Novel cancer immunotherapy antibody compositions

Abstract

The present invention provides novel compositions and methods related to or derived from anti-PD-L1 antibodies having ADCC and / or CDC activity. More specifically, the present invention discloses fully human antibodies that bind PD-L1, PD-L1 binding antibody fragments and derivatives, and PD-L1 binding polypeptides comprising these fragments.

Description

[0001] This application is a divisional application of the invention patent application with application number "201980054723.0" and invention title "Novel Cancer Immunotherapy Antibody Composition".

[0002] Cross-reference to related applications

[0003] This application claims priority to and the benefit of U.S. Provisional Patent Application No. 62 / 720,015, filed August 20, 2018, the entire contents of which are incorporated herein by reference. Technical Field

[0004] This invention relates to antigen-binding peptides that bind to human PD-L1, pharmaceutical compositions, and their uses. The invention also relates to expression systems for generating these antigen-binding peptides or antibodies. The antigen-binding peptides or pharmaceutical compositions described herein are beneficial for treating pathological conditions in subjects in need, such as mammalian cancers, infections, etc. Background Technology

[0005] On the surface of immune cells are costimulatory and inhibitory receptors that interact with membrane-bound, soluble ligands. These receptors regulate the potency, duration, and type of the immune response by altering the thresholds and durations of immune cell activation or inhibition. These are collectively referred to as immune checkpoints. Many checkpoint molecules are members of the B7 superfamily or the tumor necrosis factor (TNF) superfamily.

[0006] The B7 family contains both inhibitory and stimulatory co-receptors. For example, on one hand, the binding of programmed (cell) death receptor 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) to their respective ligands (PD-L1 and PD-L2, B7-1 and B7-2, respectively) leads to inhibition of regulatory T cell activation or proliferation, unresponsiveness, exhaustion, and apoptosis. On the other hand, the binding of differentiation cluster 28 (CD28) and inducible T cell co-stimulatory molecule (ICOS) receptors to their respective ligands leads to increased proliferation and cytokine production. Conversely, TNF family co-stimulatory receptors contain only stimulatory molecules that promote proliferation and effector differentiation, such as OX40, 4-1BB, CD40, CD27, and their ligands. In addition, there are other co-receptors belonging to one of these two families, such as Tim-3, LAG-3, and Ceacam-1.

[0007] Over the past few decades, it has been established that various cancers generate an immunosuppressive environment within the tumor through a variety of mechanisms. One mechanism of relapse is the suppression of ectopic expression of inhibitory immune checkpoint ligands (especially PDL1) on tumor-mediated T cells. There is also increasing evidence that blocking this tumor-mediated immunosuppression can desuppress tumor-mediated T cells, allowing them to kill the tumor (A dachi K, Tamada K. Cancer Sci. 2015; 106(8):945-50; Rafiq S, et al., Nat Biotechnol. 2018 Aug 13; Hargadon KM, et al., Int Immunopharmacol. 2018; 62:29-39.). Blocking can be achieved through antibodies or a variety of other methods. This differs from traditional anticancer antibody therapy, which binds to cancer cells, recruits complement-dependent cytotoxicity (CDC) and antibody-dependent cytotoxicity (ADCC), and directly kills tumor cells.

[0008] CTLA-4 antibody is the first FDA-approved immunotherapy based on immune checkpoint blockade. Other blocking targets, such as PD1 and related molecules, offer more diverse opportunities to enhance anti-tumor immunity in clinical practice. Summary of the Invention

[0009] This invention provides an antigen-binding polypeptide that binds to PD-L1 (or, interchangeably, referred to as "anti-PD-L1 polypeptide" or "PD-L1 binding polypeptide"), preferably human PD-L1; the polypeptide has one or more of the following characteristics: (a) the ability to bind to PD-L1 and inhibit its interaction with PD-L1; and (b) having an isotype or constant region capable of triggering ADCC and / or CDC. The resulting antibody kills tumor cells through two synergistic pathways—T cell desuppression and direct cytotoxicity. The polypeptide of this invention can be used alone or in combination with (a) antibodies targeting other immunosuppressive pathways; (b) chemotherapy or radiotherapy; (c) other mechanisms blocking immunosuppressive pathways, such as aptamers or RNAi; or (d) other immunotherapeutic agents, such as cytokines, targeted therapies, etc., for the treatment of tumors.

[0010] In one aspect, the present invention provides an antigen-binding polypeptide, such as an antibody, fragment, derivative, or analogue thereof, which is an isotype of IgG1 and binds to the PD-L1 epitope, preferably having at least 10 -6The M-binding affinity is "substantially composed of," in this context, at least 80%, or more preferably, 85%, 90%, 95%, or even 100%, of the same heavy chain variable region domain selected from the group consisting of the following amino acid sequences: SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:26, SEQ ID NO:30, SEQ ID NO:34, SEQ ID NO:38, SEQ ID NO:42, SEQ ID NO:46, SEQ ID NO:50, SEQ ID NO:54, SEQ ID NO:58, SEQ ID NO:62, SEQ ID NO:66, SEQ ID NO:70, SEQ ID NO:74, SEQ ID NO:78, SEQ ID NO:82, SEQ ID NO:86, SEQ ID NO:90, SEQ ID NO:94, SEQ ID NO:98, SEQ ID NO:102, SEQ ID NO: ...70, SEQ ID NO:86, SEQ ID NO:90, SEQ ID NO:94, SEQ ID NO:98, SEQ ID NO:102, SEQ ID NO:70, SEQ ID NO:74, SEQ ID NO:78, SEQ ID NO:82, SEQ ID NO:86, SEQ ID NO:90, SEQ ID NO:94, SEQ ID NO:98, SEQ ID NO:102, SEQ ID NO:70, SEQ ID NO:74, SEQ ID NO:78, SEQ ID NO:86, SEQ NO:106 and combinations thereof; and a light chain variable region domain having substantially 80%, or more preferably 85%, 90%, 95%, or even 100%, identical to sequences selected from the group consisting of the following amino acid sequences: SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:32, SEQ ID NO:36, SEQ ID NO:40, SEQ ID NO:44, SEQ ID NO:48, SEQ ID NO:52, SEQ ID NO:56, SEQ ID NO:60, SEQ ID NO:64, SEQ ID NO:68, SEQ ID NO:72, SEQ ID NO:76, SEQ ID NO:80, SEQ ID NO:84, SEQ ID NO:88, SEQ ID NO:92, SEQ ID NO:96, SEQ ID NO:100, SEQ ID NO:104, SEQ ID NO:106 ... NO:108 and its combinations.

[0011] In a preferred embodiment, the antigen-binding polypeptide or antibody of the present invention comprises a pair of heavy chain variable regions and light chain variable regions, the respective sequences of which are substantially composed of the following pairs: (a) SEQ ID NO:18 and SEQ ID NO:20; (b) SEQ ID NO:42 and SEQ ID NO:44; or (c) SEQ ID NO:34 and SEQ ID NO:36.

[0012] In other preferred embodiments, the antigen-binding polypeptide or antibody of the present invention comprises a pair of heavy chain variable regions and a light chain variable region, the respective sequences of which are substantially composed of the following pairs: (a) SEQ ID NO:22 and SEQ ID NO:24; (b) SEQ ID NO:2 and SEQ ID NO:4; (c) SEQ ID NO:62 and SEQ ID NO:64; or (d) SEQ ID NO:82 and SEQ ID NO:84.

[0013] In other preferred embodiments, the antigen-binding polypeptide or antibody of the present invention comprises a pair of heavy chain variable regions and light chain variable regions, the respective sequences of which are substantially composed of the following pairs: (a) SEQ ID NO:70 and SEQ ID NO:72; (b) SEQ ID NO:50 and SEQ ID NO:52; (c) SEQ ID NO:102 and SEQ ID NO:104; or (d) SEQ ID NO:30 and SEQ ID NO:32.

[0014] In other preferred embodiments, the antigen-binding polypeptide or antibody of the present invention comprises a pair of heavy chain variable regions and light chain variable regions, the respective variable region sequences of which are substantially composed of the following pairs: (a) SEQ ID NO:6 and SEQ ID NO:8; (b) SEQ ID NO:10 and SEQ ID NO:12; (c) SEQ ID NO:14 and SEQ ID NO:16; (d) SEQ ID NO:26 and SEQ ID NO:28; (e) SEQ ID NO:38 and SEQ ID NO:40; (f) SEQ ID NO:46 and SEQ ID NO:48; (g) SEQ ID NO:54 and SEQ ID NO:56; or (h) SEQ ID NO:58 and SEQ ID NO:60.

[0015] In other preferred embodiments, the antigen-binding polypeptide or antibody of the present invention comprises a pair of heavy chain variable regions and a light chain variable region, the respective variable region sequences of which are substantially composed of the following pairs: (a) SEQ ID NO:66 and SEQ ID NO:68; (b) SEQ ID NO:74 and SEQ ID NO:76; (c) SEQ ID NO:78 and SEQ ID NO:80; (d) SEQ ID NO:86 and SEQ ID NO:88; (e) SEQ ID NO:90 and SEQ ID NO:92; (f) SEQ ID NO:94 and SEQ ID NO:96; (g) SEQ ID NO:98 and SEQ ID NO:100; or (h) SEQ ID NO:106 and SEQ ID NO:108.

[0016] Preferably, the antigen-binding polypeptide is wholly human or humanized. In a preferred embodiment, the antigen-binding polypeptide further comprises a human constant region. In one embodiment, the human constant region is IgG1. In some embodiments, the antibody of the present invention further comprises a second pair of heavy chain variable regions and light chain variable regions, for example, which are substantially identical to the first pair.

[0017] In a preferred embodiment, the anti-PD-L1 peptide binds to PD-L1, blocking the interaction between PD-L1 and PD1. This may be because the epitope on PD-L1 that binds to it is located at or near the PD1 interaction interface, or because the conformation of the PD1 interaction interface has undergone an allosteric change.

[0018] In another aspect, the present invention provides a nucleic acid molecule encoding the aforementioned polypeptide. The nucleic acid molecule may be a DNA molecule or an RNA molecule. In a preferred embodiment, the nucleic acid molecule is a DNA molecule encoding the heavy chain variable region and the light chain variable region of the antigen-binding polypeptide or antibody of the present invention, the DNA sequence being substantially composed of the following pairs: (a) SEQ ID NO:17 and SEQ ID NO:19; (b) SEQ ID NO:33 and SEQ ID NO:35; (c) SEQ ID NO:41 and SEQ ID NO:43.

[0019] In other preferred embodiments, the nucleic acid molecule is a DNA molecule encoding the heavy chain variable region and the light chain variable region of the antigen-binding polypeptide or antibody of the present invention, the DNA sequence being substantially composed of the following pairs: (a) SEQ ID NO:21 and SEQ ID NO:23; (b) SEQ ID NO:1 and SEQ ID NO:3; (c) SEQ ID NO:61 and SEQ ID NO:63; or (d) SEQ ID NO:81 and SEQ ID NO:83.

[0020] In other preferred embodiments, the nucleic acid molecule is a DNA molecule encoding the heavy chain variable region and the light chain variable region of the antigen-binding polypeptide or antibody of the present invention, the DNA sequence being substantially composed of the following pairs: (a) SEQ ID NO:69 and SEQ ID NO:71; (b) SEQ ID NO:49 and SEQ ID NO:51; (c) SEQ ID NO:101 and SEQ ID NO:103; or (d) SEQ ID NO:29 and SEQ ID NO:31.

[0021] In other preferred embodiments, the nucleic acid molecule is a DNA molecule encoding the heavy chain variable region and the light chain variable region of the antigen-binding polypeptide or antibody of the present invention, the DNA sequence being substantially composed of the following pairs: (a) SEQ ID NO:5 and SEQ ID NO:7; (b) SEQ ID NO:9 and SEQ ID NO:11; (c) SEQ ID NO:13 and SEQ ID NO:15; (d) SEQ ID NO:25 and SEQ ID NO:27; (e) SEQ ID NO:37 and SEQ ID NO:39; (f) SEQ ID NO:45 and SEQ ID NO:47; (g) SEQ ID NO:53 and SEQ ID NO:55; or (h) SEQ ID NO:57 and SEQ ID NO:59.

[0022] In other preferred embodiments, the nucleic acid molecule is a DNA molecule encoding the heavy chain variable region and the light chain variable region of the antigen-binding polypeptide or antibody of the present invention, the DNA sequence being substantially composed of the following pairs: (a) SEQ ID NO:65 and SEQ ID NO:67; (b) SEQ ID NO:73 and SEQ ID NO:75; (c) SEQ ID NO:77 and SEQ ID NO:79; (d) SEQ ID NO:85 and SEQ ID NO:87; (e) SEQ ID NO:89 and SEQ ID NO:91; (f) SEQ ID NO:93 and SEQ ID NO:95; (g) SEQ ID NO:97 and SEQ ID NO:99; or (h) SEQ ID NO:105 and SEQ ID NO:107.

[0023] In another aspect, the present invention provides a pharmaceutical composition comprising an antigen-binding polypeptide, such as the anti-PD-L1 antibody, fragment, derivative, or analog disclosed herein. The pharmaceutical composition further comprises a pharmaceutically acceptable excipient, carrier, or diluent.

[0024] In a related aspect, the present invention provides a method for treating a pathological condition in a subject in need, the method comprising administering to the subject a therapeutically effective amount of the anti-PD-L1 peptide or antibody disclosed herein. The method may further include the step of administering a second and different therapeutic antibody, which resists at least one cell surface antigen indicating the condition. The condition being treated may be mammalian cancer, infection, etc. In various embodiments, the anti-PD-L1 peptide may be an antibody, an antibody fragment, an antibody derivative, or an antibody analog.

[0025] Preferably, the range of mammalian cancers to be treated is selected from the group consisting of: ovarian cancer, colon cancer, breast cancer, lung cancer, myeloma, neuroblastoma, monocytic leukemia, B-cell leukemia, T-cell leukemia, B-cell lymphoma, T-cell lymphoma, mast cell tumor, melanoma, bladder cancer, gastric cancer, liver cancer, urothelial carcinoma, skin cancer, kidney cancer, head and neck cancer, pancreatic cancer, and combinations of the above cancers. More broadly, any cancer in which at least a significant portion of the tumor cells express detectable levels of PD-L1 is considered a target for treatment with the compositions of the present invention.

[0026] In another aspect, the present invention provides a method for preventing a similar condition in a subject of need, the method comprising administering to the subject a preventatively effective amount of the pharmaceutical composition of the present invention. The method may further include the step of administering a vaccine against the condition. In one embodiment, the condition is cancer.

[0027] In another aspect, the present invention provides a mammalian expression system that produces an antigen-binding polypeptide, such as an antibody, fragment, derivative or analogue thereof, that binds to the PD-L1 epitope described in the present invention. Attached Figure Description

[0028] Figure 1 The illustration depicts the use of solid-phase phage screening technology, specifically, according to an embodiment of the present invention, by indirectly coating an immune tube with a test protein to screen for antigen-binding peptides.

[0029] Figure 2 The illustration depicts the use of solid-phase phage screening technology, specifically, according to an embodiment of the present invention, by directly coating an immune tube with a test protein to screen for antigen-binding peptides.

[0030] Figure 3 This is a chart that lists data characterizing the ability of a representative single-stranded variable fragment (scfv) to bind hPDL1 in an indirect ELISA binding assay obtained using embodiments of the present invention. “NC” represents a negative control.

[0031] Figure 4 This chart lists data characterizing the ability of representative single-stranded variable fragments (scfvs) obtained through embodiments of the present invention to bind hPDL1 in FACS binding assays. "PC" represents a positive control, using hPDL1 / 293T cells stained with anti-hPDL1-APC (10 μg / ml). "NC" represents a negative control, using unstained hPDL1 / 293T cells.

[0032] Figure 5 This chart presents data characterizing the ability of various single-stranded variable fragments (scfvs) obtained through embodiments of the present invention to block the interaction between hPD1 and hPDL1 in receptor blocking assays (with hPDL1-coated test plates). "PC" represents a positive control with added biotin-hPD1-Fc. "NC" represents a negative control with only buffer added.

[0033] Figure 6This chart presents data characterizing the ability of various single-stranded variable fragments (scfvs) obtained through embodiments of the present invention to block the interaction between hPD1 and hPDL1 in receptor blocking assays (with hPD1-coated test plates). "PC" represents a positive control with added biotin-hPDL1-Fc. "NC" represents a negative control with only buffer added.

[0034] Figure 7 This invention describes the ability of a single-stranded variable fragment (scfv) obtained through embodiments of the present invention to bind hPDL1-Fc, mPDL1-Fc (mouse PDL1), and hIgG1 in a direct ELISA assay.

[0035] Figure 8A and 8B Display via SDS-PAGE( Figure 8A ) and size exclusion chromatography ( Figure 8B The full-length antibody 4-1E8 was characterized.

[0036] Figure 9A and 9B Display via SDS-PAGE( Figure 9A ) and size exclusion chromatography ( Figure 9B The full-length antibody 3-1B11 was characterized.

[0037] Figure 10A and 10B Display via SDS-PAGE( Figure 10A ) and size exclusion chromatography ( Figure 10B The full-length antibody 3-1E4 was characterized.

[0038] Figure 11A This diagram shows an ELISA assay for quantitatively measuring the binding of the antibody of the present invention to hPDL1. Figure 11B and 11C Display, as shown Figure 11A The results of quantitative binding analysis of hPDL1 with a portion of the full-length antibody examples of the present invention, shown in ELISA form.

[0039] Figure 12A and 12B The image shows quantitative FACS results of some full-length antibody examples of the present invention, which bind to hPDL1-expressing 293T cells (top image) and hPDL1-negative 293T cells (bottom image).

[0040] Figure 13A This diagram shows Form 1 of the receptor blocking assay (RBA). Figure 13B In RBA form 1 ( Figure 13A): The results of the receptor blocking assay of the lead antibody candidate of the present invention are shown by coating with hPDL1-Fc and adding Biotin-hPD1-Fc.

[0041] Figure 14A This diagram shows the form 2 of the receptor blocking assay. Figure 14B In RBA form 2 ( Figure 14A (: hPD1-Fc coated and Biotin-hPDL1-Fc added), showing the results of the receptor blocking assay of the lead antibody candidate of the present invention.

[0042] Figure 15 It is a chart that lists data characterizing multiple full-length antibodies obtained through embodiments of the present invention.

[0043] Figures 16A-16D Describe the affinity of the lead antibody candidate using BIAcore for PD-L1: Figure 16A The BIAcore form applied in the embodiments of the present invention is illustrated schematically; Figure 16B The results of using BIAcore to test the affinity of the lead antibody candidate for PD-L1 are listed; Figure 16C The BIAcore affinity assay response curve of the 4-1E8 antibody was described; and Figure 16D The response curve of the BIAcore affinity assay for the 3-1B11 antibody is described.

[0044] Figure 17A The epitope identification method used in the embodiments of the present invention is illustrated schematically. Figure 17B The epitope identification of lead antibody candidates in an embodiment of the present invention is illustrated schematically. Figure 17C List of uses Figure 17A The matrix shown represents the epitope identification matrix for lead antibody candidates.

[0045] Figures 18A-18D FACS test results: Control group ( Figure 18A ), "4-1E8" in this invention

[0046] ( Figure 18B ), "3-1E4" Figure 18C ) and "3-1B11" Figure 18D The binding ability of the antibody to 293T cells (top) transfected with the rhesus monkey PDL1-GFP expression construct and parental 293T cells (bottom).

[0047] Figures 19A-19D FACS test results: Control group ( Figure 19A ), "4-1E8" in this invention

[0048] ( Figure 19B), "3-1E4" Figure 19C ) and "3-1B11" Figure 19D The binding ability of the antibody to 293T cells transfected with the rhesus monkey PDL1 expression construct (top) and parental 293T cells (bottom).

[0049] Figure 20 This invention illustrates representative EC50 results from IL-2 generation experiments in embodiments of the present invention.

[0050] Figure 21 The peptide example shown is numbered "4-1E8" and its ADCC activity is compared with that of the commercially available anti-PDL1 antibody atezolizumab.

[0051] Figures 22A-22C The peptide example shown is numbered "4-1E8" and is numbered "3-1B11" ( Figure 22A ) and "3-1E4" Figure 22B The ADCC activity of the embodiments of ) is compared, and the important data points are summarized in the chart ( Figure 22C )middle.

[0052] Figure 23A , 23B 23C provides three sets of experimental data comparing the IL-2 production capacity of PBMCs co-cultured with PDL1+MDA-MB-231 tumor cells in the presence of the lead antibody of the present invention and in the presence of a commercially available anti-PDL1 antibody.

[0053] Figure 24 The results provide a comparison of the IFNγ production capacity of CD8 T cells co-cultured with PDL1+MDA-MB-231 tumor cells in the presence of the lead antibody of the present invention and in the presence of a commercially available anti-PDL1 antibody.

[0054] Figure 25A and 25B This invention illustrates the results of a mixed lymphocyte reaction of a lead antibody in an embodiment of the present invention.

[0055] Figure 26A and 26B This invention number "4-1E8" is displayed. Figure 26A ) and "3-1B11" Figure 26B Antibody binding specificity.

[0056] Figure 27A and 27B The invention's E8 (shown compared to CD80 alone (solid line) and the second reagent alone (dashed line) demonstrates superior performance. Figure 27A ) and B11 ( Figure 27B The ability of antibodies to block the binding of CD80 and PD-L1-expressing cells (gray curve).

[0057] Figure 28 The half-life of the antibody example of the present invention was determined using Tg32 mice.

[0058] Unless otherwise stated, technical terms are used in accordance with their usual usage.

[0059] As used herein, “a” or “an” can refer to one or more. As used herein, when used in conjunction with the word “comprising,” “a” or “an” can refer to one or more. As used herein, “another” can refer to at least a second or more. Further, unless the context requires otherwise, singular terms include plurals, and plural terms include singulars.

[0060] As used in this invention, whether explicitly stated or not, "about" refers to index values, including integers, fractions, and percentages. The term "about" generally indicates a range of values ​​(e.g., ±5 to 10% of the listed values) that a person skilled in the art would consider equivalent to the listed values ​​(e.g., having the same function or result). In some cases, the term "about" may include values ​​rounded to the nearest significant figure. Unless otherwise specified, "about" is ±10% of the listed values.

[0061] An "antigen-binding polypeptide" is a polypeptide that contains a portion that binds to an antigen. Examples of antigen-binding polypeptides include antibodies, antibody fragments (e.g., the antigen-binding portion of an antibody), antibody derivatives, and antibody analogs.

[0062] Antigen-binding peptides or antigen-binding proteins can have the structure of, for example, naturally occurring antibodies (also known as "immunoglobulins"). Each naturally occurring antibody consists of two pairs of identical polypeptide chains, each pair having a "light" chain (approximately 25 kDa) and a "heavy" chain (approximately 50-70 kDa). The variable region of each light / heavy chain pair forms the antibody-binding site, so a complete antibody has two binding sites.

[0063] The variable regions of naturally occurring antibody chains exhibit the same general structure, namely, relatively conserved backbone regions (FRs) linked by three hypervariable regions also known as complementarity-determining regions or CDRs. From the N-terminus to the C-terminus, both the light and heavy chains contain FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 domains. The amino acid distribution in each domain is consistent with that described by Kabat et al. in *Sequences of Proteins of Immunological Interest, 5*.th The definition is consistent with that in Ed., US Dept. of Health and Human Services, PHS, NIH, NIH Publication No. 91-3242, 1991. Other numbering systems for amino acids on the immunoglobulin chain include IMGT (international ImMunoGeneTics information system; Lefran c et al., Dev. Comp. Immunol. 29:185-203; 2005) and AHo (Honegger and Pluckthun, J. Mol. Biol. 309(3):657-670; 2001).

[0064] Antibodies can be obtained from sources such as serum or plasma containing immunoglobulins with different antigenic properties. If these antibodies undergo affinity purification, they can be enriched for specific antigenic properties. Such enriched antibody formulations typically consist of less than about 10% antibodies with specific binding activity against a particular antigen. By subjecting these formulations to several rounds of affinity purification, the proportion of antibodies with specific binding activity against the antigen can be increased. Antibodies prepared in this manner are often referred to as "monospecific." Monospecific antibody formulations can consist of approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 99.9% antibodies with specific binding activity against a particular antigen.

[0065] As used in this invention, the terms "antibody" or "Ab" (and their plural forms) broadly refer to any immunoglobulin (Ig) molecule composed of four polypeptide chains (two heavy (H) chains and two light (L) chains), or any functional fragment, mutant, variant, derivative, or analog thereof that retains the essential characteristics of an Ig molecule and specific epitope binding characteristics. These fragments, mutants, variants, derivatives, or analogs of antibodies are known in the art and include, in particular, Fab, F(ab'), F(ab')2, Fv, single-chain antibodies (scFv), single-domain antibodies (sdAbs), complementarity-determining region (CDR) fragments, chimeric antibodies, biantibodies, triantibodies, tetraantibodies, and polypeptides containing at least a portion of an immunoglobulin sufficient to bind a specific antigen to the polypeptide. Antibody fragments, derivatives, and analogs can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies.

[0066] Fab fragments are those with V L V H C L and CH1 The domain is a monovalent segment; the F(ab')2 segment is a divalent segment with two Fab segments connected by disulfide bonds in their hinge region; the Fd segment has V H and C H1 The Fv fragment has a V-shaped structure on the antibody single arm. L and V H Structural domain; dAb fragment has V H Structural domain, V L Domain or V H or V L Antigen-binding fragments of the domain (see, for example: US Pat. Nos. 6,846,634; 6,696,245, US App. Pub. 20 / 0202512; 2004 / 0202995; 2004 / 0038291; 2004 / 0009507; 2003 / 0039958, and Ward et al., Nature 341:544-546, 1989).

[0067] Single-chain antibody (scFv) is a type of antibody that is produced in V L and V H Antibodies are antibodies that form a continuous protein chain by linkers (e.g., synthetic sequences of amino acid residues) linked together. These linkers are long enough to allow the protein chain to fold itself, forming a single monovalent antigen-binding site (see, for example: Bird et al., 1988, Science 242:423-26 and Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-83). Bivalent antibodies are two-peptide chains, each containing a V-type antigen linked by a linker. H and V L The linker is too short to allow two domains on the same chain to pair, thus allowing each domain to pair with a complementary domain on another polypeptide chain (see, for example: Holliger et al., 1993, Proc. Natl. Acad. Sci. USA 90:6444-48 and Poljak et al., 1994, Structure 2:1121-23). ​​If the two polypeptide chains of a biantibody are identical, then the resulting biantibody will have two identical antigen-binding sites. Polypeptide chains with different sequences can be used to prepare biantibodies with two different antigen-binding sites. Similarly, triantibodies and tetraantibodies are antibodies containing three and four polypeptide chains, respectively, forming three and four antigen-binding sites, which can be the same or different.

[0068] Using the systems described by Kabat et al., Lefranc et al., and / or Honegger and Pluckthun, the complementarity-determining regions (CDRs) and backbone regions (FRs) of a given antibody can be identified. One or more CDRs can be covalently or nonvalently incorporated into a molecule, making it an antigen-binding protein. Antigen-binding peptides can incorporate CDR(s) as part of a larger polypeptide chain, can covalently link CDR(s) to another polypeptide chain, or can nonvalently incorporate CDR(s). CDRs allow antigen-binding proteins to bind specifically to a particular target antigen.

[0069] Antigen-binding peptides can have one or more binding sites. If there is more than one binding site, these binding sites can be the same as or different from each other. For example, naturally occurring human immunoglobulins typically have two identical binding sites, while "bispecific" or "bifunctional" antibodies have two different binding sites.

[0070] The term "human antibody" or "humanized antibody" as used in this invention encompasses all antibodies derived from human immunoglobulin sequences having one or more variable and constant regions. In one embodiment, all variable and constant regions are derived from human immunoglobulin sequences (either fully human antibodies or humanized antibodies). These antibodies can be prepared in various ways, including by immunizing mice with a target antigen, the mice being genetically modified to express antibodies encoding genes derived from human heavy and / or light chains. Humanized antibodies possess a sequence different from that of antibodies derived from non-human species through substitution, deletion, and / or addition of one or more amino acids, thus making them less likely to induce an immune response and / or induce a milder immune response when administered to human subjects compared to antibodies from non-human species. In one embodiment, certain amino acids located in the backbone and constant regions of the heavy and / or light chains of a non-human species antibody are mutated to produce a humanized antibody. In another embodiment, the constant region of a human antibody is fused to the variable region of a non-human species antibody. In another embodiment, one or more amino acid residues in one or more CDR sequences of a non-human antibody are modified to reduce the potential immunogenicity of the non-human antibody when administered to human subjects. The modified amino acid residues are not critical to the immune-specific binding of the antibody to the antigen, or the modification to the amino acid sequence is conserved, so that the binding of the humanized antibody to the antigen is not significantly worse than that of the non-human antibody. Examples of how to prepare humanized antibodies can be found in US Patents US Pat. Nos. 6,054,297, 5,886,152, and 5,877,293.

[0071] As used in this invention, the term "chimeric antibody" refers to a class of antibodies comprising one or more regions from one antibody and one or more regions from at least another antibody. In one embodiment, CDRs from multiple human anti-PD-L1 antibodies are mixed and paired in a chimeric antibody.

[0072] Activated T cells express PD1 on their cell surface. PD-L1 binding to PD1 activates PD1 and inhibits PD1. + T cells. The “neutralizing antibody” or “inhibitory antibody” used in this invention refers to an antibody that blocks PD1 activation, i.e., by employing the assays described in the embodiments of this invention, an excess of anti-PD-L1 antibody reduces the activation level by at least about 20%. In various embodiments, the antigen-binding protein reduces PD1 activation by at least 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, and 99.9%.

[0073] Fragments or analogues of antibodies can be readily prepared by those skilled in the art following the teachings of this specification and using techniques known in the art. Preferably, the amino and carboxyl terms of the fragments or analogues are located near the boundaries of functional domains. Structural and functional domains can be determined by comparing nucleotide and / or amino acid sequence data with public or private sequence databases. Computerized contrastive methods can be used to identify sequence motifs or predict protein conformational domains appearing in proteins with other known structures and / or functions. Methods for identifying protein sequences folded into known three-dimensional structures are known. See, Bowie et al., 1991, Science 253:164.

[0074] As used in this invention, if an antigen-binding polypeptide binds to an antigen with a dissociation constant of 100 nanomolars or less, then the antigen-binding polypeptide “specifically binds” to the antigen (e.g., human PD-L1).

[0075] The "antigen-binding domain," "antigen-binding region," or "antigen-binding site" used in this invention are parts of an antigen-binding protein that contain amino acid residues (or other groups) that interact with the antigen and contribute to the specificity and affinity of the antigen-binding protein for the antigen. For an antibody to specifically bind to an antigen, it must contain at least a portion of at least one of its CDR domains.

[0076] As used in this invention, an "epitope" refers to the portion of a molecule that is bound by an antigen-binding protein (e.g., an antibody). An epitope may comprise discontinuous portions of the molecule (e.g., in a polypeptide chain, discontinuous amino acid residues in the primary sequence of the polypeptide, but which are close enough to each other in the tertiary and quaternary structures of the polypeptide for binding by an antigen-binding protein).

[0077] As used herein, the terms "polynucleotide," "oligonucleotide," and "nucleic acid" are used interchangeably throughout the text to include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), DNA or RNA analogs derived from nucleotide analogs (e.g., peptide nucleic acids and unnaturally occurring nucleotide analogs), and mixtures thereof. Nucleic acid molecules may be single-stranded or double-stranded. In one embodiment, the nucleic acid molecule of the present invention comprises consecutive open reading frames encoding antibodies, fragments thereof, derivatives, mutants, or variants thereof.

[0078] The “vector” used in this invention is a nucleic acid that can be used to introduce another nucleic acid linked to it into a cell. One type of vector is a “plasmid,” which refers to a linear or circular double-stranded DNA molecule into which an additional nucleic acid fragment can be attached. Another type of vector is a viral vector (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), in which an additional DNA fragment can be introduced into the viral genome. Some vectors are capable of autonomous replication in the introduced host cell (e.g., bacterial vectors containing bacterial origins of replication and free mammalian vectors). Other vectors (e.g., non-free mammalian vectors) are integrated into the host cell’s genome after introduction into the host cell, and thus replicated along with the host genome. An “expression vector” is a vector that can direct the expression of selected polynucleotides.

[0079] As used in this invention, if a regulatory sequence affects the expression of a nucleotide sequence (e.g., the level, timing, or site of expression), then that nucleotide sequence is “operably linked” to the regulatory sequence. A “regulatory sequence” is a nucleic acid that affects the expression (e.g., the level, timing, or site of expression) of a nucleic acid to which it is operably linked. For example, the regulatory sequence may act directly on the nucleic acid it regulates, or through the function of one or more other molecules (e.g., a polypeptide linked to the regulatory sequence and / or the nucleic acid). Examples of regulatory sequences include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). Further examples of regulatory sequences are described, for example, in Goeddel, 1990, GeneExpression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. and Baron et al., 1995, Nucleic Acids Res. 23:3605-06.

[0080] Preferably, the broad spectrum of mammalian cancers treated by the compositions of the present invention are selected from the group consisting of: ovarian cancer, colon cancer, breast cancer, lung cancer, myeloma, neuroblastoma, monocytic leukemia, B-cell leukemia, T-cell leukemia, B-cell lymphoma, T-cell lymphoma, mast cell tumor, melanoma, bladder cancer, gastric cancer, liver cancer, urothelial carcinoma, skin cancer, kidney cancer, head and neck cancer, pancreatic cancer, and combinations of the above cancers. More broadly, any cancer in which at least some tumor cells express a detectable amount of PD-L1 can be treated with the compositions of the present invention.

[0081] The polypeptides disclosed herein can be produced using any standard method known in the art. In one embodiment, the polypeptide is produced by a recombinant DNA method, which involves inserting a nucleic acid sequence encoding the polypeptide (e.g., cDNA) into a recombinant expression vector and expressing the DNA sequence under conditions that promote expression.

[0082] The nucleic acids encoding any of the various polypeptides disclosed in this invention can be chemically synthesized. To improve expression in cells, codon selection can be optimized. This codon selection will depend on the cell type chosen. Specific codon selection patterns have been developed for Escherichia coli and other bacteria, as well as mammalian, plant, yeast, and insect cells. See, for example: Mayfield et al., Proc. Natl. Acad. Sci. USA. 2003 100(2):438-42; Sinclair et al. Protein Expr. Purif. 2002(1):96-105; Connell N D. Curr. Opin. Biotechnol. 2001 12(5):446-9; Makrides et al. Microbiol. Rev. 1996 60(3):512-38 and Sharp et al. Yeast. 1991 7(7):657-78.

[0083] General techniques for nucleic acid manipulation are described in, for example: Sambrook et al., Molecular Cloning: A Laboratory Manual, Vols. 1-3, Cold Spring Harbor Laboratory Press, 2nd ed., 1989, or F. Ausubel et al., Current Protocols in Molecular Biology (Green Publishing and Wiley-Interscience: New York, 1987) and are periodically updated therein, incorporated herein by reference. DNA encoding polypeptides is operatively linked to appropriate transcriptional or translational regulatory elements derived from mammalian, viral, or insect genes. These regulatory elements include transcription promoters, optional operator sequences controlling transcription, sequences encoding suitable mRNA ribosome binding sites, and sequences controlling the termination of transcription and translation. The ability to replicate in the host is typically conferred by the origin of replication, and selection genes facilitating the identification of transformants are also incorporated.

[0084] The recombinant DNA in this invention may also contain any type of protein tag sequence, which may facilitate protein purification. Examples of protein tags include, but are not limited to, histidine tags, FLAG tags, myc tags, HA tags, or GST tags. Suitable cloning and expression vectors for bacterial, fungal, yeast, and mammalian cell hosts can be found in Cloning Vectors: A Laboratory Manual (Elsevier, NY, 1985).

[0085] The expression constructs of this invention are introduced into host cells using methods suitable for host cells. Various methods for introducing nucleic acids into host cells are well known in the art, including but not limited to, electroporation; transfection using calcium chloride, rubidium chloride, calcium phosphate, dextran (DEAE-dextran), or other substances; gene gun method; lipid transfection; and infection (wherein the vector is an infectious agent). Suitable host cells include prokaryotic cells, yeast, mammalian cells, or bacterial cells.

[0086] The protein disclosed in this invention can also be produced using a cell translation system. For this purpose, the nucleic acid encoding the polypeptide must be modified to enable in vitro transcription to produce mRNA and to allow cell-free translation of the mRNA in a specific cell-free system used (eukaryotic, such as a mammalian or yeast cell-free translation system; or prokaryotic, such as a bacterial cell-free translation system).

[0087] PD-L1-binding peptides can also be produced through chemical synthesis (e.g., by the method described in Solid Phase Peptide Synthesis, 2nd ed., 1984, The Pierce Chemical Co., Rockford, Ill). Protein modifications can also be produced through chemical synthesis.

[0088] The peptides disclosed herein can be purified using protein isolation / purification methods generally known in the field of protein chemistry. Non-limiting examples include: extraction, recrystallization, salting out (e.g., using ammonium sulfate or sodium sulfate), centrifugation, dialysis, ultrafiltration, adsorption chromatography, ion exchange chromatography, hydrophobic chromatography, normal-phase chromatography, reversed-phase chromatography, gel filtration, gel permeation chromatography, affinity chromatography, electrophoresis, countercurrent distribution, or any combination thereof. After purification, the peptides can be exchanged with different buffers and / or concentrated using any of the various methods known in the art, including but not limited to filtration and dialysis.

[0089] The purified peptides are preferably at least 85% pure, more preferably at least 90% or 95% pure, and most preferably at least 98% pure. Regardless of the exact purity value, the peptides are adequately purified for use as pharmaceutical products.

[0090] Post-translational modifications of peptides

[0091] In some embodiments, the binding peptides of the present invention may further comprise post-translational modifications. Exemplary post-translational protein modifications include phosphorylation, acetylation, methylation, ADP-riboylation, ubiquitination, glycosylation, carbonylation, ubiquitin-like formation, biotinylation, or the addition of peptide side chains or hydrophobic groups. Thus, the modified soluble peptides may contain non-amino acid elements, such as lipids, polysaccharides, or monosaccharides, as well as phosphates. A preferred form of glycosylation is sialylation, which links one or more sialic acid groups to the peptide. Sialic acid groups improve protein solubility and serum half-life, while also reducing the protein's potential immunogenicity. See Raju et al. Biochemistry. 2001 31; 40(30):8868-76. The effects of these non-amino acid elements on peptide function can be tested to understand their antagonistic effects on PD-L1 or PD-1 function, such as their inhibitory effects on angiogenesis or tumor growth.

[0092] In one embodiment, the modification of the host polypeptide comprises linking the host soluble polypeptide to a non-protein polymer. In a specific embodiment, the polymer is polyethylene glycol (“PEG”), polypropylene glycol, or polyoxyethylene, as specified in U.S. Patents US Pat. Nos. 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192, or 4,179,337.

[0093] In one embodiment, the PEGylated peptides of the present invention preferably retain at least 25%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% of the unmodified protein-related biological activity. In one embodiment, biological activity refers to its ability to bind PD-L1, via KD, k on or k off The rate was evaluated. In one specific embodiment, the PEGylated binding peptide protein exhibited increased binding to human PD-L1 compared to its unPEGylated counterpart. In another embodiment, bioactivity refers to the blocking of PD-L1 / PD1 interaction.

[0094] Treatment, Vaccines & Administration

[0095] This disclosure further provides methods for treating or preventing diseases that respond to inhibition of PD-L1 bioactivity. Preferred embodiments are diseases characterized by excessive cell proliferation and persistent infection. The application technique and dosage depend on the specific peptide type and the specific disease being treated. Because regulatory agencies require that the pyrogen content of the protein reagents used for treatment be at acceptablely low levels, the therapeutic formulations of this invention are distinguished from other formulations because they are substantially free of pyrogens, or at least contain no more than an acceptable level of pyrogens as determined by an appropriate regulatory agency (such as the U.S. FDA).

[0096] The pharmaceutical formulations of the present invention may contain at least one pharmaceutically acceptable diluent, carrier, or excipient. The excipients included in the formulation will have different uses, for example, depending on the type of gene construct or effector cell used and the method of administration. Commonly used excipients include, but are not limited to: saline, buffered saline, glucose, water for injection, glycerol, ethanol and combinations thereof, stabilizers, solubilizers and surfactants, buffers and preservatives, fortifying agents, fillers, and lubricants.

[0097] In another embodiment of the invention, the pharmaceutical preparation of the invention is administered to a patient. Exemplary administration methods include, but are not limited to, intravenous injection. Other methods include, but are not limited to, intratumoral, intradermal, subcutaneous (sc, sq, sub-Q, Hypo), intramuscular (im), intraperitoneal (ip), intraarticular, intramedullary, intracardiac, intra-articular (joint), intrasynovial (synovial fluid area), intracranial, intraspinal, and intrasheath (spinal fluid). Any known device for parenteral injection or infusion of this preparation may be used to achieve these administrations. As used herein, the term “treat, treating, treatment” has its usual and conventional meaning and includes one or more of the following meanings: blocking, improving, or reducing the severity and / or frequency of symptoms of a subject’s disease (e.g., tumor), and / or inhibiting the growth, division, spread, or proliferation of cancer cells in the subject, or cancer progression (e.g., the appearance of new tumors). Treatment means blocking, improving, reducing, or inhibiting by about 5% to about 100% compared to a subject who has not used the methods of the invention. Preferably, compared with subjects who did not use the method of the present invention, the inhibition, improvement, reduction or suppression is about 100%, 99%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10% or 5%.

[0098] The present invention also provides a kit comprising one or more containers containing a large number of gene constructs encoding the polypeptides of the present invention, and pharmaceutically acceptable excipients. The kit may also include instructions for use. Associated with the kit may be a notification in a format prescribed by a government agency regulating the production, use, or sale of pharmaceuticals or biological products, reflecting the approval of the manufacturing, using, or selling agency for human use. Example

[0099] Screening antigen-binding peptides using phage display technology

[0100] Indirect Envelope :refer to Figure 1PDL1-binding single-stranded variable fragments (scFvs) were identified using standard phage display technology. A human-derived original scFv library was generated via PCR-based reconstruction from B cells of 50 healthy donors. The hPDL1-Fc fusion protein and irrelevant Fc fusion proteins were indirectly immobilized onto immunotubes coated with anti-human IgG Fc antibodies, followed by solid-phase immunopanning. To select strong binders, irrelevant Fc fusion proteins were first used to deplete Fc-binding scFvs, and then unbound phages that could bind to the hPDL1-Fc fusion protein were selected. The eluted phages were amplified in bacteria. These processes were repeated 3-4 rounds, with phage titers and complexity determined after the second round. Once the phages were sequentially enriched (in rounds 3 and 4), the binding ability of individual phage clones to hPDL1 was assessed using an ELISA assay.

[0101] Direct wrapping : Directly coat the Fc protein onto the immunotube without using anti-human Fc antibodies ( Figure 2 ).

[0102] Phage binding ELISAs:

[0103] ELISAs were performed using the same strategy as panning. For clones selected indirectly, plates were first coated with anti-human Fc antibody, followed by the Fc protein. For clones selected directly, plates were directly coated with the Fc protein. In indirect ELISA assays, the binding affinity of phages to hPDL1-Fc and to irrelevant Fc proteins (or hIgG1) was assessed in parallel assays. Phages showing low binding affinity to irrelevant Fc proteins and high binding affinity to hPDL1 were selected for further sequencing and secondary screening. Data are as follows: Figure 3 As shown. Most clones exhibited low nonspecific binding (less than 0.2 against the Fc protein signal value (1:10 dilution) background). The binding affinity of phages to hPDL1-Fc, mPDL1-Fc (mouse PDL1), and hIgG1 was assessed in parallel assays using a direct ELISA approach. Phage studies showed that none of the lead molecules of this invention significantly bound to mouse PDL1, i.e., no lead molecule exhibited significant cross-reactivity with mouse PDL1. Data are as follows. Figure 7 As shown.

[0104] sequencing

[0105] Preliminary sequencing of the heavy chain CDR3 region identified unique clones. These will be confirmed later with the complete sequence. A small subset of clones share the same CDR3 region but exhibit significant differences in other parts of their sequences.

[0106] Secondary screening using FACS

[0107] Bacteriophages, phage lysates, or lysates of bacteria expressing scFvs were tested to determine their ability to preferentially bind to 293T cells expressing hPDL1 rather than parental 293T cells. The mean fluorescence intensity (MFI) ratio was used to identify positive clones. Data are as follows: Figure 4 As shown, most clones exhibited high ratios and could be identified as positive clones.

[0108] Identification of blocking agents

[0109] The ability of test phages, phage lysates, or lysates of bacteria expressing scFvs to block the interaction between hPD1 and hPDL1 was assessed. Binding assays were established by coating test plates with hPD1-Fc or hPDL1-Fc. Binding of biotin-labeled ligands (hPDL1 or hPD1) was detected using a standard method with streptavidin-HRP. The disappearance of binding in the presence of scFvs was used to identify potential blockers. Results are as follows: Figure 5 and 6 As shown.

[0110] Generation and characterization of Fc fusion proteins:

[0111] Because scFvs are relatively unstable, some scFvs were converted into Fc fusions and expressed in mammalian cells. These fusion proteins were purified using a protein A column, and their ability to block PD1-PDL1 interactions and their ability to bind to 293T cells expressing PDL1 were tested.

[0112] Generation of full-length antibodies:

[0113] Using standard methods familiar to those skilled in the art, the VH and VL regions of a single scFv clone were amplified by PCR to construct a full-length antibody gene, which was then cloned into a suitable expression vector. The full-length antibody protein was generated by transient transfection of suspension-cultured 293T cells using standard methods familiar to those skilled in the art, and purified using a protein A column.

[0114] Characterization of full-length antibodies

[0115] SDS-PAGE and volume exclusion chromatography (results as follows) Figure 8A , 8B Characterization of exemplary full-length antibodies (as shown in 9A, 9B, 10A, and 10B) and quantification of their potency: (a) determination of their specific binding to hPDL1 by ELISA (results are shown in 9A, 9B, 10A, and 10B). Figure 11B and 11C(a) As shown in the figure; (b) Its specific binding to 293T cells expressing hPDL1 and untreated 293T cells (results are shown in the figure). Figure 12A and 12B (a) and (c) block the PD1-PDL1 interaction in two blocking assays. Results data for the exemplary lead antibody candidates in Forms 1 and 2 show that... Figure 13B and 14B The results of 27 antibody implementation schemes of this invention are shown in [the data]. Figure 15 middle.

[0116] The affinity of PD-L1 interaction was determined using BIAcore:

[0117] The affinity of the lead antibody candidate for PD-L1 was detected using BIAcore. Figure 16B-16D In short, biotinylated hPDL1 is captured onto the sensor chip surface via streptavidin. The antibody flows through the chip, and reaction parameters are calculated using a single-cycle kinetic method based on the stability of the interaction. KD values ​​are evaluated using BIAcoreX100 evaluation software 2.0 with a bivalent analyte binding model.

[0118] PD-L1 binding in rhesus monkeys was measured by FACS.

[0119] (A) Rhesus monkey PDL1-GFP expression construct was transiently transfected into 293T cells. Implementation schemes 4-1E8, 3-1E4, and 3-1B11 were tested and compared with the control group. Results are as follows: Figures 18A-18D As shown: all three antibodies bind to PDL1 in rhesus monkeys.

[0120] (B) Rhesus monkey PDL1 expression construct was transiently transfected into 293T cells. Implementation schemes 4-1E8, 3-1E4, and 3-1B11 were tested and compared with the control group. Results are as follows: Figures 19A-19D As shown: All three antibody implementation schemes bind to PDL1 in rhesus monkeys.

[0121] IL2 induction and EC50 assay

[0122] Peripheral blood mononuclear cells (PBMCs) were isolated from human blood using a Ficoll gradient, followed by erythrocyte lysis using standard methods. The RPMI+ medium for this experiment was prepared as follows: 10% FBS, 1% anti-antibody (Gibco), and 1% non-essential amino acids (Gibco) were added to ATCC-modified RPMI medium (Gibco). After isolation from blood, the PBMCs were resuspended in 10-20 ml of RPMI+ medium. +The cells were cultured overnight at 37°C and 5% CO2. Next, PBMCs were seeded at a concentration of 100,000 PBMCs / 96 wells in 96-well tissue culture plates (Corning); the final volume per well was 200 μL. Staphylococcal enterotoxin B (SEB) was added at a concentration of 1 ng / ml, or a lead antibody was added at a concentration of 20 μg / ml (for selection) or at a concentration ranging from 50 μg / ml to 0.003 μg / ml. Control groups included cells without SEB (e.g., no stimulation), cells containing SEB alone, or cells containing SEB and isotype controls (e.g., baseline).

[0123] After incubation at 37°C and 5% CO2 for 76 hours, PBMCs were centrifuged at 1200 rpm (spun down) for 15 minutes at room temperature. The supernatant was collected and stored at -20°C. IL2 ELISA was performed using a commercially available IL2-ELISA kit (Biolegend or Thermofisher) according to the manufacturer's instructions. The supernatant was diluted 1 / 20–1 / 80 for ELISA. Absorbance was measured using a Spectramax3 M3 microplate reader (Molecular Devices), and data were analyzed using Graphpad software. The lead antibody candidate was compared with a commercially available anti-PD1 antibody. Results are as follows: Figure 20 As shown. In the co-culture assay of tumor cells and MDA-MB-231 cells (see...) Figures 23A-23C ), in de-inhibiting IL2 (see Figures 23A-23C ) aspects and desuppression of IFNγ (see Figure 24 In terms of PDL1 antibody, 4-1E8 consistently outperformed 3-1B11 and 3-1E4. However, in similar co-culture assays with T cells and MDA-MB-231 cells, all three antibodies were as good or better than commercially available PDL1 antibodies such as atezolizumab (Atezo) and durva (Durva).

[0124] ADCC activity

[0125] like Figure 21 As shown in Figure 22, all three lead antibodies exhibited strong ADCC activity, while atezolizumab (designed to be ADCC-negative) showed no activity. In the three embodiments of the invention, 4-1E8 exhibited the highest ADCC activity.

[0126] Mixed lymphocyte reaction

[0127] Peripheral blood mononuclear cells (PBMCs) were isolated from human blood using a Ficoll gradient, followed by erythrocyte lysis using standard methods. Cells were cultured in serum-free RPMI 1640 at 37°C for 1 hour. Non-adherent cells were removed, and the remaining mononuclear cells were cultured in RPMI 1640 supplemented with 5% human AB serum, 2 ng / ml GM-CSF, and 10 ng / ml IL4 (BD Biosciences). Fresh medium containing cytokine supplements was added every 2 to 3 days. On day 6, 20 ng / ml TNFα (BD Biosciences) was added to induce maturation of dendritic cells, and the cells were cultured for 24 hours.

[0128] Dendritic cells were collected, phenotypically analyzed, and frozen for later use. CD4 T cells were isolated from PBMCs using magnetic beads (Dynal) according to the manufacturer's instructions. CD4 T cells and allogeneic dendritic cells were co-cultured at a ratio of 1:2.5 in 96-well flat-bottom plates (Costar) using RPMI 1640 medium supplemented with 10% human AB serum. Dendritic cells were treated with 100 mg / ml mitomycin C (Sigma) before addition. T cells were diluted with CFSE (or a similar dye) to detect proliferation. IFNg release was measured using a commercially available IFNg-ELISA kit according to the manufacturer's instructions. Absorbance was measured using a Spectramax3 M3 microplate reader (Molecular Devices), and data were analyzed using GraphPad software. In these studies, the lead antibody candidate of the present invention performed comparably to other commercially available anti-PD1 and anti-PDL1 antibodies. Example results are shown below. Figure 25A and 25B As shown.

[0129] Combination specificity

[0130] Expi293 cell lines stably expressing multiple B7 family members and their receptors were generated. The ability of anti-PDL1 antibodies was detected by FACS using fluorescent anti-human IgG. Exemplary lead antibody candidate results are shown below. Figure 26A and 26B As shown.

[0131] Blocking CD80-PDL1 binding

[0132] DLD1 cells expressing PDL1 were designed and used to detect binding to biotin-labeled CD80-Fc cells, with or without anti-PDL1 antibodies, followed by the use of fluorescent streptavidin. Results data for exemplary lead antibody candidates of the present invention are as follows: Figure 27A and 27B As shown.

[0133] Half-life measurement

[0134] Serum half-life was measured using male homozygous Tg32 mice (B6.Cg-Fcgrttm1Dcr Tg(FCGRT)32Dcr / DcrJ, Jacksonlabs). 2 mg / kg antibody was administered intravenously on day 0, and blood was drawn at multiple time points on day 1 and thereafter. Plasma was prepared, and antibody titers were determined using a sandwich ELISA. Titers were normalized to day 1 titers. Anti-antibody reactions were also measured, and high-titer samples were removed from the analysis because they often exhibit abrupt changes in ELISA. Results data for exemplary lead antibody candidates of the present invention are as follows: Figure 28 As shown. The half-lives of different antibodies range from 6.9 days (3-1E4, see Example 9 below for detailed sequence) to 10.5 days (3-1B11, see Example 11 below for detailed sequence) and 12.3 days (4-1E8, see Example 5 below for detailed sequence).

[0135] polypeptide sequence

[0136] Examples of the PD-L1 binding polypeptide sequence of the present invention are listed below:

[0137] Example 1: Antibody number: 4-1A2

[0138] VH

[0139] DNA (SEQ ID NO:1)

[0140] CAGGTTCAGCTGGTGCAGTCTGGGACTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGATACACCTTCACCAGTTATGATATCAACTGGGTGCGACAGGCCACTGGACAAGGGCTTGAGTGGATGGGATGGATCAACCCTAACAGTGGTGGCACAAACT ATGCACAGAAGTTTCAGGGCAGGGTCACCATGACCACAGACACTTCTACGGGCACAGCCTACATGGAGCTGAGGAGCCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGATTTTTATGGGGTTCGGGGAGTTATGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

[0141] Amino acid (SEQ ID NO:2)

[0142] QVQLVQSGTEVKKPGASVKVSCKASGYTFTSYDINWVRQATGQGLEWMGWINPNSGGTNYAQKFQGRVTMTTDTSTGTAYMELRSLRSDDTAVYYCARFLWGSGSYDYWGQGTLVTVSS

[0143] VL

[0144] DNA (SEQ ID NO:3)

[0145] GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTCGCAACTTACTACTGTCAACAGACTTACACATTCCCGCACACTTTTGCCCAGGGGACCAACCTGGAGATCAAA

[0146] Amino acid (SEQ ID NO:4)

[0147] DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYTFPHTFAQGTNLEIK

[0148] Example 2: Antibody number: 4-1A12

[0149] VH

[0150] DNA (SEQ ID NO:5)

[0151] CAAGTCCAGCTGGTACAATCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTACCAGCTATGGTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAGCGCTTACAATGGTAACACAAACTATGCACAGAAGCTCCAGGGCAGAGTCACCATGACCACAGACACATCCACGAGCACAGCCTACATGGAGCTGAGGAGCCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGAGATTGGATACAGCTATGGTTACCCCTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

[0152] Amino acid (SEQ ID NO:6)

[0153] QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDWIQLWLPLDYWGQGTLVTVSS

[0154] VL

[0155] DNA (SEQ ID NO:7)

[0156] GACATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAACAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGGTGCATCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTCACAGTTCCCCCCTCACTTTCGGCGGAGGGACCAAGGTGGACATCAAA

[0157] Amino acid (SEQ ID NO:8)

[0158] DIQLTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYGASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSHSSPLTFGGGTKVDIK

[0159] Example 3: Antibody number: 4-1B9

[0160] VH

[0161] DNA (SEQ ID NO:9)

[0162] GAAGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAGTAATAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAAAGATTTGATCCCGTTGCGAGATAGTAGGGGGGGGTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGGGAGT

[0163] Amino acid (SEQ ID NO:10)

[0164] EVQLVQSGGGLVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDLIPLRDSRGGYYYGMDVWGQGTTVTVSS

[0165] VL

[0166] DNA (SEQ ID NO:11)

[0167] TCTTCTGAGCTGACTCAGGACCCTGCTGTGTCTGTGGCCTTGGGACAGACAGTCAGGATCACATGCCAAGGAGACAGCCTCAGAGACTATTATGCAAGCTGGTACCAGCAGAAGCCAGGACAGGCCCCTGTACTTGTCATCTATGGTAAAAACAACCGGCCCTCAGGAATCCCAGACCGATTCTCTGGCTCCAGCTCAGGAAACACAGCTTCCTTGACCATCACTGGGACTCAGGCGGAAGATGAGGCTGACTATTACTGTAACTCCCGTGACAGCGGTGCTTACCATTATGTCTTCGGAACTGGGACCAAGGTCACCGTCCTA

[0168] Amino acids (SEQ ID NO:12)

[0169] SSELTQDPAVSVALGQTVRITCQGDSLRDYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGTQAEDEADYYCNSRDSGAYHYVFGTGTKVTVL

[0170] Example 4: Antibody number: 4-1B12

[0171] VH

[0172] DNA (SEQ ID NO:13)

[0173] CAAATCCAGCTGGTGCAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAGTAATAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAAAGGAAGTATTATAGGGGATGGTGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCA

[0174] Amino acid (SEQ ID NO:14)

[0175] QIQLVQSGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGSIIGDGAFDIWGQGTMVTVSS

[0176] VL

[0177] DNA (SEQ ID NO:15)

[0178] GATATTGTGATGACCCAGTCTCCACTCTCCCTGCCCGTCACCCTTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGACCCTCCTGCATAATGGATTCAACTTTTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAACTCCTGATGTATTTGGCCTCTAGCCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCGGGCACAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGGTACACACTGGCCGTACACTTTTGGCCAGGGGACCAAGCTGGATATCAAA

[0179] Amino acid (SEQ ID NO:16)

[0180] DIVMTQSPLSLPVTLGEPASISCRSSQTLLHNGFNFLDWYLQKPGQSPQLLMYLASSRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGTHWPYTFGQGTKLDIK

[0181] Example 5: Antibody number: 4-1E8 (E8)

[0182] VH

[0183] DNA (SEQ ID NO:17)

[0184] CAAATCCAGCTGGTACAATCTGGGGCTGAGGTGAAGATGCCTGGGGCCTCAGTGACGATTTCCTGCGAGGCGTCTGGATACAACTTCATCAGCTACTATATACACTGGGTGCGACAGGCCCCTGGACAAGGCCTTGAGTGGATGGGATTCGTCGTCCCTAGTGGTGGTGCCGCAGGCTACACACAGAAGTTCCAGGGCAGACTCACCGTGACCAGGGACACGTCCACGAGCACAGTCTACATGGACCTGAACAGCCTGACATCTGACGACACGGCCGTGTATTACTGTGTGCGAGAAATGAGTGGTGGCTGGTTTGATTTCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCG

[0185] Amino acid (SEQ ID NO:18)

[0186] QIQLVQSGAEVKMPGASVTISCEASGYNFISYYIHWVRQAPGQGLEWMGFVVPSGGAAGYTQKFQGRLTVTRDTSTSTVYMDLNSLTSDDTAVYYCVREMSGGWFDFWGQGTLVTVSS

[0187] VL

[0188] DNA (SEQ ID NO:19)

[0189] GACATCGTGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGGCATTAGAAATGATTTAGGCTGGTATCAGCAAAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCACTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTGTCAGCAATATTATAGTACTCCTCTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAA

[0190] Amino acid (SEQ ID NO:20)

[0191] DIVMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPLTFGPGTKVDIK

[0192] Example 6: Antibody No.: 4-1G7

[0193] VH

[0194] DNA (SEQ ID NO:21)

[0195] GAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTACCAGCTATGGTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAGCGCTTACAATGGTAACACAAACTATGCACAGAAGCTCCAGGGCAGAGTCACCATGACCACAGACACATCCACGAGCACAGCCTACATGGAGCTGAGGAGCCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGAGCCTCACCGGTACAGCAGCCCATATGGTGGGCGGAGTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

[0196] Amino acid (SEQ ID NO:22)

[0197] EVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARASPVQQPIWWAEYWGQGTLVTVSS

[0198] VL

[0199] DNA (SEQ ID NO:23)

[0200] CAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTCCTGCACTGGAACCAGCAGTGACGTTGGTGGTTATAACTATGTCTCCTGGTACCAACAGCACCCAGGCAAAGCCCCCAAACTCATGATTTCTGATGTCAGTAAGCGGCCCTCAGGGGTTTCTAATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACCATCTCTGGGCTCCAGGCTGAGGACGAGGCTGATTATTACTGCAGCTCATATACAAGCAACTACACTTTGGTATTCGGCGGAGGGACCAAGCTGACCGTCCTA

[0201] Amino acids (SEQ ID NO:24)

[0202] QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMISDVSKRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSNYTLVFGGGTKLTVL

[0203] Example 7: Antibody No.: 4-1H10

[0204] VH

[0205] DNA (SEQ ID NO:25)

[0206] CAGCTGCAGCTACAGCAGTCCGGAGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTCCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAAGGATCATCCCTATCCTTGGTATAGCAAACTACGCACAGAAGTTCCAGGGCAGAGTCACGATTACCGCGGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAAGACACGGCCGTGTATTACTGTGCGAGTCATGGTCGGGCAGCAGCTGGTAGGTACGCTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA

[0207] Amino acid (SEQ ID NO:26)

[0208] QLQLQQSGAEVKKPGSSVKVSCKAPGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCASHGRAAAGRYAMDVWGQGTTVTVSS

[0209] VL

[0210] DNA (SEQ ID NO:27)

[0211] AATTTTATGCTGACTCAGCCCCACTCTGTGTCGGATTCTCCGGGGAAGACGGTAACCATCTCCTGCACCCGCAGCAGTGGCAGCATTGCCAGCAACTATGTGCAGTGGTACCAGCAGCGCCCGGGCAGTGCCCCCACCACTGTGATCTATGACGATAAGCAAAGACCCTCTGGGGTCCCTGATCGGTTCTCGGGCTCCATCGACAGCTCCTCCAACTCTGCCTCCCTCACCATCTCTGGACTGACGACTGAGGACGAGGCTGACTACTACTGTCAGTCCTTTGATGGCAGCAGTGTCATCTTCGGCGGAGGGACCAAGCTGACCGTCCTG

[0212] Amino acid (SEQ ID NO:28)

[0213] NFMLTQPHSVSDSPGKTVTISCTRSSGSIASNYVQWYQQRPGSAPTTVIYDDKQRPSGVPDRFSGSIDSSSNSASLTISGLTTEDEADYYCQSFDGSSVIFGGGTKLTVL

[0214] Example 8: Antibody number: 3-1H2

[0215] VH

[0216] DNA (SEQ ID NO:29)

[0217] CAGGTTCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGAGTCACGATTACCGCGGACGAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAAAGGAGCGTTTCTATGATAGTAGTGGTTATTACGATGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCA

[0218] Amino acid (SEQ ID NO:30)

[0219] QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARKERFYDSSGYYDAFDIWGQGTMVTVSS

[0220] VL

[0221] DNA (SEQ ID NO:31)

[0222] CAGTCTGCCCTGACTCAGCCTCGCTCAGTGTCCGGGTCTCCTGGGCAGTCAGTCACCATCTCCTGCACTGGAACCAGCAATGATGTTGGTGGTTATAACTATGTCTCCTGGTACCAACAGCACCCAGGCAAAGCCCCCAAACTCATGATTTATGATGTCACTAAGCGGCCCTCAGGGGTCCCTGATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACCGTCTCTGGCCTCCAGCCTGAGGATGAGGCTGACTATTATTGCGCCTCTTATGGAGGCAGGAACAATTTGCTTTTTGGCGGAGGGACTCAACTGACCGTCTTA

[0223] Amino acids (SEQ ID NO:32)

[0224] QSALTQPRSVSGSPGQSVTISCTGTSNDVGGYNYVSWYQQHPGKAPKLMIYDVTKRPSGVPDRFSGSKSGNTASLTVSGLQPEDEADYYCASYGGRNNLLFGGGTQLTVL

[0225] Example 9: Antibody number: 3-1E4

[0226] VH

[0227] DNA (SEQ ID NO:33)

[0228] CAAATCCAGCTGGTACAATCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGAGTCACGATTACCGCGGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCCGGAGGGGGAGCAGTGGCGGACAATAGTTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

[0229] Amino acid (SEQ ID NO:34)

[0230] QIQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAGGGAVADNSYWGQGTLVTVSS

[0231] VL

[0232] DNA (SEQ ID NO:35)

[0233] GACATCCGGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGGCATTAGAAATGATTTAGGCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTACAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGCACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCTACAAGATTACAATTACCCTCGAACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA

[0234] Amino acid (SEQ ID NO:36)

[0235] DIRMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQDYNYPRTFGQGTKVEIK

[0236] Example 10: Antibody No.: 3-1A8

[0237] VH

[0238] DNA (SEQ ID NO:37)

[0239] CAAATCCAGCTGGTACAATCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGAGTCACGATTACCGCGGACGAATCCACGAGCACGGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGACGGTTCGTATAGCAGCAGCTGGTACTCGTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

[0240] Amino acid (SEQ ID NO:38)

[0241] QIQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDGSYSSSWYSFDYWGQGTLVTVSS

[0242] VL

[0243] DNA (SEQ ID NO:39)

[0244] CAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTCCTGCACTGGAACCAGCAGTGACGTCGGTGGTTATAACTATGTCTCCTGGTACCAACAGCACCCAGGCAAAGCCCCCAAACTCATGATTTATGATGTCAGTAATCGGCCCTCAGGGGTTTCTAATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACCATCTCTGGGCTCCAGGCTGAGGACGAGGCTGATTATTACTGCTCCTCATATGCAGGTGATATTAGTTATGTACTGTTCGGCGGCGGGACCAAGCTGACCGTCCTA

[0245] Amino acid (SEQ ID NO:40)

[0246] QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYAGDISYVLFGGGTKLTVL

[0247] Example 11: Antibody number: 3-1B11 (B11)

[0248] VH

[0249] DNA (SEQ ID NO:41)

[0250] GAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACTTTTAGTGACTATGACATGATCTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAGTAATAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAAAGAGTTCTTTGGTGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCA

[0251] Amino acid (SEQ ID NO:42)

[0252] EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYDMIWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKEFFGAFDIWGQGTMVTVSS

[0253] VL

[0254] DNA (SEQ ID NO:43)

[0255] TCTTCTGAGCTGACTCAGGACCCTGCTGTGTCGGTGGCCTTGGGACAGACAGTCACGATCACATGCCAAGGAGACAGCCTCAATTACTATTATGCAAACTGGTTCCAGCTGAAGCCAGGGCAGGCCCCTGTACTTGTCCTCTTTGGTAAAAACAACCGGCCCTCAGGGATCCCAGACCGATTCTCTGGCTCCTACTCGGGAAGCACAGCTTCCTTGACCATCACTGGGGCTCAGGCGGAAGATGACGCTGACTATTACTGTAATTCGCGGGACAGCGGTGGTAATCCTTGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTA

[0256] Amino acids (SEQ ID NO:44)

[0257] SSELTQDPAVSVALGQTVTITCQGDSLNYYYANWFQLKPGQAPVLVLFGKNNRPSGIPDRFSGSYSGSTASLTITGAQAEDDADYYCNSRDSGGNPWVFGGGTKLTVL

[0258] Example 12: Antibody No.: 4-1F3

[0259] VH

[0260] DNA (SEQ ID NO:45)

[0261] CAAATCCAGCTGGTACAATCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAAGGATCATCCCTATCCTTGGTATAGCAGACTACGCACAGAAGTTCCAGGGCAGAGTCACGATTACCGCGGACAAATCCACGAGCACAGCCTACATGGAACTGAGTAGCCTGGGATCTGAGGACACGGCCGTGTATTTTTGTGCGAGAGAGGGGGGATCCTTTAGGCACTTTGACTTCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

[0262] Amino acid (SEQ ID NO:46)

[0263] QIQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIADYAQKFQGRVTITADKSTSTAYMELSSLGSEDTAVYFCAREGGSFRHFDFWGQGTLVTVSS

[0264] VL

[0265] DNA (SEQ ID NO:47)

[0266] CAGCCTGTGCTGACTCAGCCACCCTCAGTCTCTGGGGCCCCAGGGCAGAGGGTCACCATCTCCTGCGCTGGGAGCGACCCCAACATCGGGACAGGTCATGATGTGCACTGGTACCAGCAACTTCCAGGAACAGCCCCCAAACTCGTCATCTATGGTAACACCAATCGGCCCTCAGGGGTCCCTGAGCGATTCACTGCCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCACTGGGCTCCAGGCTGAGGATGAGGCTGATTATTACTGCCAGGCCTACGACAGGAGCCTGCGTGGTTATGTCTTCGGGACTGGGACCAAGGTCACCGTCCTG

[0267] Amino acid (SEQ ID NO:48)

[0268] QPVLTQPPSVSGAPGQRVTISCAGSDPNIGTGHDVHWYQQLPGTAPKLVIYGNTNRPSGVPERFTASKSGTSASLAITGLQAEDEADYYCQAYDRSLRGYVFGTGTKVTVL

[0269] Example 13: Antibody No.: 4-1G5

[0270] VH

[0271] DNA (SEQ ID NO:49)

[0272] CAAATCCAGCTGGTACAGTCTGGTGCTGAAGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGACTTCTGGTTACACCTTTACCAGCTATGGTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAGCGCTTACAATGGTAACACAAACTATGCACAGAAGCTCCAGGGCAGAGTCACCATGACCACAGACACATCCACGAGCACAGCCTACATGGAGCTGAGGAGCCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGAACTACAGGTGACGAGTGGCTACGATTGGCTATAAATGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

[0273] Amino acid (SEQ ID NO:50)

[0274] QIQLVQSGAEVKKPGASVKVSCKTSGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARTTGDEWLRLAINDYWGQGTLVTVSS

[0275] VL

[0276] DNA (SEQ ID NO:51)

[0277] GATATTGTGATGACACAGTCTCCCCTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCTGCGCCTCATGCATCCTAATGGACTCAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTAATCTTTTTGGGTTCTCAGCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGCATTTATTACTGCATGCAAGCTCTAGAACCTCCGTACACTTTTGGCCAGGGGACCAAGCTGGAGATCAAA

[0278] Amino acid (SEQ ID NO:52)

[0279] DIVMTQSPLSLPVTPGEPASISCRSSLRLMHPNGLNYLDWYLQKPGQSPQLLIFLGSQRASGVPDRFSGSGSGTDFTLKISRVEAEDVGIYYCMQALEPPYTFGQGTKLEIK

[0280] Example 14: Antibody number: 4-1C9

[0281] VH

[0282] DNA (SEQ ID NO:53)

[0283] CAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGAGTCACGATTACCGCGGACGAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGATCCCGGGTATAGCAGTGGCTGGAAAGATGATGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCA

[0284] Amino acid (SEQ ID NO:54)

[0285] QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDPGYSSGWKDDAFDIWGQGTMVTVSS

[0286] VL

[0287] DNA (SEQ ID NO:55)

[0288] GAAATTGTGATGACACAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGATACAGCCTCCCTCTCCTGCAGGGCCAGTCAGACTGTTAGCAGCAACTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATACATCCAACAGGGCCGCTGGCATCCCGGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGTAGCCTAGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTACGGTAGCTCACTCTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA

[0289] Amino acids (SEQ ID NO:56)

[0290] EIVMTQSPGTLSLSPGDTASLSCRASQTVSSNYLAWYQQKPGQAPRLLIYDTSNRAAGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQYGSSLWTFGQGTKVEIK

[0291] Example 15: Antibody No.: 11 - A4

[0292] VH

[0293] DNA (SEQ ID NO:57)

[0294] CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGAGTCACGATTACCGCGGACGAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGCGGGGCAGCAGCTGGTAGCCCTTTGGTACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

[0295] Amino acid (SEQ ID NO:58)

[0296] QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARAGQQLVALWYYWGQGTLVTVSS

[0297] VL

[0298] DNA (SEQ ID NO:59)

[0299] CAGTCTGCCCTGACTCAGCCTCCCTCCGCGTCCGGGTCTCGTGGACAGTCAGTCTCCATCTCCTGCAGTGGAAGTCGCAGTGACATTGGATATTATAACTATGTCTCCTGGTATCAACAACACCCAGGCAAAGCCCCCAAACTCATCATTTTTGACGTCAATAAGCGGCCCTCAGGGGTCCCTGATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACCGTCTCTGGCCTCCAGCCTGAGGATGAGGCTGACTATTATTGCGCCTCTTATGGAGGCAGGAACAATTTGCTTTTTGGCGGAGGGACTCAACTGACCGTCTTA

[0300] Amino acid (SEQ ID NO:60)

[0301] QSALTQPPSASGSRGQSVSISCSGSRSDIGYYNYVSWYQQHPGKAPKLIIFDVNKRPSGVPDRFSGSKSGNTASLTVSGLQPEDEADYYCASYGGRNNLLFGGGTQLTVL

[0302] Example 16: Antibody No.: 21 - A1

[0303] VH

[0304] DNA (SEQ ID NO:61)

[0305] CAGGTGCAACTGCAGGAGTCGGGCCCAGGACTGGTGGAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGTAGTTTCTACTGGAGCTGGATCCGGCAGCCCCCAGGGAAGGGACTGGAGTGGATTGGCTATATCAATTACAGTGGGAGCACCAACTACAACCCCTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTACTGTGCGAGACAGATATTATGGTTCGGGGAGTTAAGGTGGTTCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

[0306] Amino acid (SEQ ID NO:62)

[0307] QVQLQESGPGLVEPSETLSLTCTVSGGSISSFYWSWIRQPPGKGLEWIGYINYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARQILWFGELRWFDPWGQGTLVTVSS

[0308] VL

[0309] DNA (SEQ ID NO:63)

[0310] CAGTCTGCCCTGACTCAGCCTCCCTCCGCGTCCGGGTCTCCTGGACAGTCAGTCACCATCTCCTGCACTGGAACCAGCAGTGACATTGGTGGTTATAACTATGTCTCCTGGTACCAACTGCGCCCAGGCAAAGCCCCCAAACTCATGATTTATGACGTCACCAAGCGGCCCTCAGGGGTCCCTGATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACCGTCTCTGGGCTCCAGGCTGAGGATGAGGCTGATTATTACTGCAGCTCATATGCAGGCAGCAACAATGTGGTATTCGGCGGAGGGACCAAGCTGACCGTCCTA

[0311] Amino acid (SEQ ID NO: 64)

[0312] QSALTQPPSASGSPGQSVTISCTGTSSDIGGYNYVSWYQLRPGKAPKLMIYDVTKRPSGVPDRFSGSKSGNTASLTVSGLQAEDEADYYCSSYAGSNNVVFGGGTKLTVL

[0313] Example 17: Antibody No.: 21-H12

[0314] VH

[0315] DNA (SEQ ID NO: 65)

[0316] CAAGTCCAGCTGGTACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGAGTCACGATTACCGCGGACGAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAAATCCCTACGGTTTCAACTGGTTCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

[0317] Amino acid (SEQ ID NO: 66)

[0318] QVQLVQSGAEVKKPGASVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARNPYGFNWFDPWGQGTLVTVSS

[0319] VL

[0320] DNA (SEQ ID NO: 67)

[0321] AATTTTATGCTGACTCAGCCCCACTCTGTGTCGGAGTCTCCGGGGAAGACGGTAACCATCTCCTGCACCCGCAGCAGTGGCAGCATTGCCAGCAACTATGTGCAGTGGTACCAGCAGCGCCCGGGCAGTGCCCCCACCACTGTGATCTATGAGGATAACCAAAGACCCTCTGGGGTCCCTGATCGGTTCTCTGGCTCCATCGACAGCTCCTCCAACTCTGCCTCCCTCACCATCTCCGGACTGAAGACTGAGGACGAGGCTGACTACTACTGTCAGTCTTATGATGGCTTCAATCAGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTA

[0322] Amino acids (SEQ ID NO:68)

[0323] NFMLTQPHSVSESPGKTVTISCTRSSGSIASNYVQWYQQRPGSAPTTVIYEDNQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDGFNQVFGGGTKLTVL

[0324] Example 18: Antibody number: 7-D12

[0325] VH

[0326] DNA (SEQ ID NO:69)

[0327] CAAATGCAGCTGGTACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGAGTCACGATTACCGCGGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAACCGGTAGTAGTGGTTATGTACGTTGGAGCAACTGGTTCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

[0328] Amino acid (SEQ ID NO:70)

[0329] QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARTGSSGYVRWSNWFDPWGQGTLVTVSS

[0330] VL

[0331] DNA (SEQ ID NO:71)

[0332] GACATCCAGATGACCCAGTCTCCCTCCACCCTGTCTGCATTTGTAGGAGACAGAGTCACCATCACTTGCCGGGCCAGTGAGAGTATTAGTAGGTGGTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAACTCCTAATCTCTAAGACGTCTAATTTAGAAAGCGGGGTCCCGTCAAGGTTCAGTGGCGCTGGATCTGGGACAGATTTCACTCTCACCATTAGCAGTCTGCAACCTGAGGATTTTGCAACTTACTTCTGTCAACAGGGTTCCAAAATGCCTCCGACTTTCGGCGGAGGGACCAAGGTGGAGATCAAG

[0333] Amino acid (SEQ ID NO:72)

[0334] DIQMTQSPSTLSAFVGDRVTITCRASESISRWLAWYQQKPGKAPKLLISKTSNLESGVPSRFSGAGSGTDFTLTISSLQPEDFATYFCQQGSKMPPTFGGGTKVEIK

[0335] Example 19: Antibody number: 9-E3

[0336] VH

[0337] DNA (SEQ ID NO:73)

[0338] CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGAGTCACGATTACCGCGGACGAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGGGGCCTACGGTGGTAACTCCGCTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

[0339] Amino acid (SEQ ID NO:74)

[0340] QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGAYGGNSAFDYWGQGTLVTVSS

[0341] VL

[0342] DNA (SEQ ID NO:75)

[0343] CAGTCTGTGCTGACGCAGCCGCCCTCAGTGTCTGGGGCCCCAGGGCAGAGGGTCACCATCTCCTGCACTGGGAGCAGCTCCAACATCGGGGCAGGTTATGATGTACACTGGTACCAGCAGCTTCCAGGAACAGCCCCCAAACTCCTCATGTACAGTAATGATCAGCGGCCCTCAGGGGTCACTGAGCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCAGTCTGAAGATGAGGGTGATTACTACTGCCAGTCCTATGACAGAAGCCTGAGAGGTTCGGTCTTCGGCGGAGGGACCAAGCTGACCGTCCTC

[0344] Amino acids (SEQ ID NO:76)

[0345] QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLMYSNDQRPSGVTERFSGSKSGTSASLAISGLQSEDEGDYYCQSYDRSLRGSVFGGGTKLTVL

[0346] Example 20: Antibody No.: 10 - A6

[0347] VH

[0348] DNA (SEQ ID NO:77)

[0349] GAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTTTCCTGCAAGGCTTCTGGTTACACCTTTACCAGCTATGGTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAGCGCTTACAATGGTAACACAAACTATGCACAGAAGCTCCAGGGCAGAGTCACCATGACCACAGACACATCCACGAGCACAGCCTACATGGAGCTGAGGAGCCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGAGATTCCATAGCAGCAGCTGGTACTCCGTTCGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

[0350] Amino acids (SEQ ID NO:78)

[0351] EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDSIAAAGTPFDYWGQGTLVTVSS

[0352] VL

[0353] DNA (SEQ ID NO:79)

[0354] AATTTTATGCTGACTCAGCCCCACTCTGTGTCGGAGTCTCCGGGGAAGACGGTCACCATCTCCTGCACCCGCAGCAGTGGCATCATTGCCAGCAAATATGTGCACTGGTACCAGCAGCGCCCGGGCAGTGCCCCCACCACTGTGATCTATGAGGATAACCAAAGACCGTCTGGGGTCCCTGATCGATTCTCTGGCTCCATCGACAACTCCTCCAACTCTGCCTCCCTCACCATCTCTGGACTGCAGACTGAGGACGAGGCTGACTACTACTGTCAGTCTCATGACGGCATCAATCAGGTTTTCGGCGGAGGGACCAAGGTCACCGTCCTA

[0355] Amino acid (SEQ ID NO:80)

[0356] NFMLTQPHSVSESPGKTVTISCTRSSGIIASKYVHWYQQRPGSAPTTVIYEDNQRPSGVPDRFSGSIDNSSNSASLTISGLQTEDEADYYCQSHDGINQVFGGGTKVTVL

[0357] Example 21: Antibody number: 12 - A4

[0358] VH

[0359] DNA (SEQ ID NO:81)

[0360] GAGGTGCAGCTGGTGGAGTCCCGGGGAGGCTTGGTACAGCCGGGGGGGTCCCTGAGACTCTCCTGTGTAACTTCTGGATTCAGCTTTAACAACTATGCCATGAACTGGGTCCGCCAGGCTCCGGGGAAGGGGCTGGAGTGGGTCTCAGCTGTTAGTGGTAGTGGTGGTACCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTTTGTGCAGATGGACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAAAGGACTTTTCCCTACGATTTTTGGAGTAGGAGCAATGTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

[0361] Amino acid (SEQ ID NO:82)

[0362] EVQLVESRGGLVQPGGSLRLSCVTSGFSFNNYAMNWVRQAPGKGLEWVSAVSGSGGTTYYADSVKGRFTISRDNSKNTLFVQMDSLRAEDTAVYYCAKGLFPTIFGVGAMFDYWGQGTLVTVSS

[0363] VL

[0364] DNA (SEQ ID NO:83)

[0365] TCTTCTGAGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTTGTTCTGGAAGCAGCTCCAACATCGGAAGTAATGCTGTTAACTGGTATCAGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATGATAATAATCACCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCAGTCTGAGGATGAGGCTGATTATTATTGTGCAGCATGGGATGACACCATTCCTGGTGTGCTATTCGCCGGAGGGACCAAGCTGACCGTCCTA

[0366] Amino acids (SEQ ID NO:84)

[0367] SSELTQPPSASGTPGQRVTISCSGSSSNIGSNAVNWYQQLPGTAPKLLIYDNNHRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDDTIPGVLFAGGTKLTVL

[0368] Example 22: Antibody No.: 14 - G10

[0369] VH

[0370] DNA (SEQ ID NO:85)

[0371] GAAGTGCAGCTGGTGGAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGAGTCACGATTACCGCGGACGAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGGTGTTTCTTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA

[0372] Amino acid (SEQ ID NO:86)

[0373] EVQLVESGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGVSYYYGMDVWGQGTTVTVSS

[0374] VL

[0375] DNA (SEQ ID NO:87)

[0376] CAGGCTGTGCTGACTCAGCCACCCTCGGTGTCCGTGTCCCCAGGACAGACAGCCATCATCTCCTGTTCTGGACATAAATTGGGTGATAAGTATGTTTCCTGGTATCAACAGCAGCCAGGCCAGTCCCCTGTGCTGGTCCTCTTTCAGGATACCAAGCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAACTCTGGGAACACAGCCACTCTGACCATCAGCGCGACCCAGGCTGCGGATGAGGCTGACTATTACTGTCAGGCGGGGGACACCAAGTCTGTGATCTTCGGCGGCGGGACCAAGCTGACCGTCCTA

[0377] Amino acid (SEQ ID NO:88)

[0378] QAVLTQPPSVSVSPGQTAIISCSGHKLGDKYVSWYQQQPGQSPVLVLFQDTKRPSGIPERFSGSNSGNTATLTISATQAADEADYYCQAGDTKSVIFGGGTKLTVL

[0379] Example 23: Antibody number: 22 - A6

[0380] VH

[0381] DNA (SEQ ID NO:89)

[0382] CAGGTTCAGGTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGGCAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGAGTCACGATTACCGCGGACGAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGGATACAGCTATGGTTCAGGACACCTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

[0383] Amino acid (SEQ ID NO:90)

[0384] QVQVVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGYSYGSGHLDYWGQGTLVTVSS

[0385] VL

[0386] DNA (SEQ ID NO:91)

[0387] GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGGCGAGTCAGGACATTAGCAACTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTACGATGCATCCAATTTGGAAACAGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCGCTCTCACCATCAGCAGTCTCCAACCTGAAGATTTTGCAACTTATTACTGTCTACAGCATAATAGTTACCCTCGGACTTTTGGCCAGGGGACCAAGCTGGAGATCAAA

[0388] Amino acid (SEQ ID NO:92)

[0389] DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFALTISSLQPEDFATYYCLQHNSYPRTFGQGTKLEIK

[0390] Example 24: Antibody No.: 35 - B1

[0391] VH

[0392] DNA (SEQ ID NO:93)

[0393] GAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGATACACCTTCACCGGCTACTATATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATGAACCCTAACAGTGGTGACACAGCCTATACACAGAACTTCCAGGGCAGAGTCACCATGACCAGGAACCCCTCCATAAGCACAGCCTACATGGAGCTGAGCAACCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGGCCGGGGGTTCGCGGAGAAGCCCCTTGGGTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

[0394] Amino acid (SEQ ID NO:94)

[0395] EVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWMNPNSGDTAYTQNFQGRVTMTRNPSISTAYMELSNLRSEDTAVYYCARGRGFAEKPLGYWGQGTLVTVSS

[0396] VL

[0397] DNA (SEQ ID NO:95)

[0398] GATATTGTGATGACTCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGGGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTATTTTATCCAGCTCCAATAATAAGAACTATTTAGCTTGGTACCAGCAGAAACCAGGTCAGCCTCCTAAGCTGCTCATTTACTGGGCATCTACCCGGGAATCCGGGGTCCCTGACCGGTTCAGCGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTGTCAGCAATATTATAGTACTCCTCCGACATTCGGCCAAGGGACCAAGGTGGAAATCAAA

[0399] Amino acid (SEQ ID NO:96)

[0400] DIVMTQSPDSLAVSLGGRATINCKSSQSILSSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPPTFGQGTKVEIK

[0401] Example 25: Antibody number: 3-1F4

[0402] VH

[0403] DNA (SEQ ID NO:97)

[0404] GAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGAGTCACGATTACCGCGGACGAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGGGCCCCTCGAGGGCAGTGGCTGGTTCACTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

[0405] Amino acid (SEQ ID NO:98)

[0406] EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARAPRGQWLVHYFDYWGQGTLVTVSS

[0407] VL

[0408] DNA (SEQ ID NO:99)

[0409] GAAATTGTGTTGACGCAGTCTCCAGCCACCCTCTCTCTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCTGGGCCAGTCAGGATGTTAGCAACTACTTAGCCTGGTACCAACAGAAGCCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGCGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAACGTAGCAACTGGCCTCTCACTTTCGGCGGCGGGACCAAGGTGGAGCTCAAA

[0410] Amino acid (SEQ ID NO:100)

[0411] EIVLTQSPATLSLSPGERATLSCWASQDVSNYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGGGTKVELK

[0412] Example 26: Antibody No.: 4-1B3

[0413] VH

[0414] DNA (SEQ ID NO:101)

[0415] CAGGTTCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTACCAGCTATGGTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAGCGCTTACAATGGTAACACAAACTATGCACAGAAGCTCCAGGGCAGAGTCACCATGACCACAGACACATCCACGAGCACAGCCTACATGGAGCTGAGGAGCCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGAGAGTCCTACTCGTCCGCAGGTATTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

[0416] Amino acid (SEQ ID NO:102)

[0417] QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARESYSSAGIDYWGQGTLVTVSS

[0418] VL

[0419] DNA (SEQ ID NO:103)

[0420] GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGACCCTCCTGCATAGTAATGGATTCAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAACTCCTGATGTATTTGGGCTCTAGCCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCGGGCACAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAACTCTACAAACTCCTCCGGCTTTCGGCGGAGGGACCAAGGTGGAGATCAAA

[0421] Amino acids (SEQ ID NO: 104)

[0422] DIVMTQSPLSLPVTPGEPASISCRSSQTLLHSNGFNYLDWYLQKPGQSPQLLMYLGSSRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQTLQTPPAFGGGTKVEIK

[0423] Example 27: Antibody No.: 21 - G1

[0424] VH

[0425] DNA (SEQ ID NO: 105)

[0426] CAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGACGATTTCCTGCGAGGCGTCTGGATACAACTTCATCAGCTACTATATACACTGGGTGCGACAGGCCCCTGGACAAGGCCTTGAGTGGATGGGATTCGTCGTCCCTAGTGGTGGTGCCGCAGGCTACACACAGAAGTTCCAGGGCAGACTCACCGTGACCAGGGACACGTCCACGAGCACAGTCTACATGGACCTGAACAGCCTGACATCTGACGACACGGCCGTGTATTACTGTGTGCGAGAAATGAGTGGTGGCTGGTTTGATTTCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCG

[0427] Amino acid (SEQ ID NO:106)

[0428] QVQLVQSGAEVKKPGASVTISCEASGYNFISYYIHWVRQAPGQGLEWMGFVVPSGGAAGYTQKFQGRLTVTRDTSTSTVYMDLNSLTSDDTAVYYCVREMSGGWFDFWGQGTLVTVSS

[0429] VL

[0430] DNA (SEQ ID NO:107)

[0431] GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA

[0432] Amino acid (SEQ ID NO:108)

[0433] DIQMTQSPSSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPITFGQGTRLEIK

[0434] It will be apparent to those skilled in the art that various modifications and variations can be made to this invention without departing from its scope or spirit. Other embodiments of the invention will be apparent to those skilled in the art upon reference to the specification and practice of this disclosure. We intend that the specification and embodiments be considered merely illustrative, and that the following claims will set forth the true scope and spirit of the invention.

[0435] Throughout this application, references have been made to various publications, patents, and / or patent applications in order to provide a more comprehensive description of the prior art relating to this invention. The publications of these publications, patents, and / or patent applications are incorporated herein by reference in their entirety, just as each individual publication, patent, and / or patent application is specifically and individually indicated as incorporated by reference.

Claims

1. An antigen-binding polypeptide that binds to a human PD-L1 epitope, comprising a heavy chain variable region and a light chain variable region, wherein the sequence of the heavy chain variable region is composed of SEQ ID NO: 42, and the sequence of the light chain variable region is composed of SEQ ID NO:

44.

2. The antigen-binding polypeptide according to claim 1, wherein the antigen-binding polypeptide is a fully human antibody or a humanized antibody.

3. The antigen-binding polypeptide of claim 2, wherein the antigen-binding polypeptide further comprises a human constant region, wherein the human constant region has ADCC and / or CDC activity.

4. A nucleic acid molecule encoding the antigen-binding polypeptide of claim 1, wherein the nucleic acid molecule is a DNA molecule or an RNA molecule.

5. A nucleic acid molecule encoding the antigen-binding polypeptide of claim 1, wherein the nucleic acid molecule is a DNA molecule, wherein the nucleic acid molecule comprises a DNA sequence encoding a heavy chain variable region of the antigen-binding polypeptide and a DNA sequence encoding a light chain variable region of the antigen-binding polypeptide, wherein the DNA sequence encoding the heavy chain variable region of the antigen-binding polypeptide consists of a sequence having at least 80% identical to SEQ ID NO:41, and the DNA sequence encoding the light chain variable region of the antigen-binding polypeptide consists of a sequence having at least 80% identical to SEQ ID NO:

43.

6. An antibody that binds to a human PD-L1 epitope, comprising the same heavy chain variable region and light chain variable region as those described in claim 1.

7. A pharmaceutical composition comprising an antigen-binding polypeptide according to any one of claims 1-3 or an antibody according to claim 6, and a pharmaceutically acceptable excipient, carrier, or diluent.

8. Use of the antigen-binding polypeptide of any one of claims 1-3 or the antibody of claim 6 in the preparation of a medicament for treating cancer in a subject in need, wherein the cancer is selected from the group consisting of: ovarian cancer, colon cancer, breast cancer, lung cancer, neuroblastoma of the central nervous system, mast cell tumor, melanoma, bladder cancer, gastric cancer, liver cancer, urothelial carcinoma, skin cancer, kidney cancer, head and neck cancer, pancreatic cancer, and combinations thereof.

9. Use of the pharmaceutical composition of claim 7 in the preparation of a medicament for treating cancer in a subject in need, said treatment comprising... Administer a therapeutically effective amount of the pharmaceutical composition of claim 7 to the subject; or The subject is given a therapeutically effective amount of the pharmaceutical composition of claim 7, in combination with (a) an antibody targeting other immunosuppressive pathways; (b) chemotherapy or radiotherapy; (c) other mechanisms of blocking immunosuppressive pathways; or (d) other immunotherapeutic agents. The cancers mentioned therein are selected from the group consisting of: ovarian cancer, colon cancer, breast cancer, lung cancer, neuroblastoma of the central nervous system, mast cell tumor, melanoma, bladder cancer, stomach cancer, liver cancer, urothelial carcinoma, skin cancer, kidney cancer, head and neck cancer, pancreatic cancer, and combinations thereof.

10. The use according to any one of claims 8-9, wherein the cancer has at least some tumor cells expressing a detectable amount of PD-L1.

11. A mammalian expression system for producing the antigen-binding polypeptide according to any one of claims 1-3.

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