 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 Phe Cys Gln Gln Gly Tyr Thr Leu Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 141 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 141 Gln 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 Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Met Ile His Pro Asn Val Gly Ser Thr Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Ser Lys Ala Thr Met Thr Arg Asp Lys Ser Ser Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Arg Tyr Gly Ser Ser Pro Tyr Tyr Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 142 <211> 107 <212> PRT <213> artificial sequence <220> <223> synthetic <400> 142 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 Ser Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 143 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 143 Gln 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 Ser Tyr 20 25​​​​​​​​ Lys Ser 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 Ser Trp Tyr Gly Ser Ser Pro Tyr Tyr Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 144 <211> 107 <212> PRT <213> artificial sequence <220> <223> synthetic <400> 144 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 Ser Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 145 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 145 Gln 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 Ser Tyr<e 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Met Ile His Pro Thr Gly Val Ser Thr Asp Tyr Asn Glu Lys Phe 50 55 60 Lys Ser Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Asp Tyr Gly Ser Ser Pro Tyr Tyr Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 146 <211> 107 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 146 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 Ser Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Lys Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60[[ID='36']] 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 Phe Cys Gln Gln Gly Asp Thr Leu Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 147 <211> 120 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 147 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 Ser Asp 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 Asn Ser Phe Leu Arg Leu Arg Ser Tyr Phe Asp His Trp Gly Gln 100 105 110 Gly Thr Thr Leu Thr Val Ser Ser 115 120 <210> 148 <211> 107 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 148 Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly 1 5 10 15 Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Asn Val Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45 Phe Trp Ala Ser Thr Arg His Ile Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Val Gln Ala 65 70 75 80 Glu Asp Leu Ala Leu Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 149 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 149 Gln 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 Ser Tyr 20 25 30 Gly Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45 Gly Trp Cys Ile Tyr Ile Gly Asn Asp Tyr Thr Asn Tyr Asn Glu Lys 50 55 60 Phe Lys Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala 65 70 75 80 Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Ala Tyr Tyr Gly Ser Arg Val Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 150 <211> 106 <212> PRT <213> artificial sequence <220> <223> synthetic <400> 150 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Phe Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Lys Tyr 20 25 30 Ile Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 His Tyr Thr Ser Thr Leu Gln Pro Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Arg Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Asn Leu Tyr Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 151 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 151 Gln 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 Ser Tyr 20 25 30 Gly Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met<00,03011>35 40 45 Gly Trp Cys Ile Tyr Ile Gly Asn Asp Tyr Thr Asn Tyr Asn Glu Lys 50 55 60 Phe Lys Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala 65 70 75 80 Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Ala Tyr Tyr Gly Ser Arg Val Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 152 <211> 104 <212> PRT <213> artificial sequence <220> <223> synthetic <400> 152 Glu Ile Val Leu Thr Gln Ser Pro Val Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Gln Ser Ile Ser Asp Tyr Leu His Trp 20 25 30 Tyr Leu Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Lys Cys Ala 35 40 45 Ser Gln Ser Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser 50 55 60 Gly Ser Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe 65 70 75 80 Ala Val Tyr Tyr Cys Gln Asn Gly His Ser Phe Pro Tyr Thr Phe Gly 85 90 95 Gly Gly Thr Lys Val Glu Ile Lys 100 <210> 153 <211> 121 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 153 Gln 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 Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Met Ile His Pro Asn Ser Gly Gly Asn Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Ser 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 Ser Trp Tyr Gly Ser Ser Pro Tyr Tyr Phe Asp Tyr Trp Gly<​​​​​​​​​ <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 154 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 Ser Cys Arg Ala Ser Gln Asp Ile Asp Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Lys Tyr Thr Ser Arg Leu His 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 Phe Cys Gln Gln Gly Tyr Thr Leu Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 155 <211> 327 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 155 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser 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 Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 100 105 110 Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 305 310 315 320 Leu Ser Leu Ser Leu Gly Lys 325 <210> 156 <211> 330 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 156 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 Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys 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 Ile 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 Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 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 Gln Gln 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 Lys 325 330 <210> 157 <211> 330 <212> PRT <213> artificial sequence <220> <223> synthetic <400> 157 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 Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile 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 Ile 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 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 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 Gln Gln 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> 158 <211> 698 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 158 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Phe 20 25 30 Leu Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Thr Ile Ser Gly Gly Gly Arg Asp Thr Tyr Tyr Val Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 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 Ala Lys Arg Thr Thr Tyr Ser Met Asp Tyr Trp Gly Gln Gly Thr Ser 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro 210 215 220 Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe 225 230 235 240 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 245 250 255 Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe 260 265 270 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 275 280 285 Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 290 295 300 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 305 310 315 320 Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala 325 330 335 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln 340 345 350 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 355 360 365 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 370 375 380 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 385 390 395 400 Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu 405 410 415 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 420 425 430 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Gly Gly Gly Gly 435 440 445 Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu 450 455 460 Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly 465 470 475 480 Tyr Thr Phe Thr Ser Tyr Tyr Met His Trp Val Arg Gln Ala Pro Gly 485 490 495 Gln Gly Leu Glu Trp Met Gly Ile Ile Asn Pro Ser Gly Gly Arg Thr 500 505 510 Ser Tyr Ala Gln Met Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr 515 520 525 Ser Thr Ser Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp 530 535 540 Thr Ala Val Tyr Tyr Cys Ala Arg Asp Arg Glu Glu Gln Trp Pro Val 545 550 555 560 Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 565 570 575 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp 580 585 590 Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp 595 600 605 Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Arg Arg Tyr Leu 610 615 620 Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 625 630 635 640 Ser Ala Ser Asn Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 645 650 655 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu 660 665 670 Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ile Ile Pro Pro Thr 675 680 685 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 690 695 <210> 159 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 159 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Leu Ala Ser Gln Thr Ile Gly Thr Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Asp Ala Ala Thr Ser Leu Ala Asp 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 Phe Tyr Ser Ile Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 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 Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln 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 Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 160 <211> 698 <212> PRT <213> artificial sequence <220> <223> synthetic <400> 160 Gln 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 Ser Tyr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Phe Pro Gly Ser Gly Asn Ser Lys Tyr Asn Glu Asn Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Ala Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Glu Thr Tyr Asp Tyr Gly Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro 210 215 220 Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe 225 230 235 240 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 245 250 255 Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe 260 265 270 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 275 280 285 Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 290 295 300 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 305 310 315 320 Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala 325 330 335 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln 340 345 350 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 355 360 365 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 370 375 380 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 385 390 395 400 Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu 405 410 415 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 420 425 430 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Gly Gly Gly Gly 435 440 445 Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu 450 455 460 Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly 465 470 475 480 Tyr Thr Phe Thr Ser Tyr Tyr Met His Trp Val Arg Gln Ala Pro Gly 485 490 495 Gln Gly Leu Glu Trp Met Gly Ile Ile Asn Pro Ser Gly Gly Arg Thr 500 505 510 Ser Tyr Ala Gln Met Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr 515 520 525 Ser Thr Ser Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp 530 535 540 Thr Ala Val Tyr Tyr Cys Ala Arg Asp Arg Glu Glu Gln Trp Pro Val 545 550 555 560 Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 565 570 575 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp 580 585 590 Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp 595 600 605 Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Arg Arg Tyr Leu 610 615 620 Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 625 630 635 640 Ser Ala Ser Asn Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 645 650 655 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu 660 665 670 Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ile Ile Pro Pro Thr 675 680 685 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 690 695 <210> 161 <211> 214 <212> PRT <213> artificial sequence <220> <223> synthetic <400> 161 Asp Ile Gln Met Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Asn 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Ala Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ser Tyr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 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 Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln 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 Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 162 <211> 713 <212> PRT <213> artificial sequence <220> <223> synthetic <400> 162 Gln 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 Ser Tyr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Phe Pro Gly Ser Gly Asn Ser Lys Tyr Asn Glu Asn Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Ala Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Glu Thr Tyr Asp Tyr Gly Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro 210 215 220 Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe 225 230 235 240 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 245 250 255 Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe 260 265 270 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 275 280 285 Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 290 295 300 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 305 310 315 320 Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala 325 330 335 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln 340 345 350 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 355 360 365 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 370 375 380 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 385 390 395 400 Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu 405 410 415 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 420 425 430 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Gly Gly Gly Gly 435 440 445 Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu 450 455 460 Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly 465 470 475 480 Tyr Thr Phe Thr Ser Tyr Tyr Met His Trp Val Arg Gln Ala Pro Gly 485 490 495 Gln Gly Leu Glu Trp Met Gly Ile Ile Asn Pro Ser Gly Gly Arg Thr 500 505 510 Ser Tyr Ala Gln Met Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr 515 520 525 Ser Thr Ser Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp 530 535 540 Thr Ala Val Tyr Tyr Cys Ala Arg Asp Arg Glu Glu Gln Trp Pro Val 545 550 555 560 Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 565 570 575 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 580 585 590 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile 595 600 605 Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg 610 615 620 Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Arg Arg Tyr Leu Asn 625 630 635 640 Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser 645 650 655 Ala Ser Asn Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly 660 665 670 Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp 675 680 685 Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ile Ile Pro Pro Thr Phe 690 695 700 Gly Gln Gly Thr Lys Val Glu Ile Lys 705 710 <210> 163 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 163 Asp Tyr Tyr Met Asn 1 5 <210> 164 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 164 Val Ile Asn Pro Tyr Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe Lys 1 5 10 15 Gly <210> 165 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 165 Asp Asp Gly Tyr Tyr Val His Tyr Phe Asp Tyr 1 5 10 <210> 166 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 166 His Tyr Tyr Met Asn 1 5 <210> 167 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 167 Leu Ile Asn Pro Tyr Asn Gly Asp Thr Ala Tyr Asn Gln Lys Phe Lys 1 5 10 15 Asp <210> 168 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 168 Thr Arg Asp Asp Gly Tyr Tyr Val Glu His 1 5 10 <210> 169 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 169 Thr Ala Tyr Thr Ile His 1 5 <210> 170 <211> 17 <212> PRT <213> Artificia...

Claims

1. An anti-tumor antagonist, comprising: A first targeting domain that specifically binds to PD-1 or LAG-3; and a second targeting domain, wherein the second targeting domain comprises a scFv that specifically binds to TIGIT; in, The first targeting domain specifically binds to PD-1, and the first targeting domain comprises: (1) an immunoglobulin HCVR comprising three complementarity determining regions (HCDRs) HCDR1, HCDR2 and HCDR3, Wherein, the amino acid sequence of HCDR1 is shown in SEQ ID NO: 79, Wherein, the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 80, Wherein, the amino acid sequence of the HCDR3 is shown in SEQ ID NO: 81; and (2) An immunoglobulin LCVR comprising three complementarity determining regions (LCDRs) LCDR1, LCDR2 and LCDR3, Wherein, the amino acid sequence of LCDR1 is shown in SEQ ID NO: 93, The amino acid sequence of LCDR2 is shown in SEQ ID NO: 94, and Wherein, the amino acid sequence of the LCDR3 is as shown in SEQ ID NO: 95; or the first targeting domain specifically binds to LAG-3, and the first targeting domain comprises: (1) an immunoglobulin HCVR comprising three complementarity determining regions (HCDRs) HCDR1, HCDR2 and HCDR3, Wherein, the amino acid sequence of HCDR1 is shown in SEQ ID NO: 163, Wherein, the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 164, and wherein the amino acid sequence of the HCDR3 is shown in SEQ ID NO: 165; and (2) An immunoglobulin LCVR comprising three complementarity determining regions (LCDRs) LCDR1, LCDR2 and LCDR3, Wherein, the amino acid sequence of LCDR1 is shown in SEQ ID NO: 172, The amino acid sequence of LCDR2 is shown in SEQ ID NO: 173, and Wherein, the amino acid sequence of LCDR3 is shown in SEQ ID NO: 174; and The second targeting domain comprises: (1) an immunoglobulin HCVR comprising three complementarity determining regions (HCDRs) HCDR1, HCDR2 and HCDR3, Wherein, the amino acid sequence of the HCDR1 is shown in SEQ ID NO: 23, Wherein, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 24, and Wherein, the amino acid sequence of the HCDR3 is shown in SEQ ID NO: 25; and (2) An immunoglobulin LCVR comprising three complementarity determining regions (LCDRs) LCDR1, LCDR2 and LCDR3, Wherein, the amino acid sequence of LCDR1 is shown in SEQ ID NO: 45, The amino acid sequence of LCDR2 is shown in SEQ ID NO: 46, and The amino acid sequence of LCDR3 is shown in SEQ ID NO:

47.

2. The anti-tumor antagonist according to claim 1, wherein The first targeting domain comprises: An immunoglobulin HCVR, the amino acid sequence of which is shown in SEQ ID NO: 106; and The immunoglobulin LCVR has an amino acid sequence as shown in SEQ ID NO:

107.

3. The anti-tumor antagonist according to claim 1, wherein The first targeting domain comprises: An immunoglobulin HCVR, the amino acid sequence of which is shown in SEQ ID NO: 180; and The immunoglobulin LCVR has an amino acid sequence as shown in SEQ ID NO:

181.

4. The anti-tumor antagonist according to claim 1, wherein The second targeting domain comprises: An immunoglobulin HCVR, the amino acid sequence of which is shown in SEQ ID NO: 66; and The immunoglobulin LCVR has an amino acid sequence as shown in SEQ ID NO:

67.

5. The anti-tumor antagonist according to claim 1, further comprising an immunoglobulin scaffold having an amino terminal and a carboxyl terminal, in, The first targeting domain is connected to the amino terminus of the immunoglobulin scaffold, and the second targeting domain is connected to the carboxyl terminus of the immunoglobulin scaffold via a peptide linker, and Wherein, the peptide linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 188-191.

6. The anti-tumor antagonist according to claim 1, comprising a heavy chain and a light chain, wherein the amino acid sequence of the heavy chain is as shown in SEQ ID NO: 160 or 162; and the amino acid sequence of the light chain is as shown in SEQ ID NO:

161.

7. The anti-tumor antagonist according to claim 1, comprising a heavy chain and a light chain, wherein the amino acid sequence of the heavy chain is as shown in SEQ ID NO: 192 or 193; and the amino acid sequence of the light chain is as shown in SEQ ID NO:

194.

8. Use of the anti-tumor antagonist according to any one of claims 1 to 7 for preparing a medicament for treating solid tumors.

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