Humanized anti-PD-L1 antibody

Abstract

The invention provides isolated binding molecules that bind to and block PD-L1, vectors containing nucleic acid molecules encoding the amino acid sequences of the binding molecules, host cells containing the vectors, methods of making the binding molecules, pharmaceutical compositions containing the binding molecules, and methods of using such antibodies, antibody fragments and derivatives and polypeptides, including methods of treating diseases that require the stimulation of an immune response, such as cancer.

Description

Related Applications

[0001] This application claims priority to U.S. Provisional Application No. 62 / 835,764, filed April 18, 2019, the entire disclosure of which is incorporated herein by reference. (Incorporation of Sequence Listing) This application contains a Sequence Listing, which has been submitted via EFS-Web in ASCII format, is entitled "QLSF003PCT_ST25.txt," is 158 KB, and was created on April 17, 2020. The contents of this Sequence Listing are hereby incorporated by reference in their entirety. BACKGROUND OF THE INVENTION

[0002] Programmed cell death protein 1 (PD-1) is a 288-amino acid cell surface protein molecule encoded by the PDCD1 gene in humans. PD-1 is a type I transmembrane protein containing an immunoglobulin variable type amino-terminal extracellular domain, a transmembrane domain, and a cytoplasmic tail containing a tyrosine-based inhibitory motif and an immunoreceptor tyrosine-based switch motif (Ishida et al. 1992). PD-1 is expressed on pro-B cells and activated T cells but not on resting T cells in vivo. PD-1 acts as a checkpoint protein on immune cells. It negatively regulates the immune system by binding to its ligand, PD-L1, and suppressing effective T cell function.

[0003] PD-1 / PD-L1 interaction plays an important role in autoimmunity and cancer immunity. PD-1 expression has been observed in various cancers, including melanoma and non-small cell lung cancer. Many studies suggest that cancer cells overexpress PD-L1, which helps them evade immune attack by utilizing the PD-1 / PD-ligand (PDL) pathway. Based on the conclusions of these studies, several checkpoint blockade inhibitors against the PD-1 / PDL pathway have been developed.

[0004] PD-1 / PD-L1 blockade has achieved significant clinical success in fighting cancer. Several immunotherapies, such as Pembrolizumab (Keytruda) and Nivolumab (Opdivo) targeting PD-1 and Atezolizumab (Tecentriq), Avelumab (Bavencio), and Durvalumab (Imfinzi) targeting PD-L1, are effective in blocking the binding between PD-1 and PD-L1, thereby reversing T cell dysfunction. However, in many advanced cancers, the objective response rate for monotherapy is only 20% (Xu-Monette et al. 2017).

[0005] As a result, improving therapeutic agents that target PD-1 and PD-L1 is an area of intense interest. Also, many other immunotherapies are currently being tested alone and in combination in clinical trials. Thus, there remains a need for more effective immunotherapies that target the PD-1 / PD-L1 pathway, used alone or in combination with other therapeutic agents in drug treatment regimens. Summary of the Invention

[0006] The present invention provides isolated monoclonal anti-PD-L1 antagonist antibodies that specifically bind to human PD-L1 and their antigen-binding site sites.

[0007] In one embodiment of the present invention, the isolated monoclonal anti-PD-L1 antagonist antibody, or antigen-binding portion thereof, comprises a heavy chain variable region CDR3 consisting of SEQ ID NO: 43. In some embodiments, the monoclonal anti-PD-L1 antagonist antibody, or antigen-binding portion thereof, further comprises a heavy chain variable region CDR1 consisting of SEQ ID NO: 31 and a heavy chain variable region CDR2 consisting of SEQ ID NO: 37. In a preferred embodiment, the monoclonal anti-PD-L1 antagonist antibody, or antigen-binding portion thereof, further comprises (a) a light chain variable region CDR1 consisting of SEQ ID NO: 13, (b) a light chain variable region CDR2 consisting of SEQ ID NO: 19, and (c) a light chain variable region CDR3 consisting of SEQ ID NO: 25.

[0008] In one embodiment, the antibody or portion thereof consists of a light chain variable region amino acid sequence that is at least 95% identical to SEQ ID NO: 1 and a heavy chain variable region amino acid sequence that is at least 95% identical to SEQ ID NO: 7. In another embodiment, the antibody or portion thereof consists of a heavy chain variable region amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 7.

[0009] In another aspect of the present invention, the isolated monoclonal anti-PD-1 antagonist antibody, or antigen-binding portion thereof, comprises a heavy chain variable region CDR3 consisting of SEQ ID NO: 44. In some embodiments, the monoclonal anti-PD-L1 antagonist antibody, or antigen-binding portion thereof, further comprises a heavy chain variable region CDR1 consisting of SEQ ID NO: 32 and a heavy chain variable region CDR2 consisting of SEQ ID NO: 38. In a preferred embodiment, the monoclonal anti-PD-L1 antagonist antibody, or antigen-binding portion thereof, further comprises (a) a light chain variable region CDR1 consisting of SEQ ID NO: 14, (b) a light chain variable region CDR2 consisting of SEQ ID NO: 20, and (c) a light chain variable region CDR3 consisting of SEQ ID NO: 26.

[0010] In one embodiment, the antibody or portion thereof comprises a light chain variable region amino acid sequence at least 95% identical to SEQ ID NO: 2 and a heavy chain variable region amino acid sequence at least 95% identical to SEQ ID NO: 8. In another embodiment, the antibody or portion thereof comprises a heavy chain variable region amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 8.

[0011] In another aspect of the present invention, the isolated monoclonal anti-PD-1 antagonist antibody, or antigen-binding portion thereof, comprises a heavy chain variable region CDR3 consisting of SEQ ID NO: 45. In some embodiments, the monoclonal anti-PD-L1 antagonist antibody, or antigen-binding portion thereof, further comprises a heavy chain variable region CDR1 consisting of SEQ ID NO: 33, and a heavy chain variable region CDR2 consisting of SEQ ID NO: 39. In a preferred embodiment, the monoclonal anti-PD-L1 antagonist antibody, or antigen-binding portion thereof, further comprises (a) a light chain variable region CDR1 consisting of SEQ ID NO: 15, (b) a light chain variable region CDR2 consisting of SEQ ID NO: 21, and (c) a light chain variable region CDR3 consisting of SEQ ID NO: 27.

[0012] In one embodiment, the antibody or portion thereof consists of a light chain variable region amino acid sequence that is at least 95% identical to SEQ ID NO: 3 and a heavy chain variable region amino acid sequence that is at least 95% identical to SEQ ID NO: 9. In another embodiment, the antibody or portion thereof consists of a heavy chain variable region amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 9.

[0013] In another aspect of the present invention, the isolated monoclonal anti-PD-1 antagonist antibody, or antigen-binding portion thereof, comprises a heavy chain variable region CDR3 consisting of SEQ ID NO: 46. In some embodiments, the monoclonal anti-PD-L1 antagonist antibody, or antigen-binding portion thereof, further comprises a heavy chain variable region CDR1 consisting of SEQ ID NO: 34, and a heavy chain variable region CDR2 consisting of SEQ ID NO: 40. In a preferred embodiment, the monoclonal anti-PD-L1 antagonist antibody, or antigen-binding portion thereof, further comprises (a) a light chain variable region CDR1 consisting of SEQ ID NO: 16, (b) a light chain variable region CDR2 consisting of SEQ ID NO: 22, and (c) a light chain variable region CDR3 consisting of SEQ ID NO: 28.

[0014] In one embodiment, the antibody or portion thereof consists of a light chain variable region amino acid sequence that is at least 95% identical to SEQ ID NO: 4 and a heavy chain variable region amino acid sequence that is at least 95% identical to SEQ ID NO: 10. In another embodiment, the antibody or portion thereof consists of a heavy chain variable region amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 10.

[0015] In another aspect of the present invention, the isolated monoclonal anti-PD-L1 antagonist antibody, or antigen-binding portion thereof, comprises a heavy chain variable region CDR3 consisting of SEQ ID NO: 47. In some embodiments, the monoclonal anti-PD-L1 antagonist antibody, or antigen-binding portion thereof, further comprises a heavy chain variable region CDR1 consisting of SEQ ID NO: 35, and a heavy chain variable region CDR2 consisting of SEQ ID NO: 41. In a preferred embodiment, the monoclonal anti-PD-L1 antagonist antibody, or antigen-binding portion thereof, further comprises (a) a light chain variable region CDR1 consisting of SEQ ID NO: 17, (b) a light chain variable region CDR2 consisting of SEQ ID NO: 23, and (c) a light chain variable region CDR3 consisting of SEQ ID NO: 29.

[0016] In one embodiment, the antibody or portion thereof consists of a light chain variable region amino acid sequence that is at least 95% identical to SEQ ID NO: 5 and a heavy chain variable region amino acid sequence that is at least 95% identical to SEQ ID NO: 11. In another embodiment, the antibody or portion thereof consists of a heavy chain variable region amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 11.

[0017] In another aspect of the present invention, the isolated monoclonal anti-PD-L1 antagonist antibody, or antigen-binding portion thereof, comprises a heavy chain variable region CDR3 consisting of SEQ ID NO: 48. In some embodiments, the monoclonal anti-PD-L1 antagonist antibody, or antigen-binding portion thereof, further comprises a heavy chain variable region CDR1 consisting of SEQ ID NO: 36, and a heavy chain variable region CDR2 consisting of SEQ ID NO: 42. In a preferred embodiment, the monoclonal anti-PD-L1 antagonist antibody, or antigen-binding portion thereof, further comprises (a) a light chain variable region CDR1 consisting of SEQ ID NO: 18, (b) a light chain variable region CDR2 consisting of SEQ ID NO: 24, and (c) a light chain variable region CDR3 consisting of SEQ ID NO: 30.

[0018] In one embodiment, the antibody or portion thereof consists of a light chain variable region amino acid sequence that is at least 95% identical to SEQ ID NO: 6 and a heavy chain variable region amino acid sequence that is at least 95% identical to SEQ ID NO: 12. In another embodiment, the antibody or portion thereof consists of a heavy chain variable region amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 12.

[0019] In another embodiment of the invention, the isolated monoclonal anti-PD-L1 antagonist antibody, or antigen-binding portion thereof, comprises a heavy chain variable region consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 65-67, and a light chain variable region consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 49-51.

[0020] In another embodiment of the invention, the isolated monoclonal anti-PD-L1 antagonist antibody, or antigen-binding portion thereof, comprises a heavy chain variable region consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 68-70, and a light chain variable region consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 55-58.

[0021] In another embodiment of the invention, the isolated monoclonal anti-PD-L1 antagonist antibody, or antigen-binding portion thereof, comprises a heavy chain variable region consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 71-76, and a light chain variable region consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 59-61.

[0022] In another embodiment of the present invention, the isolated monoclonal anti-PD-L1 antagonist antibody, or antigen-binding portion thereof, comprises a heavy chain variable region consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 77-78, and a light chain variable region consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 62-63.

[0023] In another embodiment of the invention, the isolated monoclonal anti-PD-L1 antagonist antibody, or antigen-binding portion thereof, comprises a heavy chain variable region consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 79-80, and a light chain variable region consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 64.

[0024] In another embodiment of the invention, the isolated monoclonal anti-PD-L1 antagonist antibody, or antigen-binding portion thereof, comprises a heavy chain variable region consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 81-83, and a light chain variable region consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 52-54.

[0025] In another embodiment of the present invention, the isolated monoclonal antibody, or antigen-binding portion thereof, comprises a light chain variable region consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 84-99, and a heavy chain variable region consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 100-118.

[0026] The disclosed antibodies of the present invention can be further modified to make them suitable for human therapy by minimizing immunogenicity. Preferred antibodies include, but are not limited to, chimeric and humanized antibodies. The affinity, stability, and specificity of the disclosed antibodies can also be further optimized using techniques known to those skilled in the art. Other formats include oligomerization, drug conjugation, and fusion of the disclosed antibodies with other functional proteins.

[0027] The disclosed antibodies of the present invention can be, for example, full-length antibodies, e.g., of the IgG1, IgG2, IgG3, or IgG4 isotype. Alternatively, the disclosed antibodies can be antibody fragments, e.g., Fab, Fab', and F(ab')2 fragments, diabodies, triabodies, tetrabodies, single-chain variable region fragments (scFv), disulfide-stabilized variable region fragments (dsFv), and half antibodies. Alternatively, the disclosed antibodies can be bispecific antibodies.

[0028] In another embodiment of the invention, the antibody or antigen-binding fragment thereof is -8 M to 1 x 10 -10 M, or 1.32 x 10 -9M to 2.68 x 10 -10 It has affinity for PD-L1 ranging from M to M. In some embodiments, the anti-PD-L1 antagonist antibody, or antigen-binding portion thereof, binds to and blocks human PD-L1. Thus, the antibody, or antigen-binding portion thereof, can stimulate an anti-tumor immune response. In some embodiments, the anti-PD-L1 antagonist antibody, or antigen-binding portion thereof, binds to and blocks non-human primate PD-L1.

[0029] In another aspect, the invention provides a composition comprising an isolated anti-PD-L1 antagonist monoclonal antibody, or an antigen-binding portion thereof. In another aspect, the invention provides pharmaceutical compositions comprising an isolated anti-PD-L1 antagonist monoclonal antibody, or an antigen-binding portion thereof, and a pharmaceutically acceptable carrier. Also provided are compositions comprising an immunoconjugate of the invention and a pharmaceutically acceptable carrier. In further aspects of the invention, there are also provided vectors comprising the isolated nucleic acid molecules encoding antibodies, or antigen-binding portions thereof, and host cells comprising expression vectors comprising the nucleic acid molecules.

[0030] The invention further provides methods for stimulating an immune response using the anti-PD-L1 antagonist antibodies of the disclosed invention. For example, in one embodiment, the disclosed invention provides a method of treatment in a subject in need thereof, comprising administering to the subject an effective amount of an antibody or antigen-binding portion of the disclosed invention. In another aspect, the disclosed invention provides a method of treating cancer in a human, comprising the step of administering to a human an anti-PD-L1 antagonist antibody or antigen-binding portion of the disclosed invention in an amount effective to treat the cancer.

[0031] In another aspect, the disclosed invention provides a method of treating an infectious disease in a human, comprising administering to the human an effective amount of an anti-PD-L1 antagonist antibody or antigen-binding portion of the disclosed invention to treat the infectious disease. Other features and advantages of the present invention will be apparent from the following detailed description and examples, which should not be construed as limiting. The contents of all references, GenBank entries, patents and published patent applications cited throughout this application are expressly incorporated by reference herein. [Brief explanation of the drawings]

[0032] Exemplary embodiments are illustrated in the referenced figures. It is intended that the embodiments and figures disclosed herein be considered illustrative and not limiting. Figure 1 shows that anti-PD-L1 lead candidates bind to PD-L1 on A431 cells. Figure 2 shows that anti-PD-L lead candidates block PD-1 interactions on HEK293 / huPD-L1 cells. Figure 3 shows that anti-PD-L1 antibody enhanced SEB-stimulated IL-2 release. Figure 4 shows PD-L1 antibody-enhanced IFN-gamma secretion in a mixed lymphocyte reaction assay, demonstrating that the lead anti-PD-L1 antibody blocked PD-1-mediated suppression. Figure 5 shows that PD-L1 blockade with the lead antibody enhanced interferon gamma production by T cells in a CMV peptide-specific recall response assay. Figure 6 shows the thermal stability of the humanized anti-PD-L1 lead antibody.

[0033] The following embodiments and aspects thereof are described and illustrated in conjunction with systems, compositions, and methods that are intended to be exemplary and illustrative, not limiting in scope.

[0034] (definition) As used herein, the terms "comprising" or "comprises" are used in reference to compositions, methods, and their respective components useful for the embodiments, but also include unspecified elements, whether useful or not. Those skilled in the art will generally understand that the terms used herein are generally intended to be "open-ended" terms (e.g., "including" should be interpreted as "including, but not limited to," "having" should be interpreted as "having at least," "includes" should be interpreted as "including, but not limited to," etc.).

[0035] Unless otherwise indicated, the terms "a," "an," "the," and similar words when used in the context of describing particular embodiments of this application (particularly in the context of the claims) may be construed to refer to the singular and the plural. The recitation of ranges of values herein is merely intended to serve as a shorthand method for stating each separate value falling within the range individually. Unless otherwise indicated herein, each value is incorporated herein as if each were individually set forth herein. All methods described herein can be performed in any suitable order unless otherwise indicated or otherwise clearly contradicted by context. Any example or exemplary language used herein with respect to certain embodiments (e.g., "such as") is intended solely to better describe the application and does not limit the scope of any otherwise claimed application. The abbreviation "eg" is derived from the Latin exempli gratia and is used herein to indicate a non-limiting example. Accordingly, the abbreviation "eg" is synonymous with the term "for example." No language in the specification should be construed as indicating any non-claimed element essential to the practice of the application.

[0036] As used herein, the term "about" refers to a measurable value, such as an amount, a time course, etc., and includes variations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or ±0.1% from the specified value.

[0037] As used herein, the term "epitope" includes any protein determinant capable of specific binding to an immunoglobulin or T-cell receptor. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics. An antibody is said to specifically bind an antigen when the equilibrium dissociation constant is ≦1 μM, preferably ≦100 nM, and most preferably ≦10 nM. "K D The term "antigen-antibody" refers to the equilibrium dissociation constant of a particular antigen-antibody interaction. The term "immune response," as used herein, can refer to the action of, for example, lymphocytes, antigen-presenting cells, phagocytes, granulocytes, and soluble macromolecules (e.g., antibodies, cytokines, and complement) produced by such cells or hepatocytes, which selectively damage, destroy, and result in the elimination from the body of invading pathogens, pathogen-infected cells or tissues, cancer cells, or, in the case of autoimmunity or pathological inflammation, normal biological cells or tissues.

[0038] As used herein, an "antigen-specific T cell response" refers to a response by a T cell that results from stimulating the T cell with an antigen for which the T cell is specific. Non-limiting examples of responses that result from antigen-specific stimulation of T cells include proliferation and cytokine production (e.g., IL-2 production).

[0039] As used herein, the term "antibody" refers to an intact immunoglobulin or a monoclonal or polyclonal antigen-binding fragment having the Fc (fragment crystallizable) region or an FcRn-binding fragment of the Fc region (referred to herein as an "Fc fragment" or "Fc region"). Antigen-binding fragments may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Antigen-binding fragments include, inter alia, Fab, Fab', F(ab'), Fv, dAb, and complementarity-determining region (CDR) fragments, single-chain antibodies (scFv), single-domain antibodies, chimeric antibodies, bispecific antibodies (diabodies), and polypeptides containing at least a sufficient portion of an immunoglobulin to confer antigen-binding properties to the polypeptide. The Fc region includes portions of the two heavy chains that contribute to two or three classes of antibodies. Fc regions may be produced by recombinant DNA techniques or by enzymatic (e.g., papain cleavage) or chemical cleavage of intact antibodies.

[0040] As used herein, the term "antibody fragment" refers to a protein fragment consisting of only a portion of an intact antibody, which generally includes the antigen-binding site of the intact antibody and thus retains antigen-binding ability. Examples of antibody fragments included in the current definition include: (i) a Fab fragment comprising the VL, CL, VH, and CH1 regions; (ii) a Fab' fragment, which is a Fab fragment having one or more cysteine residues at the C-terminus of the CH1 region; (iii) an Fd fragment comprising the VH and CH1 regions; (iv) an Fd' fragment comprising the VH and CH1 regions and one or more cysteine residues at the C-terminus of the CH1 region; (v) an Fv fragment comprising the VL and VH of a single arm of an antibody; (vi) a dAb fragment comprising the VH region (Ward et al., Nature 341, 544-546 (1989)); (vii) an isolated CDR region; (viii) an F(ab')2 fragment, a bivalent fragment comprising two Fab' fragments linked by a disulfide bond at the hinge region; and (ix) a single-chain antibody molecule (e.g., single-chain Fv, scFv) (Bird et al., Science 242:423-426 (1988), and Huston et al., PNAS (USA) 85:5879-5883 (1988)); (x) "diabodies," which have two antigen-binding sites and are composed of a heavy-chain variable region (VH) associated with a light-chain variable region (VL) of the same polypeptide chain (see, for example, EP 404,097, WO 93 / 11161, and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and (xi) "linear antibodies," which have a pair of tandem Fd segments (VH-CH1-VH-CH1), which, together with complementary light-chain polypeptides, form a pair of antigen-binding sites (Zapata et al., Protein Eng. 8(10):1057-1062(1995), and U.S. Pat. No. 5,641,870).

[0041] As used herein, "single-chain variable fragment," "single-chain antibody variable fragment," or "scFv" antibody refers to a form of antibody composed of only the variable regions of the heavy and light chains connected by a linker peptide. scFvs can be expressed as single-chain polypeptides. scFvs retain the specificity of the intact antibody from which they are derived. The light and heavy chains can be in any order, for example, VH-linker-VL or VL-linker-VH, as long as the specificity of the scFv for its target antigen is maintained.

[0042] As used herein, an "isolated antibody" refers to an antibody that is substantially free of other antibodies with different antigenic specificities (e.g., an isolated antibody that specifically binds to the PD-L1 protein may be substantially free of antibodies that specifically bind to antigens other than the PD-L1 protein). However, an isolated antibody that specifically binds to the PD-L1 protein may cross-react with other antigens, such as PD-L1 proteins from other species. Moreover, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0043] Anti-PD-L1 antagonist antibody-producing cells are, for example, hybridomas, which can be selectively cloned and further screened for desirable properties, including robust proliferation, high antibody production, and desirable antibody characteristics. Hybridomas can be expanded in vivo in syngeneic animals, animals that lack an immune system, such as nude mice, or in cell culture under in vitro conditions. Methods for selecting, cloning, and expanding hybridomas are well known to those of skill in the art.

[0044] The terms "monoclonal antibody" or "monoclonal antibody composition" as used herein refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.

[0045] As used herein, the term "recombinant human antibody" refers to any human antibody prepared, expressed, created, or isolated by recombinant methods, including, for example, (a) antibodies isolated from animals (e.g., mice) that have been genetically or chromosomally modified for human immunoglobulin genes or hybridomas (described below) generated therefrom, (b) antibodies isolated from host cells transformed to express human antibodies, e.g., from transfectomas, (c) antibodies isolated from recombinant, combinatorial human antibody libraries, and (d) antibodies prepared, expressed, created, or isolated by other methods involving splicing human immunoglobulin gene sequences into other DNA sequences. Such recombinant human antibodies have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies are subject to in vitro mutation (or, when animals transgenic for human Ig sequences are used, in vivo somatic mutation) and therefore the amino acid sequences of the VH and VL regions of the recombinant antibodies, while derived from and related to human germline VH and VL sequences, are sequences that may not naturally occur within the human antibody germline repertoire in vivo.

[0046] The term "isotype" refers to the class of antibody (e.g., IgM or IgG1) that is encoded by the heavy chain constant region genes. An antibody may be an immunoglobulin G molecule (IgG), or may be derived from an IgM, IgE, IgA, or IgD molecule. The phrases "antibody that recognizes an antigen" and "antibody specific for an antigen" are used herein synonymously with the term "antibody that specifically binds to an antigen."

[0047] As used herein, an antibody that "specifically binds to human PD-L1" refers to an antibody that binds to the human PD-L1 protein (and possibly to PD-L1 proteins of one or more non-human animal species), but does not substantially bind to non-PD-L1 proteins. Preferably, the antibody binds to the human PD-L1 protein with "high affinity," i.e., K D 1×10 -7 M or less, preferably 5 × 10 -8 M or less, and more preferably 3 × 10 -8 M or less, and more preferably 1×10 -8 M or less, preferably 5 × 10 -9 M or less, and even more preferably 1×10 -9 Joins at M or less.

[0048] As used herein, the term "does not substantially bind" to a protein or cell means that the antibody is unable to bind to the protein or cell or does not bind with high affinity, i.e., does not bind to the protein or cell. D 2×10 -6 or more, preferably 1 × 10 -5 M or more, preferably 1 × 10 -4 M or more, preferably 1 × 10 -3 M or more, and even more preferably 1×10 -2 It can mean bonding at M or more.

[0049] The term "high affinity" in reference to IgG antibodies refers to the K D is 1×10 -6 M or less, preferably 1 × 10 -7 M or less, preferably 1×10 -8 M or less, and even more preferably 1×10 -9 M or less, and even more preferably 1×10 -10 It refers to antibodies with a binding affinity of less than or equal to M. However, "high affinity" binding may differ for other antibody isotypes.

[0050] The term "pharmaceutical formulation" refers to a formulation that is prepared in a form that allows the biological activity of the active ingredient contained therein to be effective, and does not contain other components that are unacceptably toxic to the subject to whom the formulation is administered. For example, a "therapeutically effective amount" of an agent, such as a pharmaceutical formulation or cells, refers to an amount effective, at an amount and for a period of time necessary, to achieve the desired therapeutic result for the treatment of a disease, condition, or disorder, and / or the pharmacokinetic or pharmacodynamic effects of the treatment. A therapeutically effective amount may vary depending on factors such as the disease state, age, sex, and weight of the subject, and the number of cells administered. In certain embodiments, provided methods are directed to the administration of an effective amount, e.g., a therapeutically effective amount, of cells and / or compositions.

[0051] As used herein, the term "antagonist antibody" refers to an antibody that blocks or inhibits a biological response by binding to and blocking a ligand (e.g., PD-L1) to which the antibody binds. An antagonist may, for example, bind to PD-L1 and block the binding of PD-L1 to PD-1, thereby inhibiting phosphorylation of the PD-1 receptor or inhibiting signaling / cell activation. In one embodiment, the antibody of the invention is an antagonist anti-PD-L1 antibody.

[0052] A "CDR-grafted antibody" is an antibody that is composed of one or more CDRs from an antibody of a particular species or isotype and a framework from another antibody of the same or another species or isotype. A "humanized antibody" has a sequence that differs from that of an antibody derived from a non-human species by one or more amino acid substitutions, deletions, and / or additions, such that when administered to a human subject, the humanized antibody elicits less and / or a milder immune response than the non-human species antibody. In one embodiment, certain amino acids in the framework 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. In another embodiment, a humanized antibody is a CDR-grafted antibody, which consists of one or more CDRs from an antibody of a particular species or isotype and a human antibody framework. In another embodiment, one or more amino acid residues in one or more CDRs of a non-human antibody are altered to reduce the immunogenicity of the non-human antibody when administered to a human subject, either because the altered amino acid residues are not important for immunospecific binding of the antibody to an antigen, or because the changes made to the amino acid sequence are so modest that they do not significantly impair the binding of the humanized antibody to the antigen compared to the binding of the non-human antibody to the antigen. Examples of methods for making humanized antibodies can be found in US Pat. Nos. 6,054,297, 5,886,152 and 5,877,293.

[0053] The term "chimeric antibody" refers to an antibody that contains one or more regions of one antibody and one or more regions of one or more other antibodies. In one embodiment, one or more CDRs are derived from a human anti-CD47 antibody. In another embodiment, all CDRs are derived from a human anti-CD47 antibody. In another embodiment, CDRs from one or more anti-human CD47 antibodies are combined in various ways in the chimeric antibody. For example, a chimeric antibody may be composed of CDR1 from the light chain of a first human anti-CD47 antibody, CDR2 and CDR3 from the light chain of a second human anti-CD47 antibody, and CDRs from the heavy chain of a third anti-CD47 antibody. Other combinations are also possible.

[0054] The term "subject" refers to a human or non-human animal. The subject can be male or female and of any appropriate age, including infants, juveniles, adolescents, adults, and geriatric subjects. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, cows, horses, chickens, rabbits, mice, rats, amphibians, and reptiles, although mammals are preferred, e.g., non-human primates, sheep, dogs, cats, cows, and horses.

[0055] The binding of the disclosed antibodies of the invention to PD-L1 can be assessed using one or more techniques well established in the art. For example, in a preferred embodiment, the antibodies can be tested in an ELISA assay, e.g., using recombinant PD-L1 protein. Yet other suitable binding assays include, but are not limited to, flow cytometry assays in which the antibodies are reacted with a cell line expressing human PD-L1, such as Expi293 or ExpiCHO cells transfected to express PD-L1 (e.g., human PD-L1) on the cell surface. Additionally, or alternatively, the binding kinetics (e.g., K D Antibody binding, including the antibody binding value, can be tested using BIAcore binding assays, Octet Red 96 (Pall), etc.

[0056] The disclosed antibodies of the present invention bind to the human PD-L1 protein and K D 5×10 -8 Binds to human PD-L1 protein at a molecular weight of less than M and K D 2×10 -8 Binds to human PD-L1 protein at a molecular weight of less than M and K D 5×10 -9 Binds to human PD-L1 protein at a molecular weight of less than M and K D 4×10 -9 Binds to human PD-L1 protein at a molecular weight of less than M and K D 3×10 -9 Binds to human PD-L1 protein at a molecular weight of less than M and K D 2×10-9 Binds to human PD-L1 protein at a molecular weight of less than M and K D 1×10 -9 It is desirable to bond at M or less.

[0057] The present invention relates to isolated monoclonal antibodies, or antigen-binding portions thereof, that bind to and block PD-L1 and uses thereof. In certain embodiments, the disclosed antibodies of the invention are derived from identified heavy and light chain germline sequences and / or comprise identified structural features, e.g., CDR regions comprised of identified amino acid sequences. The present invention provides isolated antibodies and methods for producing such antibodies and antigen-binding portions thereof. The present invention also relates to methods of using the antibodies, such as using the disclosed anti-PD-L1 antibodies of the present invention, alone or in combination with other immunostimulatory or therapeutic antibodies, to stimulate an immune response. Accordingly, methods of using the disclosed anti-PD-L1 antagonist antibodies of the present invention are also provided, including, but not limited to, treating human cancer. Various aspects of the present invention relate to antibodies and antibody fragments, pharmaceutical compositions, nucleic acids, recombinant expression vectors, and host cells for producing such antibodies and fragments. Also included in the present invention are methods of using the antibodies of the present invention to detect human PD-L1, inhibit PD-L1 activity in vitro or in vivo, and prevent or treat diseases such as cancer.

[0058] The complementarity-determining regions (CDRs) are known as hypervariable regions in both the light and heavy chain variable regions. The more highly conserved regions of the variable regions are called framework regions (FRs). The complementarity-determining regions (CDRs) and framework regions (FRs) of an antibody may be identified using the systems described by Kabat et al., supra, Lefranc et al., supra, and / or Honegger and Plückthun, supra. Also familiar to those skilled in the art is the numbering system described by Kabat et al. (1991, NIH Publication 91-3242, National Technical Information Service, Springfield, Va.). In this regard, Kabat et al. established a numbering system for variable region sequences that is applicable to any antibody. One skilled in the art can unambiguously assign this "Kabat numbering" system to any variable region amino acid sequence without relying on experimental data other than the sequence itself.

[0059] In certain embodiments, the invention provides anti-PD-L1 antagonist antibodies or antigen-binding portions thereof. In one embodiment, the mouse antibody or portion thereof comprises: (a) a light chain variable region CDR1 consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18; (b) a light chain variable region CDR2 consisting of SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24; (c) a light chain variable region CDR3 consisting of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, or SEQ ID NO: 30; (d) a heavy chain variable region CDR1 consisting of SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, or SEQ ID NO: 36; (e) a heavy chain variable region CDR2 consisting of SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, or SEQ ID NO: 42; and (f) a heavy chain variable region CDR3 consisting of SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, or SEQ ID NO: 48.

[0060] In one embodiment, the present invention provides a monoclonal antibody, or antigen-binding portion thereof, that binds to a PD-L1 epitope, comprising a light chain variable region amino acid sequence that is at least 95% identical to SEQ ID NO: 1, 49, 50, or 51, and a heavy chain variable region amino acid sequence that is at least 95% identical to SEQ ID NO: 7, 65, 66, or 67.

[0061] In another embodiment, the invention provides a monoclonal antibody, or antigen-binding portion thereof, that binds to a PD-L1 epitope, comprising a light chain variable region amino acid sequence that is at least 95% identical to SEQ ID NO: 2, 55, 56, 57, or 58, and a heavy chain variable region amino acid sequence that is at least 95% identical to SEQ ID NO: 8, 68, 69, or 70.

[0062] In yet another embodiment, the invention provides a monoclonal antibody, or antigen-binding portion thereof, that binds to a PD-L1 epitope, comprising a light chain variable region amino acid sequence that is at least 95% identical to SEQ ID NO: 3, 59, 60, or 61, and a heavy chain variable region amino acid sequence that is at least 95% identical to SEQ ID NO: 9, 71, 72, 73, 74, 75, or 76.

[0063] In yet another embodiment, the invention provides a monoclonal antibody, or antigen-binding portion thereof, that binds to a PD-L1 epitope, comprising a light chain variable region amino acid sequence that is at least 95% identical to SEQ ID NO: 4, 62, or 63, and a heavy chain variable region amino acid sequence that is at least 95% identical to SEQ ID NO: 10, 77, or 78.

[0064] In yet another embodiment, the invention provides a monoclonal antibody, or antigen-binding portion thereof, that binds to a PD-L1 epitope, comprising a light chain variable region amino acid sequence that is at least 95% identical to SEQ ID NO: 5 or 64, and a heavy chain variable region amino acid sequence that is at least 95% identical to SEQ ID NO: 11, 79, or 80.

[0065] In yet another embodiment, the invention provides a monoclonal antibody, or antigen-binding portion thereof, that binds to a PD-L1 epitope, comprising a light chain variable region amino acid sequence that is at least 95% identical to SEQ ID NO: 6, 52, 53, or 54, and a heavy chain variable region amino acid sequence that is at least 95% identical to SEQ ID NO: 12, 81, 82, or 83. Given that the Fabs of these antibodies can each bind to human PD-L1, the VH and VL sequences can be "mixed and matched" to create other anti-PD-L1 binding molecules of the invention. When VH and VL chains are mixed and matched, it may be desirable to replace a VH sequence from a particular VH / VL pairing with a structurally similar VH sequence. Similarly, it may be desirable to replace a VL sequence from a particular VH / VL pairing with a structurally similar VL sequence.

[0066] In some embodiments, the humanized anti-PD-L1 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 65-67, and a light chain variable region consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 49-51. Preferred heavy and light chain combinations include, but are not limited to, the following: (a) a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 65 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 49; (b) a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 66 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 50; (c) A heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 67 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 51.

[0067] In some embodiments, the humanized anti-PD-L1 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 68-70, and a light chain variable region consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 55-58. Preferred heavy and light chain combinations include, but are not limited to, the following: (a) a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 68 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 55; (b) a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 69 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 56; (c) A heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 70 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 57.

[0068] In some embodiments, the humanized anti-PD-L1 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 71-76, and a light chain variable region consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 59-61. Preferred heavy and light chain combinations include, but are not limited to, the following: (a) a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 71 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 59; (b) a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 72 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 60; (c) A heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 73 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 61.

[0069] In some embodiments, the humanized anti-PD-L1 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 77-78, and a light chain variable region consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 62-63. Preferred heavy and light chain combinations include, but are not limited to, the following: (a) a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 77 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 62; (b) A heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 78 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 63.

[0070] In some embodiments, the humanized anti-PD-L1 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 79-80, and a light chain variable region consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 64. Preferred heavy and light chain combinations include, but are not limited to, the following: (a) A heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 79 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 64.

[0071] In some embodiments, the humanized anti-PD-L1 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 81-83, and a light chain variable region consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 52-54. Preferred heavy and light chain combinations include, but are not limited to, the following: (a) a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 81 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 52; (b) a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 82 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 53; (c) A heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 83 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 54.

[0072] In some embodiments, the humanized anti-PD-L1 antagonist antibody, or antigen-binding portion thereof, comprises a light chain consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 84-99, and a heavy chain consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 100-118.

[0073] In one embodiment, the invention provides an anti-PD-L1 antibody or antigen-binding fragment thereof, comprising a heavy chain comprising a CDR3 region set forth in SEQ ID NO:43, and a heavy chain variable region consisting of an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in any one of SEQ ID NOs:7, or 65-67.

[0074] In one embodiment, the invention provides an anti-PD-L1 antibody or antigen-binding fragment thereof, comprising a heavy chain comprising a CDR3 region set forth in SEQ ID NO:44, and a heavy chain variable region consisting of an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in any one of SEQ ID NOs:8, or 68-70.

[0075] In one embodiment, the invention provides an anti-PD-L1 antibody or antigen-binding fragment thereof, comprising a heavy chain comprising a CDR3 region set forth in SEQ ID NO: 45, and a heavy chain variable region consisting of an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence set forth in any one of SEQ ID NOs: 9, or 71-76.

[0076] In one embodiment, the invention provides an anti-PD-L1 antibody or antigen-binding fragment thereof, comprising a heavy chain comprising a CDR3 region set forth in SEQ ID NO: 46, and a heavy chain variable region consisting of an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence set forth in any one of SEQ ID NOs: 10, or 77-78.

[0077] In one embodiment, the invention provides an anti-PD-L1 antibody or antigen-binding fragment thereof, comprising a heavy chain comprising a CDR3 region set forth in SEQ ID NO:47, and a heavy chain variable region consisting of an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence set forth in any one of SEQ ID NOs:11, or 79-80.

[0078] In one embodiment, the invention provides an anti-PD-L1 antibody or antigen-binding fragment thereof, comprising a heavy chain comprising a CDR3 region set forth in SEQ ID NO:48, and a heavy chain variable region consisting of an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence set forth in any one of SEQ ID NOs:12, or 81-83.

[0079] In one embodiment, the invention provides an anti-PD-L1 antibody or antigen-binding fragment thereof, comprising a light chain comprising a CDR3 region set forth in SEQ ID NO:25, and a light chain variable region having an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in any one of SEQ ID NOs:1, or 49-51.

[0080] In one embodiment, the invention provides an anti-PD-L1 antibody or antigen-binding fragment thereof, comprising a light chain comprising a CDR3 region set forth in SEQ ID NO:26, and a light chain variable region having an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in any one of SEQ ID NOs:2, or 55-58.

[0081] In one embodiment, the invention provides an anti-PD-L1 antibody or antigen-binding fragment thereof, comprising a light chain comprising the CDR3 region set forth in SEQ ID NO:27, and a light chain variable region having an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in any one of SEQ ID NOs:3, or 59-61.

[0082] In one embodiment, the invention provides an anti-PD-L1 antibody or antigen-binding fragment thereof, comprising a light chain comprising a CDR3 region set forth in SEQ ID NO:28, and a light chain variable region having an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in any one of SEQ ID NOs:4, or 62-63.

[0083] In one embodiment, the invention provides an anti-PD-L1 antibody or antigen-binding fragment thereof, comprising a light chain comprising a CDR3 region set forth in SEQ ID NO:29, and a light chain variable region having an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in any one of SEQ ID NOs:5 or 64.

[0084] In one embodiment, the invention provides an anti-PD-L1 antibody or antigen-binding fragment thereof, comprising a light chain comprising a CDR3 region set forth in SEQ ID NO: 30, and a light chain variable region having an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in any one of SEQ ID NOs: 6, or 52-54.

[0085] Thus, in certain embodiments, the CDR3 region may be held constant while variability is introduced into the remaining CDR and / or framework regions of the heavy and / or light chain, and the antibody or antigen-binding fragment thereof retains the ability to bind PD-L1 and retains the parent functional properties, such as binding affinity.

[0086] In one embodiment, substitutions made within heavy or light chains that are at least 95% homologous (or at least 96% homologous, or at least 97% homologous, or at least 98% homologous, or at least 99% homologous) are conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced with another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially alter the functional properties of the protein. When two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence homology or similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those skilled in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24: 307-331, incorporated herein by reference. Examples of groups of amino acids with side chains with similar chemical properties include: (1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine, (2) aliphatic-hydroxyl side chains: serine and threonine, (3) amide-containing side chains: asparagine and glutamine, (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan, (5) basic side chains: lysine, arginine, and histidine, (6) acidic side chains: aspartic acid and glutamic acid, and (7) sulfur-containing side chains: cysteine and methionine. In addition to chemically conserved amino acids, substitutions can include amino acids that occur at analogous positions in related evolutionarily conserved human variable heavy chain sequences, human variable light chain sequences, and orthologous sequences from non-human species. Unless otherwise stated or implicit from context, the following terms and terms include the meanings provided below. Unless otherwise expressly stated or apparent from context, these following terms and terms do not exclude the meanings they have acquired in the relevant art. Definitions are provided for the purpose of helping to describe particular embodiments and are not intended to limit the claimed invention, which is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0087] All documents in this specification are incorporated by reference to the same extent as if each individual document or patent application was specifically and individually indicated to be incorporated by reference. The following description contains information that may be useful in understanding the present invention. This is not an admission that any information provided herein is prior art or relevant to the presently claimed invention, or that any document expressly or implicitly incorporated is prior art. Working Example:

[0088] The following examples are not intended to limit the scope of the claims to the present invention, but rather to illustrate certain embodiments. Variations of the illustrated methods that occur to those skilled in the art are intended to fall within the scope of the present invention.

[0089] Vector construction: The vector pcDNA3.4TOPO (Invitrogen) was ligated with a short polylinker containing EcoRI, XhoI, and NotI. The resulting plasmid was digested with EcoRI and NotI restriction enzymes and purified by gel electrophoresis. For heavy chain cloning, a prepared vector was assembled using Gibson assembly with a gblock encoding the VH region (IDT) and a human IgG2 gblock encoding the XhoI site at the junction of the J chain and CH1 region. Plasmids were prepared and digested with EcoRI and XhoI to incorporate all humanized variable heavy chain (VH) regions with an IgG2 isotype. All assemblies were performed using the Gibson method (NEB). The variable heavy chain regions were generated in a similar manner using gblocks, and the Vkappa region was assembled with a gblock fragment encoding the constant region kappa (Ck).

[0090] Protein expression, purification, and binding properties: Plasmids were prepared and transfected into Expi293 or ExpiCHO cells using a transient expression system (Thermo Fisher). Briefly, plasmids were transfected into 3e6 cells / ml of cells at 1µg total plasmid DNA / ml of culture. Heavy and light chain plasmids were mixed at a 1:1 ratio. Cultures were incubated at 37°C with shaking. After 16 hours, we added transfection enhancers 1 and 2 to the cultures and continued incubation for 6 days. Supernatants were filtered, and protein titers were determined using Octet Red96 (Pall) with an IgG quantification protocol. IgG was purified using a Mab Select Sure Protein-A column on an ACTA PURE system and dialyzed overnight against PBS. The purified antibodies were characterized for antigen affinity with Octet Red 96 by subjecting the purified antibodies to an anti-human heavy chain (AHC) capture sensor and measuring the association and dissociation rates of the PDL1 histidine-tagged target at three concentrations (Table 1). Table 1. Measurements were performed on Octet to compare the monovalent binding kinetics of humanized anti-PD-L1 antibodies with benchmark controls. [Table 1]

[0091] Flow cytometry binding analysis of anti-PD-L1 antibodies to PD-L1-expressing A431 cancer cells Two days before FACS analysis, A431 cancer cells were seeded in 24-well plates, covering 30-60% of the culture surface area, and cultured in the presence or absence of 1000 U / ml recombinant human IFN-γ to stimulate PD-L1 expression. On the day of FACS analysis, wells were washed with PBS and harvested with trypsin / EDTA. Detached cells were washed twice, resuspended in FACS buffer at 5E6 cells / ml, and evenly distributed into 96-well plates at 1E5 cells / well. Cells were stained with 5 μg / ml anti-PD-L1 antibody, positive control antibody "C1-IgG1," or isotype IgG1 for 45 minutes on ice, then washed and secondary stained with goat anti-human IgG-AF647 at a 1:500 dilution. Cells were washed a final time, added with 7-AAD, and analyzed by flow cytometry (Figure 1).

[0092] PD-1 His-tag blocking assay in hPDL1 / HEK293 cells for humanized anti-PD-L1 testing For hybridoma screening, hPD-L1-expressing 293 cells were harvested with Accutase and resuspended in FACS Wash buffer at 4E6 cells / ml. To compare the blocking ability of antibodies, we mixed 30 μl of cells / well (120,000), 30 μl of 6 μg / ml rhPD-1 His-tagged protein (final concentration 2 μg / ml), and 30 μl of serially titrated anti-PD-L1 antibodies and incubated on ice for 20 minutes. After incubation, cells were washed and bound PD-1 was detected using anti-His-tagged APC mouse IgG1 (R&D Cat# IC050A). Cells were washed a final time, added 7-AAD, and analyzed by flow cytometry. The blocking ability of the humanized antibodies was compared to the same assay using rhPD-1 Fc-biotin at a final concentration of 0.5ug / ml with detection by streptavidin-APC (R&D cat#F0050) (Figure 2) (Table 2). [Table 2]

[0093] SEB stimulation assay Fresh PBMCs were diluted to 2E6 cells / ml in X-Vivo15 medium (Lonza cat# 04-744Q). SEB (Millipore cat# 324798) was added to PBMCs at 200 ng / ml (2x). Cells were antibody-treated by adding 100 μl of diluted antibody and 100 μl of the PBMC / SEB antigen mixture to a 96-well flat-bottom plate. After 48 hours of incubation at 37°C, supernatants were analyzed for IL-2 using an ELISA kit (R&D Systems) (Figure 3).

[0094] Mixed lymphocyte reaction assay PBMCs were isolated from human buffy coats using density gradient centrifugation (Miltenyi Biotec) and washed four times with PBS. CD4+ T cells were isolated from the PBMCs (Miltenyi Biotec cat#130-096-533) and resuspended at 4E6 cells / ml in X-Vivo15 medium. Monocyte-derived dendritic cells were obtained from positively selected CD14+ monocytes (Monocyte Isolation Kit II, Cat. no. 130-091-153, Miltenyi Biotec). Cells were seeded at 5E5 cells / ml in RPMI-1640 complete medium supplemented with 10% fetal bovine serum (FBS) and cultured for 7 days. The medium was supplemented with recombinant human (rh-)IL-4 (1000 U / ml) (R&D Systems) and rh granulocyte-macrophage colony-stimulating factor (rh-GMCSF) (500 U / ml) (R&D Systems) on days 0, 2, and 5. Immature DCs were harvested on day 7 and resuspended in 5 ml RPMI-1640, 10% FBS medium. Cells were incubated with 20ug / ml mitomycin C (Roche REF 10107409001) at 37°C for 1 hour with occasional mixing, washed and resuspended in X-Vivo15 medium at 4E5 cells / ml.

[0095] In a 96-well flat-bottom plate, we mixed 50ul DC cells (2E4 DCs / well), 100ul serially diluted anti-PD-L1 antibodies, a 2x control, and 50ul purified CD4+ T cells (2E5 CD4+ T cells / well) at a 1:10 ratio (DC:T cells). The plates were incubated for 5 days, and the supernatants were analyzed for IFNg release by ELISA (R&D Systems) (Figure 4).

[0096] In vitro CMV antigen recall response with anti-PDL1 treatment Thawed PBMCs from CMV+ donors (Astarte Biologics) were counted and resuspended at 2E6 cells / ml in AIM-V medium supplemented with β-mercaptoethanol (1:1000). In a 96-well flat-bottom plate, we mixed 100µl of CMV+ PBMCs (2E5 cells / well), 50µl of 5µg / ml CMV antigen (Astarte cat#1004), and 50µl of serially diluted anti-PDL1 antibody and controls. Plates were incubated for 4 days, and supernatants were analyzed for IFNg release by ELISA (R&D Systems) (Figure 5).

[0097] Protein thermal shift of humanized anti-PDL1 antibody 10µg / ml anti-PD-L1 antibody was mixed with 2µl of 50X protein thermal shift dye and PBS to a final volume of 100µl. Samples were distributed evenly across a PCR 96-tube plate in sets of 4 wells (25µl / well). Protein thermal shift reactions were measured using an Applied Biosystems StepOne Real-Time PCR instrument with a continuous temperature gradient from 22°C to 95°C at a rate of 1°C per 1 minute, 5 seconds. Tm was analyzed using the induction method (Figure 6). Sequence Listing Mouse antibodies SEQ ID NO: 1 DIQMTQSPASLSVSVGETVTITC <h2 style=";text-align:left;direction:ltr">RASENIHSNLA WYQQKQGKSPQLLVY <h2 style=";text-align:left;direction:ltr"> GATNLAD GVPSRFSGSGSGAQYSLKINSLQSEDFGSYYC <h2 style=";text-align:left;direction:ltr"> QHFWGTPPYA FGGGTKLETK Underlined and bold: CDR1, 2 and 3 (same below) defined according to the Kabat numbering system. SEQ ID NO: 2 QIVLTQSPAIMSASPGEKVTISC <h2 style=";text-align:left;direction:ltr"> SASSSVFDMY WYQQKPGSSPKPWIY <h2 style=";text-align:left;direction:ltr"> RTSNLAS GVPARFSGSGSGTSYFLTISSMEAEDAATYYC <h2 style=";text-align:left;direction:ltr"> QQYQSFPLT FGAGTKLELK SEQ ID NO: 3 DIQMTQSPSSLSASLGERVSLIC <h2 style=";text-align:left;direction:ltr"> RASQEISGYLS WLQQKSDGTIKRLIF <h2 style=";text-align:left;direction:ltr"> AASTLDP GVPKRFSGSRSGADYSLTISSLESEDFADYYC <h2 style=";text-align:left;direction:ltr"> LQYAIYPPT FGSGTKLEIK SEQ ID NO:4 QIVLTQSPAIMSASPGEKVTMTC <h2 style=";text-align:left;direction:ltr"> SASSSVSFMH WYQQKSGTSPKKWIY <h2 style=";text-align:left;direction:ltr"> DTSKLAS GVPVRFSGSGSGTSYSLTIINMEAEEDAATYYC <h2 style=";text-align:left;direction:ltr"> QQWTYYPPT FGGGTKLEIK SEQ ID NO:5 DIQMTQSPSSLSASLGERVSLTC <h2 style=";text-align:left;direction:ltr"> RASQEISVYLS WLQQKPDGTIKRLIY <h2 style=";text-align:left;direction:ltr"> AASTLDS GVPQRFRGSRSGSDYSLTISSLESEDFADYYC <h2 style=";text-align:left;direction:ltr"> VQYTSHPYT FGGGTKLEIK SEQ ID NO:6 DIQMTQSPASLSVSVGETVTITC <h2 style=";text-align:left;direction:ltr"> RASENIHSNLA WYQQKQGKSPQLLVY <h2 style=";text-align:left;direction:ltr"> GATNLAD GVPSRFSGSGSGAQYSLKINSLQSEDFGSYYC <h2 style=";text-align:left;direction:ltr"> QHFWGTPPYV FGGGTKLETK SEQ ID NO:7 QVQLQQPGSELVRPGTSVKLSCKASGYTFT <h2 style=";text-align:left;direction:ltr"> TFWMH WVKQRPGQGLEWIG <h2 style=";text-align:left;direction:ltr"> NIYPGSGTINYDEKFRS KATLTVDTSSNTAYMQVSSLTSEDSAVYYCTT <h2 style=";text-align:left;direction:ltr"> GWDGEH WGQGTTLTVSS SEQ ID NO:8 EVQLVESGGGLVKPGGSLKLSCAASGFTFS <h2 style=";text-align:left;direction:ltr"> DYGMH WIRQAPEKGLEWIA <h2 style=";text-align:left;direction:ltr"> YIGTTSSIIYYADTVKG RFTISRDNAKNTLFLQMTSLRSEDTAMYYCAR <h2 style=";text-align:left;direction:ltr"> RDYGNYYWYLDV WGTGTTVTVSS SEQ ID NO:9 EVQLQQSGAELVKPGASVKLSCTASGFNIK <h2 style=";text-align:left;direction:ltr"> DTYMH WVQQRPEQGLEWIG <h2 style=";text-align:left;direction:ltr"> RIDPMNGNSKYDPKFQD KATITADTSSNTAYLQLSSLTSEDTAVYYCTS <h2 style=";text-align:left;direction:ltr"> SKWGDY WGQGTTLTVSS SEQ ID NO: 10 EVQLQQSGAELVRPGALVKVSCKASGFNIK <h2 style=";text-align:left;direction:ltr"> DYYIH WVKQRPEQGLEWIG <h2 style=";text-align:left;direction:ltr"> WIDPDNGNTKYDPNFQG KASMTADTSSNTVNLQLSSLTSKDTAVYYCAR <h2 style=";text-align:left;direction:ltr"> YGGYGGFYTMDY WGQGTSVTVSS SEQ ID NO: 11 QVQLQQPGTELVKPGASVKLSCKASGYTFT <h2 style=";text-align:left;direction:ltr"> IYWMH WVKQRPGRGLEWIG <h2 style=";text-align:left;direction:ltr"> RIDPNSGDTKYTEKFKNRATLTVDKSSNTAYMQFSSLASDDSAVYYCAR <h2 style=";text-align:left;direction:ltr"> GGPNWDGFAY WGQGTLVTVSA SEQ ID NO: 12 QVQLQQPRSELVRPGASVKLSCKASGYTFT <h2 style=";text-align:left;direction:ltr"> TFWMH WVKQRPGQGLEWIG <h2 style=";text-align:left;direction:ltr"> NIYPGSGISNYDEKFKN KATLTVDTSSSTAYMQVSSLTSEDSAVYYCTT <h2 style=";text-align:left;direction:ltr"> GWDGEH WGQGTTLTVSS Light chain CDRs CDR1 <h2 style=";text-align:left;direction:ltr"> RASENIHSNLA SEQ ID NO: 13 <h2 style=";text-align:left;direction:ltr"> SASSSVFDMY SEQ ID NO: 14 <h2 style=";text-align:left;direction:ltr"> RASQEISGYLS SEQ ID NO: 15 <h2 style=";text-align:left;direction:ltr"> SASSSVSFMH SEQ ID NO: 16 <h2 style=";text-align:left;direction:ltr"> RASQEISVYLS SEQ ID NO: 17 <h2 style=";text-align:left;direction:ltr"> RASENIHSNLA SEQ ID NO: 18 CDR2 <h2 style=";text-align:left;direction:ltr"> GATNLAD SEQ ID NO: 19 <h2 style=";text-align:left;direction:ltr"> RTSNLAS SEQ ID NO: 20 <h2 style=";text-align:left;direction:ltr"> AASTLDP SEQ ID NO: 21 <h2 style=";text-align:left;direction:ltr"> DTSKLAS SEQ ID NO: 22 <h2 style=";text-align:left;direction:ltr"> AASTLDS SEQ ID NO: 23 <h2 style=";text-align:left;direction:ltr"> GATNLAD SEQ ID NO: 24 CDR3 <h2 style=";text-align:left;direction:ltr"> QHFWGTPPYA SEQ ID NO: 25 <h2 style=";text-align:left;direction:ltr"> QQYQSFPLT SEQ ID NO: 26 <h2 style=";text-align:left;direction:ltr"> LQYAIYPPT SEQ ID NO: 27 <h2 style=";text-align:left;direction:ltr"> QQWTYYPPT SEQ ID NO: 28 <h2 style=";text-align:left;direction:ltr"> VQYTSHPYT SEQ ID NO: 29 <h2 style=";text-align:left;direction:ltr"> QHFWGTPPYV SEQ ID NO: 30 Heavy chain CDRs CDR1 <h2 style=";text-align:left;direction:ltr"> TFWMH SEQ ID NO: 31 <h2 style=";text-align:left;direction:ltr"> DYGMH SEQ ID NO: 32 <h2 style=";text-align:left;direction:ltr"> DTYMH SEQ ID NO: 33 <h2 style=";text-align:left;direction:ltr"> DYYIH SEQ ID NO: 34 <h2 style=";text-align:left;direction:ltr"> IYWMH SEQ ID NO: 35 <h2 style=";text-align:left;direction:ltr"> TFWMH SEQ ID NO: 36 CDR2 <h2 style=";text-align:left;direction:ltr"> NIYPGSGTINYDEKFRS SEQ ID NO: 37 <h2 style=";text-align:left;direction:ltr"> YIGTTSSIIYYADTVKG SEQ ID NO: 38 <h2 style=";text-align:left;direction:ltr"> RIDPMNGNSKYDPKFQD SEQ ID NO: 39 <h2 style=";text-align:left;direction:ltr"> WIDPDNGNTKYDPNFQG SEQ ID NO: 40 <h2 style=";text-align:left;direction:ltr"> RIDPNSGDTKYTEKFKN SEQ ID NO: 41 <h2 style=";text-align:left;direction:ltr"> NIYPGSGISNYDEKFKN SEQ ID NO: 42 CDR3 <h2 style=";text-align:left;direction:ltr"> GWDGEH SEQ ID NO: 43 <h2 style=";text-align:left;direction:ltr"> RDYGNYYWYLDV SEQ ID NO: 44 <h2 style=";text-align:left;direction:ltr"> SKWGDY SEQ ID NO: 45 <h2 style=";text-align:left;direction:ltr"> YGGYGGFYTMDY SEQ ID NO: 46 <h2 style=";text-align:left;direction:ltr"> GGPNWDGFAY SEQ ID NO: 47 <h2 style=";text-align:left;direction:ltr"> GWDGEH SEQ ID NO: 48 Humanized Light Chain Variable Domain SEQ ID NO: 49 DIQMTQSPSSLSASVGDRVTITC <h2 style=";text-align:left;direction:ltr"> RASENIHSNLA WYQQKPGKAPKLLVY <h2 style=";text-align:left;direction:ltr"> GATNLAD GVPSRFSGSGSGAQYTLTISSLQPEDFATYYC <h2 style=";text-align:left;direction:ltr"> QHFWGTPPYA FGGGTKLEIK SEQ ID NO:50 DIQMTQSPSSLSASVGDRVTITC <h2 style=";text-align:left;direction:ltr"> RASENIHSNLA WYQQKPGKAPQLLVY <h2 style=";text-align:left;direction:ltr"> GATNLAD GVPSRFSGSGSGAQYTLTISSLQPEDFATYYC <h2 style=";text-align:left;direction:ltr"> QHFWGTPPYA FGGGTKLEIK SEQ ID NO:51 DIQMTQSPSSLSVSVGDRVTITC <h2 style=";text-align:left;direction:ltr"> RASENIHSNLA WYQQKPGKAPQLLVY <h2 style=";text-align:left;direction:ltr"> GATNLADGVPSRFSGSGSGAQYTLTISSLQPEDFATYYC <h2 style=";text-align:left;direction:ltr"> QHFWGTPPYA FGGGTKLEIK SEQ ID NO:52 DIQMTQSPSSLSASVGDRVTITC <h2 style=";text-align:left;direction:ltr"> RASENIHSNLA WYQQKPGKAPKLLVY <h2 style=";text-align:left;direction:ltr"> GATNLAD GVPSRFSGSGSGAQYTLTISSLQPEDFATYYCQHFWGTPPYVFGGGTKLEIK SEQ ID NO:53 DIQMTQSPSSLSASVGDRVTITC <h2 style=";text-align:left;direction:ltr"> RASENIHSNLA WYQQKPGKAPQLLVY <h2 style=";text-align:left;direction:ltr"> GATNLAD GVPSRFSGSGSGAQYTLTISSLQPEDFATYYCQHFWGTPPYVFGGGTKLEIK SEQ ID NO:54 DIQMTQSPSSLSVSVGDRVTITC <h2 style=";text-align:left;direction:ltr"> RASENIHSNLA WYQQKPGKAPQLLVY <h2 style=";text-align:left;direction:ltr"> GATNLAD GVPSRFSGSGSGAQYTLTISSLQPEDFATYYCQHFWGTPPYVFGGGTKLEIK SEQ ID NO: 55 EIVLTQSPATLSLSPGERATLSC <h2 style=";text-align:left;direction:ltr"> SASSSVFDMY WYQQKPGQSPRPWIY <h2 style=";text-align:left;direction:ltr"> RTSNLAS GIPARFSGSGSGTDFLTISSLEPEDFAVYYC <h2 style=";text-align:left;direction:ltr"> QQYQSFPLT FGQGTKLELK SEQ ID NO:56 EIVLTQSPATLSLSPGERVTLSC <h2 style=";text-align:left;direction:ltr"> SASSSVFDMY WYQQKPGQSPRPWIY <h2 style=";text-align:left;direction:ltr"> RTSNLAS GIPARFSGSGSGTDFLTISSLEPEDAAVYYC <h2 style=";text-align:left;direction:ltr"> QQYQSFPLT FGQGTKLELK SEQ ID NO:57 EIVLTQSPATLSLSPGERVTLSC <h2 style=";text-align:left;direction:ltr"> SASSSVFDMY WYQQKPGSSPRPWIY <h2 style=";text-align:left;direction:ltr"> RTSNLASGVPARFSGSGSGTDFLTISSLEPEDAAVYYC <h2 style=";text-align:left;direction:ltr"> QQYQSFPLT FGQGTKLELK SEQ ID NO:58 EIVLTQSPATLSLSPGERVTLSC <h2 style=";text-align:left;direction:ltr"> SASSSVFDMY WYQQKPGSSPRPWIY <h2 style=";text-align:left;direction:ltr"> RTSNLAS GVPARFSGSGSGTDYFLTISSMEPEDAATYYC <h2 style=";text-align:left;direction:ltr"> QQYQSFPLT FGQGTKLELK SEQ ID NO:59 DIQMTQSPSSLSASVGDRVTIIC <h2 style=";text-align:left;direction:ltr"> RASQEISGYLS WLQQKPDGTIKSLIY <h2 style=";text-align:left;direction:ltr"> AASTLDP GVPSRFSGSRSGADFTLTISSLQPEDFATYYC <h2 style=";text-align:left;direction:ltr"> LQYAIYPPT FGQGTKLEIK Sequence number 60 DIQMTQSPSSLSASVGDRVTLIC <h2 style=";text-align:left;direction:ltr"> RASQEISGYLS WLQQKPDGTIKRLIF <h2 style=";text-align:left;direction:ltr"> AASTLDP GVPSRFSGSRSGADFTLTISSLQPEDFADYYC <h2 style=";text-align:left;direction:ltr"> LQYAIYPPT FGQGTKLEIK SEQ ID NO: 61 DIQMTQSPSSLSASVGDRVTLIC <h2 style=";text-align:left;direction:ltr"> RASQEISGYLS WLQQKPDGTIKRLIF AASTLDP GVPSRFSGSRSGADYTLTISSLQPEDFADYYC LQYAIYPPT FGQGTKLEIK SEQ ID NO: 62 EIVLTQSPATLSLSPGERATLSC SASSSVSFMH WYQQKPGLAPRKLIY DTSKLAS GIPDRFSGSGSGTSYTLTISRLEPEDFAVYYC QQWTYYPPT FGQGTKVEIK SEQ ID NO: 63 QIVLTQSPATLSLSPGERATLSC SASSSVSFMH WYQQKPGLAPRKWIY DTSKLAS GVPDRFSGSGSGTSYTLTISRLEPEDFAVYYC QQWTYYPPT FGQGTKVEIK SEQ ID NO: 64 DIQMTQSPSSLSASVGDRVTITC RASQEISVYLS WLQQKPGKAPKRLIY AASTLDS GVPSRFSGSGSGSDYTLTISSLQPEDFATYYC VQYTSHPYT FGQGTKVEIK Humanized Heavy Chain Variable Domain SEQ ID NO: 65 QVQLVQSGAEVVKPGASVKLSCKASGYTFT TFWMH WVRQAPGQGLEWIG NIYPGSGTINYDEKFRS RVTLTVDTSISTAYMELSRLRSEDTAVYYCTT GWDGEH WGQGTTLTVSS SEQ ID NO: 66 QVQLVQSGAEVVKPGASVKLSCKASGYTFT TFWMH WVRQAPGQGLEWIG NIYPGSGTINYDEKFRS RATLTVDTSISTAYMEVSRLRSEDTAVYYCTT GWDGEH WGQGTTLTVSS SEQ ID NO: 67 QVQLVQSGAEVVKPGASVKLSCKASGYTFT TFWMH WVKQAPGQGLEWIG NIYPGSGTINYDEKFRS RATLTVDTSISTAYMEVSRLRSEDTAVYYCTT GWDGEH WGQGTTLTVSS SEQ ID NO: 68 EVQLVESGGGLVKPGGSLRLSCAASGFTFS DYGMH WIRQAPGKGLEWVS YIGTTSSIIYYADTVKG RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR RDYGNYYWYLDV WGQGTMVTVSS SEQ ID NO: 69 EVQLVESGGGLVKPGGSLRLSCAASGFTFS DYGMH WIRQAPGKGLEWIA YIGTTSSIIYYADTVKG RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR RDYGNYYWYLDV WGTGTMVTVSS Sequence number 70 EVQLVESGGGLVKPGGSLRLSCAASGFTFS DYGMH WIRQAPGKGLEWIA YIGTTSSIIYYADTVKG RFTISRDNAKNSLYLQMNSLRAEDTAMYYCAR RDYGNYYWYLDV WGTGTMVTVSS SEQ ID NO: 71 QVQLVQSGAEVKKPGASVKVSCKASGFNIK DTYMH WVQQAPGQRLEWMG RIDPMNGNSKYDPKFQD RVTITADTSASTAYLELSSLRSEDTAVYYCTS SKWGDY WGQGTLLTVSS SEQ ID NO: 72 QVQLVQSGAEVKKPGASVKVSCKASGFNIK DTYMH WVQQAPGQRLEWMG RIDPMNANSKYDPKFQD RVTITADTSASTAYLELSSLRSEDTAVYYCTS SKWGDY WGQGTLLTVSS SEQ ID NO: 73 QVQLVQSGAEVKKPGASVKLSCKASGFNIK DTYMH WVQQAPEQRLEWMG RIDPMNANSKYDPKFQD RVTITADTSASTAYLELSSLRSEDTAVYYCTS SKWGDY WGQGTLLTVSS SEQ ID NO: 74 QVQLVQSGAEVKKPGASVKLSCKASGFNIK DTYMH WVQQAPEQRLEWIG RIDPMNANSKYDPKFQD RATITADTSANTAYLELSSLRSEDTAVYYCTS SKWGDY WGQGTLLTVSS SEQ ID NO: 75 QVQLQQSGAEVKKPGASVKLSCTASGFNIK DTYMH WVQQAPEQRLEWIG RIDPMNGNSKYDPKFQD RATITADTSANTAYLELSSLRSEDTAVYYCTS SKWGDY WGQGTLLTVSS SEQ ID NO: 76 QVQLQQSGAEVKKPGASVKLSCTASGFNIK DTYMH WVQQAPEQRLEWIG RIDPMNANSKYDPKFQD RATITADTSANTAYLELSSLRSEDTAVYYCTS SKWGDY WGQGTLLTVSS SEQ ID NO: 77 QVQLVQSGAEVKKPGASVKVSCKASGYNIK DYYIH WVRQAPGQGLEWIG WIDPDNGNTKYDPNFQG RVTMTADTSTSTVYMELSSLRSEDTAVYYCAR YGGYGGFYTMDY WGQGTLVTVSS SEQ ID NO: 78 QVQLVQSGAEVKKPGASVKVSCKASGFNIK DYYIH WVRQAPGQGLEWIG WIDPDNGNTKYDPNFQG RATMTADTSTSTVYMELSSLRSEDTAVYYCAR YGGYGGFYTMDY WGQGTLVTVSS SEQ ID NO: 79 QVQLVQSGAEVKKPGASVKVSCKASGYTFT IYWMH WVRQAPGQGLEWIG RIDPNSGDTKYTEKFKN RATLTVDKSTSTAYMELSSLRSEDTAVYYCAR GGPNWDGFAY WGQGTLVTVSS SEQ ID NO: 80 QVQLVQSGAEVKKPGASVKVSCKASGYTFT IYWMH WVKQRPGQGLEWIG RIDPNSGDTKYTEKFKN RATLTVDKSTSTAYMEFSSLRSEDTAVYYCAR GGPNWDGFAY WGQGTLVTVSS SEQ ID NO: 81 QVQLVQSGAEVVKPGASVKLSCKASGYTFT TFWMH WVRQAPGQGLEWIG NIYPGSGISNYDEKFKN RVTLTVDTSISTAYMELSRLRSEDTAVYYCTT GWDGEH WGQGTTLTVSS SEQ ID NO: 82 QVQLVQSGAEVVKPGASVKLSCKASGYTFT TFWMH WVRQAPGQGLEWIG NIYPGSGISNYDEKFKN RATLTVDTSISTAYMEVSRLRSEDTAVYYCTT GWDGEH WGQGTTLTVSS SEQ ID NO: 83 QVQLVQSGAEVVKPGASVKLSCKASGYTFT TFWMH WVKQAPGQGLEWIG NIYPGSGISNYDEKFKN RATLTVDTSISTAYMEVSRLRSEDTAVYYCTT GWDGEH WGQGTTLTVSS Humanized Light Chain SEQ ID NO:84 DIQMTQSPSSLSASVGDRVTITC RASENIHSNLA WYQQKPGKAPKLLVY GATNLAD GVPSRFSGSGSGAQYTLTISSLQPEDFATYYC QHFWGTPPYA FGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 85 DIQMTQSPSSLSASVGDRVTITC RASENIHSNLA WYQQKPGKAPQLLVY GATNLAD GVPSRFSGSGSGAQYTLTISSLQPEDFATYYC QHFWGTPPYA FGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:86 DIQMTQSPSSLSVSVGDRVTITC RASENIHSNLA WYQQKPGKAPQLLVY GATNLAD GVPSRFSGSGSGAQYTLTISSLQPEDFATYYC QHFWGTPPYA FGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:87 DIQMTQSPSSLSASVGDRVTITC RASENIHSNLA WYQQKPGKAPKLLVY GATNLAD GVPSRFSGSGSGAQYTLTISSLQPEDFATYYCQHFWGTPPYVFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 88 DIQMTQSPSSLSASVGDRVTITC RASENIHSNLA WYQQKPGKAPQLLVY GATNLAD GVPSRFSGSGSGAQYTLTISSLQPEDFATYYCQHFWGTPPYVFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:89 DIQMTQSPSSLSVSVGDRVTITC RASENIHSNLA WYQQKPGKAPQLLVY GATNLAD GVPSRFSGSGSGAQYTLTISSLQPEDFATYYCQHFWGTPPYVFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 90 EIVLTQSPATLSLSPGERATLSC SASSSVFDMY WYQQKPGQSPRPWIY RTSNLAS GIPARFSGSGSGTDFLTISSLEPEDFAVYYC QQYQSFPLT FGQGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 91 EIVLTQSPATLSLSPGERVTLSC SASSSVFDMY WYQQKPGQSPRPWIY RTSNLAS GIPARFSGSGSGTDFLTISSLEPEDAAVYYC QQYQSFPLT FGQGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:92 EIVLTQSPATLSLSPGERVTLSC SASSSVFDMY WYQQKPGSSPRPWIY RTSNLAS GVPARFSGSGSGTDFLTISSLEPEDAAVYYC QQYQSFPLT FGQGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 93 EIVLTQSPATLSLSPGERVTLSC SASSSVFDMY WYQQKPGSSPRPWIY RTSNLAS GVPARFSGSGSGTDYFLTISSMEPEDAATYYC QQYQSFPLT FGQGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:94 DIQMTQSPSSLSASVGDRVTIIC RASQEISGYLS WLQQKPDGTIKSLIY AASTLDP GVPSRFSGSRSGADFTLTISSLQPEDFATYYC LQYAIYPPT FGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Sequence number 95 DIQMTQSPSSLSASVGDRVTLIC RASQEISGYLS WLQQKPDGTIKRLIF AASTLDP GVPSRFSGSRSGADFTLTISSLQPEDFADYYC LQYAIYPPT FGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Sequence number 96 DIQMTQSPSSLSASVGDRVTLIC RASQEISGYLS WLQQKPDGTIKRLIF AASTLDP GVPSRFSGSRSGADYTLTISSLQPEDFADYYC LQYAIYPPT FGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 97 EIVLTQSPATLSLSPGERATLSC SASSSVSFMH WYQQKPGLAPRKLIY DTSKLASGIPDRFSGSGSGTSYTLTISRLEPEDFAVYYC QQWTYYPPT FGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 98 QIVLTQSPATLSLSPGERATLSC SASSSVSFMH WYQQKPGLAPRKWIY DTSKLAS GVPDRFSGSGSGTSYTLTISRLEPEDFAVYYC QQWTYYPPT FGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Sequence number 99 DIQMTQSPSSLSASVGDRVTITC RASQEISVYLS WLQQKPGKAPKRLIY AASTLDS GVPSRFSGSGSGSDYTLTISSLQPEDFATYYC VQYTSHPYT FGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Humanized Heavy Chain Sequence number 100 QVQLVQSGAEVVKPGASVKLSCKASGYTFT TFWMH WVRQAPGQGLEWIG NIYPGSGTINYDEKFRS RVTLTVDTSISTAYMELSRLRSEDTAVYYCTT GWDGEHWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Sequence number 101 QVQLVQSGAEVVKPGASVKLSCKASGYTFT TFWMH WVRQAPGQGLEWIG NIYPGSGTINYDEKFRS RATLTVDTSISTAYMEVSRLRSEDTAVYYCTT GWDGEH WGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Sequence number 102 QVQLVQSGAEVVKPGASVKLSCKASGYTFT TFWMH WVKQAPGQGLEWIG NIYPGSGTINYDEKFRS RATLTVDTSISTAYMEVSRLRSEDTAVYYCTT GWDGEHWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Sequence number 103 EVQLVESGGGLVKPGGSLRLSCAASGFTFS DYGMH WIRQAPGKGLEWVS YIGTTSSIIYYADTVKG RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR RDYGNYWYLDV WGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Sequence number 104 EVQLVESGGGLVKPGGSLRLSCAASGFTFS DYGMH WIRQAPGKGLEWIA YIGTTSSIIYYADTVKG RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR RDYGNYWYLDVWGTGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Sequence number 105 EVQLVESGGGLVKPGGSLRLSCAASGFTFS DYGMH WIRQAPGKGLEWIA YIGTTSSIIYYADTVKG RFTISRDNAKNSLYLQMNSLRAEDTAMYYCAR RDYGNYWYLDV WGTGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 106 QVQLVQSGAEVKKPGASVKVSCKASGFNIK DTYMH WVQQAPGQRLEWMG RIDPMNGNSKYDPKFQD RVTITADTSASTAYLELSSLRSEDTAVYYCTS SKWGDYWGQGTLLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 107 QVQLVQSGAEVKKPGASVKVSCKASGFNIK DTYMH WVQQAPGQRLEWMG RIDPMNANSKYDPKFQD RVTITADTSASTAYLELSSLRSEDTAVYYCTS SKWGDY WGQGTLLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 108 QVQLVQSGAEVKKPGASVKLSCKASGFNIK DTYMH WVQQAPEQRLEWMG RIDPMNANSKYDPKFQD RVTITADTSASTAYLELSSLRSEDTAVYYCTS SKWGDYWGQGTLLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 109 QVQLVQSGAEVKKPGASVKLSCKASGFNIK DTYMH WVQQAPEQRLEWIG RIDPMNANSKYDPKFQD RATITADTSANTAYLELSSLRSEDTAVYYCTS SKWGDY WGQGTLLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 110 QVQLQQSGAEVKKPGASVKLSCTASGFNIK DTYMH WVQQAPEQRLEWIG RIDPMNGNSKYDPKFQD RATITADTSANTAYLELSSLRSEDTAVYYCTS SKWGDYWGQGTLLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 111 QVQLQQSGAEVKKPGASVKLSCTASGFNIK DTYMH WVQQAPEQRLEWIG RIDPMNANSKYDPKFQD RATITADTSANTAYLELSSLRSEDTAVYYCTS SKWGDY WGQGTLLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 112 QVQLVQSGAEVKKPGASVKVSCKASGYNIK YYYY WVRQAPGQGLEWIG WIDPDNGNTKYDPNFQG RVTMTADTSTSTVYMELSSLRSEDTAVYYCAR YGGYGGFYTMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 113 QVQLVQSGAEVKKPGASVKVSCKASGFNIK YYYY WVRQAPGQGLEWIG WIDPDNGNTKYDPNFQG RATMTADTSTSTVYMELSSLRSEDTAVYYCAR YGGYGGFYTMDY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 114 QVQLVQSGAEVKKPGASVKVSCKASGYTFT IYWMH WVRQAPGQGLEWIG RIDPNSGDTKYTEKFKN RATLTVDKSTSTAYMELSSLRSEDTAVYYCAR GGPNWDGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 115 QVQLVQSGAEVKKPGASVKVSCKASGYTFT IYWMH WVKQRPGQGLEWIG RIDPNSGDTKYTEKFKN RATLTVDKSTSTAYMEFSSLRSEDTAVYYCAR GGPNWDGFAY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 116 QVQLVQSGAEVVKPGASVKLSCKASGYTFT TFWMH WVRQAPGQGLEWIG NIYPGSGISNYDEKFKN RVTLTVDTSISTAYMELSRLRSEDTAVYYCTT GWDGEHWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Sequence number 117 QVQLVQSGAEVVKPGASVKLSCKASGYTFT TFWMH WVRQAPGQGLEWIG NIYPGSGISNYDEKFKN RATLTVDTSISTAYMEVSRLRSEDTAVYYCTT GWDGEH WGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Sequence number 118 QVQLVQSGAEVVKPGASVKLSCKASGYTFT TFWMH WVKQAPGQGLEWIG NIYPGSGISNYDEKFKN RATLTVDTSISTAYMEVSRLRSEDTAVYYCTT GWDGEHWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Claims

1. an isolated monoclonal anti-PD-L1 antibody, or antigen-binding fragment thereof; A heavy chain variable region CDR3 consisting of SEQ ID NO: 43; A heavy chain variable region CDR1 consisting of SEQ ID NO: 31; A heavy chain variable region CDR2 consisting of SEQ ID NO: 37; moreover, (a) a light chain variable region CDR1 consisting of SEQ ID NO: 13; (b) a light chain variable region CDR2 consisting of SEQ ID NO: 19; (c) a light chain variable region CDR3 consisting of SEQ ID NO: 25; A monoclonal antibody, or an antigen-binding fragment thereof, characterized in that the antibody or portion thereof specifically binds to human PD-L1.

2. a light chain variable region amino acid sequence having at least 95% identity to SEQ ID NO: 1 and a heavy chain variable region amino acid sequence having at least 95% identity to SEQ ID NO: 7; or a heavy chain variable region consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 65-67; and a light chain variable region consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 49-51; The monoclonal antibody of claim 1, or an antigen-binding fragment thereof, comprising:

3. Fab fragment, F(ab') 2 A monoclonal antibody, or an antigen-binding fragment thereof, according to claims 1 and 2, characterized in that it is an immunoglobulin G (IgG), IgM, IgE, IgA or IgD molecule, or an Fv fragment, Fv fragment, single-chain antibody, or bispecific antibody.

4. 3. A monoclonal antibody according to claims 1 and 2, characterized in that it is a chimeric or humanized antibody.

5. The monoclonal antibody according to claim 4, which is IgG1, IgG2, IgG3, or IgG4.

6. An immunoconjugate comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, linked to a therapeutic agent.

7. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof described in any one of claims 1 to 5 and a pharmaceutically acceptable carrier.

8. 8. The pharmaceutical composition of claim 7, comprising a therapeutically effective amount for stimulating an immune response, for stimulating the immune response in a subject, or comprising a therapeutically effective amount for treating cancer, for treating the cancer in a subject.

9. 5 × 10 antibodies or antigen-binding fragments thereof -8 M to 1 x 10 -10 An antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, characterized in that it has an affinity (KD) for PD-L1 in the range of up to M.

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