Humanized anti-PD-L1 antibody

Abstract

The present disclosure provides isolated binding molecules that bind to and block PD-L1, vectors comprising nucleic acid molecules encoding the amino acid sequences of the binding molecules, host cells containing the vectors, methods of preparing 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 requiring stimulation of an immune response, including cancer.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 62 / 835,764, filed on April 18, 2019, the disclosure of which is hereby incorporated by reference in its entirety.

[0003] Incorporation of Sequence Listing This application includes a sequence listing filed in ASCII format via EFS-Web, entitled "QLSF003PCT_ST25.txt", with a size of 158 KB and a creation date of April 17, 2020. The contents of the sequence listing are hereby incorporated herein by reference in their entirety. Background of the Invention

[0005] Programmed cell death protein 1 (PD-1) is a 288 amino acid cell surface protein molecule and is encoded by the PDCD1 gene in humans. PD-1 is a type I transmembrane protein that contains an immunoglobulin variable amino ectodomain terminal, a transmembrane region, and a cytoplasmic tail with an immunoreceptor 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. Programmed death-1 (PD-1) acts as a checkpoint protein on immune cells. It negatively regulates the immune system by binding to PD-L1, a ligand for the PD-1 receptor, to inhibit effective T cell function.

[0006] The PD-1 / PD-L1 interaction plays an important role in autoimmunity as well as cancer immunology. PD-L1 expression has been observed in a variety of cancers, including melanoma and non-small cell lung cancer. Many studies have shown that cancer cells overexpress PD-L1, which helps them escape immune attack by using the PD-1 / PD-ligand (PDL) pathway. Based on the conclusions of these studies, a variety of checkpoint blockade inhibitors targeting the PD-1 / PDL pathway have been developed.

[0007] PD-1 / PD-L1 blockade has achieved great clinical success in the fight against cancer. Various immunotherapies such as Pembrolizumab (Keytruda) and Nivolumab (Opdivo) targeting PD-1 and Atezolizumab (Tecentriq), Avelumab (Bavencio) and Durvalumab (Imfinzi) targeting PD-L1 can effectively block the binding between PD-1 and PD-L1, thereby reversing T cell dysfunction. However, in many advanced cancers, the objective response rate of monotherapy is only 20% (Xu-Monette et al., 2017).

[0008] Therefore, improved therapies targeting PD-1 and PD-L1 are an area of ​​great interest. In addition, multiple other immunotherapies, both alone and in combination, are now being tested in clinical trials. Therefore, there remains a need for more effective immunotherapies targeting the PD-1 / PD-L1 pathway, both alone and in combination with other therapeutic agents in drug treatment regimens. Summary of the invention

[0009] The present disclosure provides isolated anti-PD-L1 monoclonal antagonist antibodies and antigen-binding portions thereof that specifically bind to human PD-L1.

[0010] In one aspect of the invention, an isolated anti-PD-L1 monoclonal antagonist antibody or an antigen-binding portion thereof comprises a heavy chain variable region CDR3 comprising SEQ ID NO: 43. In some embodiments, the anti-PD-L1 monoclonal antagonist antibody or an antigen-binding portion thereof further comprises a heavy chain variable region CDR1 comprising SEQ ID NO: 31, and a heavy chain variable region CDR2 comprising SEQ ID NO: 37. In a preferred embodiment, the anti-PD-L1 monoclonal antagonist antibody or an antigen-binding portion thereof further comprises: (a) a light chain variable region CDR1 comprising SEQ ID NO: 13; (b) a light chain variable region CDR2 comprising SEQ ID NO: 19; and (c) a light chain variable region CDR3 comprising SEQ ID NO: 25.

[0011] In one embodiment, the antibody or portion comprises 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 comprises a heavy chain variable region comprising an 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 shown in SEQ ID NO: 7.

[0012] In another aspect of the invention, an isolated anti-PD-L1 monoclonal antagonist antibody or an antigen-binding portion thereof comprises a heavy chain variable region CDR3 comprising SEQ ID NO: 44. In some embodiments, the anti-PD-L1 monoclonal antagonist antibody or an antigen-binding portion thereof further comprises a heavy chain variable region CDR1 comprising SEQ ID NO: 32, and a heavy chain variable region CDR2 comprising SEQ ID NO: 38. In a preferred embodiment, the anti-PD-L1 monoclonal antagonist antibody or an antigen-binding portion thereof further comprises: (a) a light chain variable region CDR1 comprising SEQ ID NO: 14; (b) a light chain variable region CDR2 comprising SEQ ID NO: 20; and (c) a light chain variable region CDR3 comprising SEQ ID NO: 26.

[0013] In one embodiment, the antibody or portion comprises a light chain variable region amino acid sequence that is at least 95% identical to SEQ ID NO: 2 and a heavy chain variable region amino acid sequence that is at least 95% identical to SEQ ID NO: 8. In another embodiment, the antibody or portion comprises a heavy chain variable region comprising an 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 shown in SEQ ID NO: 8.

[0014] In another aspect of the invention, an isolated anti-PD-L1 monoclonal antagonist antibody or an antigen-binding portion thereof comprises a heavy chain variable region CDR3 comprising SEQ ID NO: 45. In some embodiments, the anti-PD-L1 monoclonal antagonist antibody or an antigen-binding portion thereof further comprises a heavy chain variable region CDR1 comprising SEQ ID NO: 33, and a heavy chain variable region CDR2 comprising SEQ ID NO: 39. In a preferred embodiment, the anti-PD-L1 monoclonal antagonist antibody or an antigen-binding portion thereof further comprises: (a) a light chain variable region CDR1 comprising SEQ ID NO: 15; (b) a light chain variable region CDR2 comprising SEQ ID NO: 21; and (c) a light chain variable region CDR3 comprising SEQ ID NO: 27.

[0015] In one embodiment, the antibody or portion comprises 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 comprises a heavy chain variable region comprising an 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 shown in SEQ ID NO: 9.

[0016] In another aspect of the invention, an isolated anti-PD-L1 monoclonal antagonist antibody or an antigen-binding portion thereof comprises a heavy chain variable region CDR3 comprising SEQ ID NO: 46. In some embodiments, the anti-PD-L1 monoclonal antagonist antibody or an antigen-binding portion thereof further comprises a heavy chain variable region CDR1 comprising SEQ ID NO: 34, and a heavy chain variable region CDR2 comprising SEQ ID NO: 40. In a preferred embodiment, the anti-PD-L1 monoclonal antagonist antibody or an antigen-binding portion thereof further comprises: (a) a light chain variable region CDR1 comprising SEQ ID NO: 16; (b) a light chain variable region CDR2 comprising SEQ ID NO: 22; and (c) a light chain variable region CDR3 comprising SEQ ID NO: 28.

[0017] In one embodiment, the antibody or portion comprises 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 comprises a heavy chain variable region comprising an 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 shown in SEQ ID NO: 10.

[0018] In another aspect of the invention, an isolated anti-PD-L1 monoclonal antagonist antibody or an antigen-binding portion thereof comprises a heavy chain variable region CDR3 comprising SEQ ID NO: 47. In some embodiments, the anti-PD-L1 monoclonal antagonist antibody or an antigen-binding portion thereof further comprises a heavy chain variable region CDR1 comprising SEQ ID NO: 35, and a heavy chain variable region CDR2 comprising SEQ ID NO: 41. In a preferred embodiment, the anti-PD-L1 monoclonal antagonist antibody or an antigen-binding portion thereof further comprises: (a) a light chain variable region CDR1 comprising SEQ ID NO: 17; (b) a light chain variable region CDR2 comprising SEQ ID NO: 23; and (c) a light chain variable region CDR3 comprising SEQ ID NO: 29.

[0019] In one embodiment, the antibody or portion comprises 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 comprises a heavy chain variable region comprising an 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 shown in SEQ ID NO: 11.

[0020] In another aspect of the invention, an isolated anti-PD-L1 monoclonal antagonist antibody or an antigen-binding portion thereof comprises a heavy chain variable region CDR3 comprising SEQ ID NO: 48. In some embodiments, the anti-PD-L1 monoclonal antagonist antibody or an antigen-binding portion thereof further comprises a heavy chain variable region CDR1 comprising SEQ ID NO: 36, and a heavy chain variable region CDR2 comprising SEQ ID NO: 42. In a preferred embodiment, the anti-PD-L1 monoclonal antagonist antibody or an antigen-binding portion thereof further comprises: (a) a light chain variable region CDR1 comprising SEQ ID NO: 18; (b) a light chain variable region CDR2 comprising SEQ ID NO: 24; and (c) a light chain variable region CDR3 comprising SEQ ID NO: 30.

[0021] In one embodiment, the antibody or portion comprises 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 comprises a heavy chain variable region comprising an 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 shown in SEQ ID NO: 12.

[0022] In another aspect of the invention, an isolated anti-PD-L1 monoclonal antagonist antibody, or an antigen-binding portion thereof, comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:65 to SEQ ID NO:67; and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:49 to SEQ ID NO:51.

[0023] In another aspect of the invention, an isolated anti-PD-L1 monoclonal antagonist antibody, or an antigen-binding portion thereof, comprises: a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:68 to SEQ ID NO:70; and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:55 to SEQ ID NO:58.

[0024] In another aspect of the invention, an isolated anti-PD-L1 monoclonal antagonist antibody, or an antigen-binding portion thereof, comprises: a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:71 to SEQ ID NO:76; and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:59 to SEQ ID NO:61.

[0025] In another aspect of the invention, an isolated anti-PD-L1 monoclonal antagonist antibody, or an antigen-binding portion thereof, comprises: a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:77 to SEQ ID NO:78; and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:62 to SEQ ID NO:63.

[0026] In another aspect of the invention, an isolated anti-PD-L1 monoclonal antagonist antibody, or an antigen-binding portion thereof, comprises: a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:79 to SEQ ID NO:80; and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:64.

[0027] In another aspect of the invention, an isolated anti-PD-L1 monoclonal antagonist antibody, or an antigen-binding portion thereof, comprises: a heavy chain variable region comprising an amino acid sequence selected from SEQ ID NO:81 to SEQ ID NO:83; and a light chain variable region comprising an amino acid sequence selected from SEQ ID NO:52 to SEQ ID NO:54.

[0028] In another aspect of the invention, an isolated monoclonal antibody or antigen binding portion thereof comprises a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:84 to SEQ ID No:99, and a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:100 to SEQ ID No:118.

[0029] The antibodies of the disclosed invention can be further engineered into a form suitable for human treatment by modifications that minimize immunogenicity. Suitable antibodies include, but are not limited to, chimeric antibodies and humanized antibodies. The affinity, stability and specificity of the disclosed antibodies can also be further optimized by techniques known to those skilled in the art. Other forms may involve oligomerization, drug conjugation and fusion of the disclosed antibodies with other functional proteins.

[0030] The antibodies of the disclosed invention may be, for example, full-length antibodies, such as IgG1, IgG2, IgG3 or IgG4 isotypes. Alternatively, the disclosed antibodies may be antibody fragments, such as Fab fragments, Fab' fragments 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 may be bispecific antibodies.

[0031] In another aspect of the present invention, the antibody or antigen-binding fragment thereof has a 5×10 -8 M to 1×10 -10 M or 1.32×10 -9 M to 2.68×10 -10 The affinity (KD) is in the range of M.

[0032] 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.

[0033] In another aspect of the present invention, a composition comprising the isolated anti-PD-L1 monoclonal antagonist antibody or an antigen-binding portion thereof is also provided.

[0034] In another aspect of the present invention, a pharmaceutical composition comprising the isolated anti-PD-L1 monoclonal antagonist antibody or antigen-binding portion thereof and a pharmaceutically acceptable transporter is also provided. A composition comprising the immunoconjugate of the present invention and a pharmaceutically acceptable transporter is also provided.

[0035] In another aspect of the present invention, a vector comprising an isolated nucleic acid molecule encoding the antibody or antigen-binding portion thereof, and a host cell comprising an expression vector comprising the nucleic acid molecule are also provided.

[0036] The present invention further provides a method of stimulating an immune response using the anti-PD-L1 antagonist antibody of the present disclosure. For example, in one embodiment, the present disclosure provides a method of treating a subject in need thereof, comprising the step of administering to the subject an effective amount of the antibody or antigen binding portion of the present disclosure.

[0037] In another aspect, the present disclosure provides a method of treating cancer in a human, comprising the step of administering to the human an amount of an anti-PD-L1 antagonist antibody or antigen-binding portion of the present disclosure effective to treat the cancer.

[0038] In another aspect, the present disclosure provides a method of treating an infectious disease in a human, comprising the step of administering to the human an amount of an anti-PD-L1 antagonist antibody or antigen-binding portion of the present disclosure effective to treat the infectious disease.

[0039] Other features and advantages of the present disclosure will be apparent from the following description and examples, which should not be construed as limiting.The contents of all references, GenBank entries, patents, and published patent applications cited in this application are expressly incorporated herein by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Exemplary embodiments are shown in the referenced drawings.The embodiments and drawings disclosed herein are to be considered for illustrative rather than restrictive purposes.

[0041] Figure 1 The anti-PD-L1 lead candidate is shown to bind to PD-L1 on A431 cells.

[0042] Figure 2 The anti-PD-L1 lead candidate is shown to block PD-1 interaction on HEK293 / huPD-L1 cells.

[0043] Figure 3It was shown that anti-PD-L1 antibody enhanced IL-2 release by SEB stimulation.

[0044] Figure 4 It was shown that PD-L1 antibody enhanced IFNγ secretion in a mixed lymphocyte reaction assay, suggesting that the lead anti-PD-L1 antibody blocked PD-1-mediated inhibition.

[0045] Figure 5 Blockade of PD-L1 by the lead antibody was shown to enhance interferon gamma production by T cells in a CMV peptide-specific recall response assay.

[0046] Figure 6 The thermal stability of the humanized anti-PD-L1 lead antibody is shown. DETAILED DESCRIPTION

[0047] The embodiments and aspects thereof are described and illustrated below in conjunction with systems, compositions, and methods which are for exemplary and illustrative purposes only and not limiting in scope.

[0048] definition

[0049] As used herein, the terms "include" or "comprising" are used to indicate compositions, methods and their respective components that are useful for the embodiments but may include unspecified elements (whether useful or not). Those skilled in the art will understand that, in general, the terms used herein are generally intended to be "open" terms (e.g., the term "include" should be interpreted as "including but not limited to", the term "have" should be interpreted as "have at least", the term "include" should be interpreted as "include but not limited to", etc.).

[0050] Unless otherwise stated, the terms "a", "an" and "said" and similar quotations used in the context of describing a specific embodiment of the present application (especially in the context of the claims) may be interpreted as covering the singular and plural. References to numerical ranges herein are intended only to be used as a shorthand method of referring to each individual value falling within the range individually. Unless otherwise stated herein, each individual value is incorporated into the specification as if it were individually quoted herein. Unless otherwise stated herein or clearly contradictory to the context, all methods described herein may be performed in any suitable order. The use of all examples or exemplary language (e.g., "such as") associated with a specific embodiment herein is intended only to better illustrate the present application and is not intended to limit the scope of the present application to which protection is otherwise claimed. The abbreviation "eg" is derived from Latin exempli gratia and is used herein to indicate non-limiting examples. Therefore, the abbreviation "eg" is synonymous with the word "for example". Any language in this specification should not be interpreted as representing any unclaimed element that is essential to the practice of the present application.

[0051] As used herein, the term "about" refers to a measurable value, such as an amount, duration, etc., and encompasses variations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or ±0.1% of the specified value.

[0052] As used herein, the term "epitope" may include any protein determinant that can specifically bind to an immunoglobulin or T cell receptor. Epitope determinants are usually composed of chemically active molecular surface groups, such as amino acids or sugar side chains, and usually have specific three-dimensional structural characteristics and specific charge characteristics. When the equilibrium dissociation constant is ≤1 μM (preferably ≤100nM, most preferably ≤10nM), the antibody is said to specifically bind to the antigen.

[0053] The term "K D ” may refer to the equilibrium dissociation constant for a specific antibody-antigen interaction.

[0054] As used herein, the term "immune response" may refer to, for example, the actions of lymphocytes, antigen presenting cells, phagocytes, granulocytes, and soluble macromolecules (including antibodies, cytokines, and complement) produced by the above cells or the liver, which can cause selective damage to the organism, destroy or eliminate invading pathogens, cells or tissues infected with pathogens, cancer cells, or normal body cells or tissues (in the case of autoimmunity or pathological inflammation) in the organism.

[0055] As used herein, "antigen-specific T cell response" may refer to a T cell response caused by stimulating T cells with a T cell-specific antigen. Non-limiting examples of T cell responses to antigen-specific stimulation include proliferation and production of cytokines (eg, production of IL-2).

[0056] The term "antibody" as used herein refers to a complete immunoglobulin or a monoclonal or polyclonal antigen-binding fragment having an Fc (fragment crystallizable) region or an FcRn binding fragment of an Fc region, referred to herein as an "Fc fragment" or "Fc region". Antigen-binding fragments can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies. Antigen-binding fragments include, among others, Fab, Fab', F(ab')2, Fv, dAb and complementary determining region (CDR) fragments, single-chain antibodies (scFv), single-domain antibodies, chimeric antibodies, diabodies and polypeptides comprising at least a portion of an immunoglobulin sufficient to confer specific antigens bound to the polypeptide. The Fc region includes portions of the two heavy chains that constitute two or three classes of antibodies. The Fc region can be produced by recombinant DNA technology or by enzymatic (e.g., papain cleavage) or by chemical cleavage of intact antibodies.

[0057] The term "antibody fragment" as used herein refers to a protein fragment that comprises only a portion of an intact antibody, generally including the antigen binding site of the intact antibody and thus retains the ability to bind antigen. Examples of antibody fragments encompassed by this definition include: (i) a Fab fragment having a VL region, a CL region, a VH region, and a CH1 region; (ii) a Fab' fragment, which is a Fab fragment having one or more cysteine ​​residues at the C-terminus of the CH1 region; (iii) a Fd fragment having a VH region and a CH1 region; (iv) a Fd' fragment having a VH region and a CH1 region and one or more cysteine ​​residues at the C-terminus of the CH1 region; (v) a Fv fragment having a single-arm VL region and a VH region of an antibody; (vi) a dAb fragment consisting of a VH region (Ward et al., Nature 341, 544-546 (1989)); (vii) an isolated CDR region; (viii) a F(ab')2 fragment (a bivalent fragment) comprising two Fab' fragments linked by a disulfide bond at the hinge region; (ix) a single-chain antibody molecule (e.g., single-chain Fv; scFv) (Bird et al., Science 341, 544-546 (1989)). 242:423-426 (1988); and Huston et al., PNAS (USA) 85:5879-5883 (1988)); (x) "diabodies" with two antigen-binding sites, comprising a heavy chain variable region (VH) connected to a light chain variable region (VL) in the same polypeptide chain (see, e.g., EP 404,097; WO 93 / 11161; and Hollinger et al., Natl. Acad. Sci. USA, 90:6444-6448 (1993)); (xi) "linear antibodies" comprising a pair of tandem Fd segments (VH-CH1-VH-CH1), which together with complementary light chain polypeptides form a pair of antigen-binding regions (Zapata et al., Protein Eng. 8(10):1057-1062 (1995); and U.S. Pat. No. 5,641,870).

[0058] As used herein, "single chain variable fragment", "single chain antibody variable fragment" or "scFv" antibody refers to an antibody format containing only the variable regions of the heavy chain (VH) and the light chain (VL) connected by a linker peptide. scFv can be expressed as a single chain polypeptide. scFv retains the specificity of the intact antibody from which it is derived. The light chain and heavy chain can be in any order, for example, VH-linker-VL or VL-linker-VH, as long as the specificity of the scFv for the target antigen is maintained.

[0059] As used herein, "isolated antibody" may refer to an antibody that is substantially free of other antibodies with different antigenic specificities (e.g., an isolated antibody that specifically binds to a 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 a human PD-L1 protein may have cross-reactivity with other antigens (such as PD-L1 proteins from other species). In addition, an isolated antibody may be substantially free of other cellular materials and / or chemicals.

[0060] Anti-PD-L1 antagonist antibody-producing cells, such as hybridomas, can be selected, cloned, and further screened to obtain desired characteristics, including robust growth, high antibody production, and desired antibody characteristics. Hybridomas can be expanded in syngeneic animals, animals lacking an immune system (e.g., nude mice), or in vitro cell culture. Methods for selecting, cloning, and expanding hybridomas are well known to those of ordinary skill in the art.

[0061] The term "monoclonal antibody" or "monoclonal antibody composition" as used herein may 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.

[0062] As used herein, the term "recombinant human antibody" may refer to all human antibodies prepared, expressed, produced or isolated by recombinant means, such as (a) antibodies isolated from animals (e.g., mice) that are transgenic or transchromosomal for human immunoglobulin genes or hybridomas prepared therefrom (as described below), (b) antibodies isolated from host cells transformed to express human antibodies, such as antibodies isolated from transfectomas, (c) antibodies isolated from recombinant, combinatorial human antibody libraries, and (d) antibodies prepared, expressed, produced or isolated by any other means involving splicing human immunoglobulin gene sequences to 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 may be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, somatic mutagenesis in vivo), so that the amino acid sequences of the VH and VL regions of the recombinant antibodies, although derived from and related to human germline VH and VL sequences, may not exist in the human antibody germline library in vivo under natural conditions.

[0063] The term "isotype" may refer to the antibody class (e.g., IgM or IgG1) encoded by the heavy chain constant region gene. The antibody may be an immunoglobulin G (IgG), IgM, IgE, IgA or IgD molecule, or derived from them.

[0064] Herein, the phrases "an antibody that recognizes an antigen" and "an antibody specific for an antigen" are used interchangeably with the term "an antibody that specifically binds to an antigen."

[0065] As used herein, an antibody that "specifically binds to human PD-L1" may refer to an antibody that binds to human PD-L1 protein (and possibly PD-L1 protein from one or more non-human species) but does not substantially bind to non-PD-L1 proteins. Preferably, the antibody binds to human PD-L1 protein with "high affinity", i.e., with a specific affinity of 1×10 -7 M or less, more preferably 5×10 -8 M or less, more preferably 3×10 -8 M or less, more preferably 1×10 -8 M or less, more preferably 5×10 -9 M or less or even more preferably 1×10 -9 M or less K D .

[0066] As used herein, the term "substantially does not bind" to a protein or cell may mean that it cannot bind or does not bind to a protein or cell with high affinity, i.e., with an affinity of 2×10 -6 M or more, more preferably 1×10 -5 M or more, more preferably 1×10 -4 M or more, more preferably 1×10 -3 M or more, even more preferably 1×10 -2 M or above K D Bind to proteins or cells.

[0067] The term "high affinity" for IgG antibodies may refer to antibodies with a 1×10 -6 M or less, preferably 1×10 -7 M or less, more preferably 1×10 -8 M or less, even more preferably 1×10 -9 M or less, even more preferably 1×10 -10 M or less K D However, "high affinity" binding may vary for other antibody isotypes. The term "pharmaceutical formulation" refers to a preparation that is in a form that permits the biological activity of the active ingredient contained therein to be effective and does not contain additional ingredients that are unacceptably toxic to a subject to which the preparation would be administered.

[0068] A "therapeutically effective amount" of an agent (e.g., a pharmaceutical preparation or cell) refers to an effective amount that achieves a desired therapeutic outcome, such as a pharmacokinetic or pharmacodynamic effect for treating a disease, condition, or disorder and / or a therapeutic effect, within the necessary dosage and time period. A therapeutically effective amount may vary depending on factors such as the disease state, age, sex, and weight of the subject, as well as the cell population being administered. In some embodiments, the methods provided herein involve administering cells and / or compositions in an effective amount (e.g., a therapeutically effective amount).

[0069] As used herein, an "antagonist antibody" is 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. For example, an antagonist can bind to PD-L1 and block the binding of PD-L1 to PD-1, thereby inhibiting phosphorylation of the PD-1 receptor or can inhibit signaling / cell activation. In one embodiment, the antibody of the invention is an anti-PD-L1 antagonist antibody.

[0070] A "CDR-grafted antibody" is an antibody that comprises one or more CDRs derived from a particular species or isotype and the framework of another antibody of the same or different species or isotype.

[0071] "Humanized antibodies" have sequences that are different from antibodies derived from non-human species by one or more amino acid substitutions, deletions and / or additions, so that when administered to human subjects, humanized antibodies are less likely to induce an immune response and / or induce a less severe immune response than non-human species antibodies. In one embodiment, specific amino acids in the framework and constant region of the heavy chain and / or light chain of non-human species antibodies are mutated to produce humanized antibodies. In another embodiment, the constant region from a human antibody is fused with the variable region of a non-human species. In another embodiment, the humanized antibody is a CDR-grafted antibody that comprises one or more CDRs derived from antibodies of a specific species or isotype and a human antibody framework. In another embodiment, one or more amino acid residues in one or more CDR sequences of a non-human antibody are changed to reduce the possible immunogenicity of the non-human antibody when it is administered to a human subject, wherein the amino acid residues that are changed are not critical for the immunospecific binding of the antibody to its antigen, or the changes made to the amino acid sequence are conservative changes, so that the binding of the humanized antibody to the antigen is not significantly inferior to the binding of the non-human antibody to the antigen. For examples of how to make humanized antibodies, see U.S. Patent Nos. 6,054,297, 5,886,152, and 5,877,293.

[0072] The term "chimeric antibody" (cAb) refers to an antibody comprising one or more regions from one antibody and one or more regions from one or more other antibodies. In one embodiment, one or more of the PD-L1 is derived from a human anti-PD-L1 antibody. In another embodiment, all CDRs are derived from human anti-PD-L antibodies. In another embodiment, CDRs from more than one human anti-PD-L1 antibody are mixed and matched in a chimeric antibody. For example, a chimeric antibody may comprise CDR1 from the light chain of a first human anti-PD-L1 antibody, CDR2 and CDR3 from the light chain of a second human anti-PD-L1 antibody, and CDRs from the heavy chain of a third anti-PD-L1 antibody. In the context of the present disclosure, cAb represents the variable region of a mouse monoclonal antibody fused to the Fc region of a human antibody. In another embodiment, other combinations are also possible.

[0073] The term "subject" may refer to any human or non-human animal. The subject may be male or female and may be of any suitable age, including infants, juveniles, adolescents, adults, and elderly 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, such as non-human primates, sheep, dogs, cats, cows, and horses, are preferred.

[0074] The binding of the antibodies of the disclosed invention to PD-L1 can be evaluated using one or more mature techniques in the art. For example, in a preferred embodiment, the antibodies can be tested by ELISA assays, for example using recombinant PD-L1 protein. Other suitable binding assays include, but are not limited to, flow cytometry assays, in which the antibodies react with cell lines expressing human PD-L1, such as Expi293 cells or ExpiCHO cells that have been transfected to express PD-L1 (e.g., human PD-L1) on their cell surfaces. In addition or alternatively, the binding of the antibodies can be tested in BIAcore binding assays, Octet Red96 (Pall), etc., including binding kinetics (e.g., K D value).

[0075] Preferably, the antibodies of the present invention bind to 5×10 -8 M or less K D Human PD-L1 protein, binding with 2×10 -8 M or less K D Human PD-L1 protein, binding with 2×10 -8 M or less K D Human PD-L1 protein, binding with 5×10 -9 M or less KD Human PD-L1 protein, binding with 4×10 -9 M or less K D Human PD-L1 protein, binding with 3×10 -9 M or less K D Human PD-L1 protein, binding with 2×10 -9 M or less K D Human PD-L1 protein, binding with 1×10 -9 M or less K D of human PD-L1 protein.

[0076] The present disclosure relates to isolated monoclonal antibodies or antigen-binding portions thereof that bind to and block PD-L1 and their uses. In a specific embodiment, the antibodies of the present disclosure are derived from identified heavy and light chain germline sequences and / or contain identified structural features, such as CDR regions containing identified amino acid sequences. The present disclosure provides isolated antibodies of the present disclosure, methods for preparing such antibodies, and antigen-binding portions thereof. The present disclosure also relates to methods of using antibodies, such as using the anti-PD-L1 antibodies of the present disclosure alone or in combination with other immunostimulatory antibodies to stimulate an immune response. Therefore, methods of using the anti-PD-L1 antagonist antibodies of the present disclosure are also provided, for example, including but not limited to treating human cancers. Various aspects of the present invention relate to antibodies and antibody fragments, pharmaceutical compositions, nucleic acids, recombinant expression vectors, and host cells for preparing such antibodies and fragments. The present invention also includes 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.

[0077] Complementarity determining regions (CDRs) are referred to as hypervariable regions in the light chain variable region and the heavy chain variable region. The more highly conserved portions of the variable region are referred to as frameworks (FRs). The complementary determining regions (CDRs) and framework regions (FRs) of a given antibody can be identified using the systems described by Kabat et al., Lefranc et al., and / or Honegger and Pluckthun above. Those skilled in the art are also familiar with 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. define a numbering system applicable to the variable region sequences of any antibody. One of ordinary skill in the art can clearly assign this "Kabat numbering" system to any variable region amino acid sequence without relying on any experimental data other than the sequence itself.

[0078] In certain embodiments, the invention provides anti-PD-L1 antagonist antibodies, or antigen-binding portions thereof. In one embodiment, the mouse antibody or portion comprises (a) a light chain variable region CDR1 comprising SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16; SEQ ID NO: 17, SEQ ID NO: 18, (b) a light chain variable region CDR2 comprising SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24; (c) a light chain variable region CDR3 comprising SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30; (d) a heavy chain variable region CDR1 comprising SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35 NO:35, SEQ ID NO:36; (e) a heavy chain variable region CDR2, which comprises SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42; (f) a heavy chain variable region CDR3, which comprises SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQID NO:46, SEQ ID NO:47, SEQ ID NO:48.

[0079] In one embodiment, the present disclosure 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, SEQ ID NO: 49, SEQ ID NO: 50, or SEQ ID NO: 51; and a heavy chain variable region amino acid sequence that is at least 95% identical to SEQ ID NO: 7, SEQ ID NO: 65, SEQ ID NO: 66, or SEQ ID NO: 67.

[0080] In another embodiment, the present disclosure 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, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, or SEQ ID NO:58; and a heavy chain variable region amino acid sequence that is at least 95% identical to SEQ ID NO:8, SEQ ID NO:68, SEQ ID NO:69, or SEQ ID NO:70.

[0081] In yet another embodiment, the present disclosure 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, SEQ ID NO:59, SEQ ID NO:60, or SEQ ID NO:61; and a heavy chain variable region amino acid sequence that is at least 95% identical to SEQ ID NO:9, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, or SEQ ID NO:76.

[0082] In yet another embodiment, the present disclosure 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, SEQ ID NO:62, or SEQ ID NO:63; and a heavy chain variable region amino acid sequence that is at least 95% identical to SEQ ID NO:10, SEQ ID NO:77, or SEQ ID NO:78.

[0083] In yet another embodiment, the present disclosure 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 SEQ ID NO:64; and a heavy chain variable region amino acid sequence that is at least 95% identical to SEQ ID NO:11, SEQ ID NO:79 or SEQ ID NO:80.

[0084] In yet another embodiment, the present disclosure 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, SEQ ID NO:52, SEQ ID NO:53, or SEQ ID NO:54; and a heavy chain variable region amino acid sequence that is at least 95% identical to SEQ ID NO:12, SEQ ID NO:81, SEQ ID No:82, or SEQ ID NO:83.

[0085] Given that each of these antibody Fabs can bind to human PD-L1, VH sequences and VL sequences can be "mixed and matched" to generate other anti-PD-L1 binding molecules of the present invention. Preferably, when VH chains and VL chains are mixed and matched, the VH sequence from a specific VH / VL pairing is replaced with a structurally similar VH sequence. Similarly, preferably, the VL sequence from a specific VH / VL pairing is replaced with a structurally similar VL sequence.

[0086] In some embodiments, the humanized anti-PD-L1 antibody or antigen-binding portion thereof comprises a heavy chain variable region comprising an amino acid sequence selected from SEQ ID NO: 65 to SEQ ID NO: 67; and a light chain variable region comprising an amino acid sequence selected from SEQ ID NO: 49 to SEQ ID NO: 51. Preferred heavy chain and light chain combinations include, but are not limited to:

[0087] (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 65 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 49;

[0088] (b) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 66 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 50;

[0089] (c) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 67 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 51;

[0090] In some embodiments, the humanized anti-PD-L1 antibody or antigen-binding portion thereof comprises a heavy chain variable region comprising an amino acid sequence selected from SEQ ID NO: 68 to SEQ ID NO: 70, and a light chain variable region comprising an amino acid sequence selected from SEQ ID NO: 55 to SEQ ID NO: 58. Preferred heavy chain and light chain combinations include, but are not limited to:

[0091] (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 68 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 55;

[0092] (b) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 69 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 56;

[0093] (c) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 70 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 57;

[0094] In some embodiments, the humanized anti-PD-L1 antibody or antigen-binding portion thereof comprises a heavy chain variable region comprising an amino acid sequence selected from SEQ ID NO: 71 to SEQ ID NO: 76, and a light chain variable region comprising an amino acid sequence selected from SEQ ID NO: 59 to SEQ ID NO: 61. Preferred heavy chain and light chain combinations include, but are not limited to:

[0095] (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 71 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 59;

[0096] (b) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 72 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 60;

[0097] (c) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 73 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 61;

[0098] In some embodiments, the humanized anti-PD-L1 antibody or antigen-binding portion thereof comprises a heavy chain variable region comprising an amino acid sequence selected from SEQ ID NO: 77 to SEQ ID NO: 78, and a light chain variable region comprising an amino acid sequence selected from SEQ ID NO: 62 to SEQ ID NO: 63. Preferred heavy chain and light chain combinations include, but are not limited to:

[0099] (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 77 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 62;

[0100] (b) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 78 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 63;

[0101] In some embodiments, the humanized anti-PD-L1 antibody or antigen-binding portion thereof comprises a heavy chain variable region comprising an amino acid sequence selected from SEQ ID NO: 79 to SEQ ID NO: 80; and a light chain variable region comprising an amino acid sequence selected from SEQ ID NO: 64. Preferred heavy chain and light chain combinations include, but are not limited to:

[0102] (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 79 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 64;

[0103] In some embodiments, the humanized anti-PD-L1 antibody or antigen-binding portion thereof comprises a heavy chain variable region comprising an amino acid sequence selected from SEQ ID NO: 81 to SEQ ID NO: 83, and a light chain variable region comprising an amino acid sequence selected from SEQ ID NO: 52 to SEQ ID NO: 54. Preferred heavy chain and light chain combinations include, but are not limited to:

[0104] (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 81 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 52;

[0105] (b) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 82 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 53;

[0106] (c) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 83 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 54;

[0107] In some embodiments, the humanized anti-PD-L1 antibody, or antigen binding portion thereof, comprises a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:84 to SEQ ID NO:99, and a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:100 to SEQ ID NO:118.

[0108] In one embodiment, the present invention provides an anti-PD-L1 antibody or an antigen-binding fragment thereof, comprising a heavy chain comprising a CDR3 region as shown in SEQ ID NO:43, and comprising a heavy chain variable region comprising an 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 shown in SEQ ID NO:7 or any one of SEQ ID NO:65 to SEQ ID NO:67.

[0109] In one embodiment, the present invention provides an anti-PD-L1 antibody or an antigen-binding fragment thereof, comprising a heavy chain comprising a CDR3 region as shown in SEQ ID NO: 44, and comprising a heavy chain variable region comprising an 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 shown in SEQ ID NO: 8 or any one of SEQ ID NO: 68 to SEQ ID NO: 70.

[0110] In one embodiment, the present invention provides an anti-PD-L1 antibody or an antigen-binding fragment thereof, comprising a heavy chain comprising a CDR3 region as shown in SEQ ID NO:45, and comprising a heavy chain variable region comprising an 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 shown in SEQ ID NO:9 or any one of SEQ ID NO:71 to SEQ ID NO:76.

[0111] In one embodiment, the present invention provides an anti-PD-L1 antibody or an antigen-binding fragment thereof, comprising a heavy chain comprising a CDR3 region as shown in SEQ ID NO: 46, and comprising a heavy chain variable region comprising an 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 shown in SEQ ID NO: 10 or any one of SEQ ID NO: 77 to SEQ ID NO: 78.

[0112] In one embodiment, the present invention provides an anti-PD-L1 antibody or an antigen-binding fragment thereof, comprising a heavy chain comprising a CDR3 region as shown in SEQ ID NO: 47, and comprising a heavy chain variable region comprising an 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 shown in SEQ ID NO: 11 or any one of SEQ ID NO: 79 to SEQ ID NO: 80.

[0113] In one embodiment, the present invention provides an anti-PD-L1 antibody or an antigen-binding fragment thereof, comprising a heavy chain comprising a CDR3 region as shown in SEQ ID NO: 48, and comprising a heavy chain variable region comprising an 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 shown in SEQ ID NO: 12 or any one of SEQ ID NO: 81 to SEQ ID NO: 83.

[0114] In one embodiment, the present invention provides an anti-PD-L1 antibody or an antigen-binding fragment thereof, comprising a light chain comprising a CDR3 region as shown in SEQ ID NO: 25, and having a light chain variable region comprising an 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 shown in SEQ ID NO: 1 or any one of SEQ ID NO: 49 to SEQ ID NO: 51.

[0115] In one embodiment, the present invention provides an anti-PD-L1 antibody or an antigen-binding fragment thereof, comprising a light chain comprising a CDR3 region as shown in SEQ ID NO: 26, and having a light chain variable region comprising an 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 shown in any one of SEQ ID NO: 2 or SEQ ID NO: 55 to SEQ ID NO: 58.

[0116] In one embodiment, the present invention provides an anti-PD-L1 antibody or an antigen-binding fragment thereof, comprising a light chain comprising a CDR3 region as shown in SEQ ID NO: 27, and having a light chain variable region comprising an 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 shown in SEQ ID NO: 3 or any one of SEQ ID NO: 59 to SEQ ID NO: 61.

[0117] In one embodiment, the present invention provides an anti-PD-L1 antibody or an antigen-binding fragment thereof, comprising a light chain comprising a CDR3 region as shown in SEQ ID NO: 28, and having a light chain variable region comprising an 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 shown in any one of SEQ ID NO: 4 or SEQ ID NO: 62 to SEQ ID NO: 63.

[0118] In one embodiment, the present invention provides an anti-PD-L1 antibody or an antigen-binding fragment thereof, comprising a light chain comprising a CDR3 region as shown in SEQ ID NO: 29, and having a light chain variable region comprising an 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 shown in any one of SEQ ID NO: 5 or SEQ ID NO: 64.

[0119] In one embodiment, the present invention provides an anti-PD-L1 antibody or an antigen-binding fragment thereof, comprising a light chain comprising a CDR3 region as shown in SEQ ID NO: 30, and having a light chain variable region comprising an 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 shown in SEQ ID NO: 6 or any one of SEQ ID NO: 52 to SEQ ID NO: 54.

[0120] Thus, in certain embodiments, the CDR3 region remains constant, while variability can be introduced into the remaining CDR and / or framework regions of the heavy and / or light chain, while the antibody or antigen-binding fragment thereof retains the ability to bind to PD-L1 and retains the functional properties of the parent, such as binding affinity.

[0121] In one embodiment, substitutions made within a heavy chain or light chain of at least 95% identity (or at least 96% identity, or at least 97% identity, or at least 98% identity, or at least 99% identity) are conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) of similar chemical properties (e.g., charge or hydrophobicity). In general, conservative amino acid substitutions do not substantially alter the functional properties of a protein. Where two or more amino acid sequences differ from one another by conservative substitutions, the percent sequence identity or similarity may be adjusted upward to correct for the conservative nature of the substitution. Means for making such adjustments are well known to those skilled in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24: 307-331, incorporated herein by reference. Examples of groups of amino acids having 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, tryptophan; (5) basic side chains: lysine, arginine, 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 may include any amino acid that occurs at a similar position in related evolutionarily conserved human variable heavy chain sequences, human variable light chain sequences, and orthologous sequences from non-human species.

[0122] Unless otherwise specified or implied from the context, the following terms and phrases include the meanings provided below. Unless otherwise explicitly stated, or obvious from the context, the following terms and phrases do not exclude the meanings that the term or phrase has acquired in the art to which it belongs. These definitions are provided to help describe specific embodiments and are not intended to limit the claimed invention, as the scope of the invention is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by ordinary technicians in the field to which the invention belongs.

[0123] All publications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The following description includes information that may be helpful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication referenced, whether expressly or implicitly, is prior art.

[0124] Working Example:

[0125] The following examples are not intended to limit the scope of the claims of the present invention, but are intended as examples of specific embodiments. Any variations in the example methods that occur to a skilled person are intended to fall within the scope of the present invention.

[0126] Vector construction:

[0127] The vector pcDNA3.4TOPO (Invitrogen) was connected to a short polylinker comprising EcoRI, XhoI and NotI. The resulting plasmid was digested with EcoRI and NotI restriction enzymes and purified by gel electrophoresis. For heavy chain cloning, we assembled the prepared vector, gblock encoding and VH region (IDT) and human IgG2 gblock encoding XhoI site at the junction of J chain and CH1 domain using the Gibson assembly method. Prepare and digest plasmids with EcoRI and XhoI to adapt to all humanized heavy variable (VH) domains with IgG2 isotypes. All assemblies were completed by the Gibson method (NEB). Light variable regions were constructed by a similar method and using gblocks to assemble the Vkappa region together by gblock fragments encoding constant kappa (Ck).

[0128] Protein expression, purification and binding characterization:

[0129] 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 1 μg total plasmid DNA / mL of culture. Heavy and light chain plasmids were mixed at a 1:1 ratio. The cultures were incubated at 37°C with shaking. After 16 hours, we added transfection enhancer 1 and transfection enhancer 2 to the cultures and continued to grow for 6 days. The supernatant was filtered and protein titers were determined using Octet Red96 (Pall) using an IgG quantification protocol. IgG was purified by Mab Select Sure Protein-A column purification on the ACTA PURE system and dialyzed overnight in PBS. The affinity of the purified antibodies for the antigen was characterized by Octet Red96 by loading the purified antibodies onto an anti-human heavy chain (AHC) capture sensor and measuring the binding and dissociation rates of the PD-L1 histidine-tagged target at three concentrations. (Table 1).

[0130] Table 1. Monovalent binding kinetics of humanized anti-PD-L1 antibodies compared to a benchmark control as determined by Octet.

[0131]

[0132]

[0133] Flow cytometric binding analysis of anti-PD-L1 antibodies to A431 cancer cells expressing PD-L1

[0134] Two days prior to FACS analysis, A431 cancer cells were seeded into 24-well plates at 30% to 60% confluence in the absence or presence of 1000U / ml recombinant human IFN-γ to stimulate PD-L1 expression. On the day of FACS analysis, the wells were washed with PBS and collected using Trypsin / EDTA. The isolated cells were washed twice, resuspended in FACS buffer at 5E6 cells / ml, and aliquoted into 96-well plates at 1E5 cells / well. The cells were stained on ice for 45 minutes using 5ug / ml anti-PD-L1 antibody, positive control antibody "C1-IgG1", or isotype IgG1, followed by washing and secondary staining with 1:500 dilution of goat anti-human IgG-AF647. After the final wash and addition of 7-AAD, the cells were analyzed by flow cytometry ( Figure 1 ).

[0135] PD-1His Tag Blocking Assay Detecting Humanized Anti-PD-L1 on hPDL1 / HEK293 Cells

[0136] To screen hybridomas, 293 cells expressing hPD-L1 were collected by Accutase and resuspended at 4E6 cells / ml using FACS wash buffer. To compare the blocking ability of the antibodies, we mixed 30ul cells / well (120,000), 30ul of 6ug / ml rhPD-1His Tag protein (2ug / ml final concentration) and 30ul of serial titrations of anti-PDL1 antibodies and incubated on ice for 20 minutes. After incubation, the cells were washed and bound PD-1 was detected using anti-His Tag-APC mouse IgG1 (R&D cat#IC050A). After the final wash and addition of 7-AAD, the cells were analyzed by flow cytometry. The blocking ability of the humanized antibodies was compared with the same assay using rhPD-1Fc-biotin at a final concentration of 0.5ug / ml and with detection using Streptavidin-APC (R&D cat#F0050) ( Figure 2 )(Table 2).

[0137]

[0138] SEB stimulation assay

[0139] Fresh PBMCs were diluted to 2E6 cells / ml in X-Vivo 15 medium (Lonza cat#04-744Q). 200ng / ml (2x) of SEB (Millipore cat#324798) was added to the PBMCs. Cells were treated with antibodies by adding 100ul of diluted antibody and 100ul of 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 ).

[0140] Mixed lymphocyte reaction assay

[0141] PBMCs were isolated from human buffy coats using density gradient centrifugation (Miltenyi Biotec) and washed 4 times with PBS. CD4+ T cells (Miltenyi Biotec cat# 130-096-533) were isolated from PBMCs and resuspended in X-Vivo 15 medium at 4E6 cells / ml.

[0142] Monocyte-derived dendritic cells were generated from positively selected CD14+ monocytes (Monocyte Isolation Kit II, Cat no. 130-091-153, Miltenyi Biotec). The cells were seeded at 5E5 cells / ml in complete RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and cultured for 7 days. On days 0, 2, and 5, 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) were supplemented to the culture. Immature DCs were collected on day 7 and resuspended in 5 ml of RPMI-1640, 10% FBS medium. Cells were incubated with 20 ug / ml mitomycin C (Roche REF 10107409001) with occasional mixing for 1 hour at 37°C, then washed and resuspended to 4E5 cells / ml in X-Vivo 15 medium.

[0143] In a 96-well flat-bottom plate, we mixed 50ul DC cells (2E4 DC / well), 100ul of serially diluted anti-PD-L1 antibody and control group at 2x concentration, and mixed 50ul purified CD4+T cells (2E5 CD4+T cells / well) at a ratio of 1:10 (DC:T cells). The plates were cultured for 5 days, and the supernatants were analyzed for IFNg release by ELISA (R&D Systems) ( Figure 4 ).

[0144] In vitro CMV antigen recall responses with anti-PDL1 therapy

[0145] Thawed PBMCs from CMV+ donors (Astarte Biologics) were counted and resuspended at 2E6 cells / ml in AIM-V medium containing β-mercaptoethanol (1:1000). In a 96-well flat-bottom plate, we mixed 100ul CMV+PBMCs (2E5 cells / well), 50ul of 5ug / ml CMV antigen (Astarte cat#1004) and 50ul of serially diluted anti-PDL1 antibody and control groups. The plates were incubated for 4 days and the supernatants were analyzed for IFNg release by ELISA (R&D Systems) ( Figure 5 ).

[0146] Protein thermal shift of humanized anti-PDL1 antibody

[0147] Ten (10) ug / ml anti-PD-L1 antibody was mixed with 2ul 50X protein thermal shift dye and PBS to a final volume of 100ul. The sample was aliquoted into a PCR 96-tube plate in four copies (25ul / well). The protein thermal shift reaction was measured on an Applied Biosystems StepOne Real-Time PCR instrument using a continuous temperature gradient from 22°C to 95°C with a change of 1°C every 1 minute and 5 seconds. The derivative method was used to analyze Tm ( Figure 6 ).

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Claims

1. An isolated anti-PD-L1 monoclonal antibody or an antigen-binding portion thereof, comprising a heavy chain variable region CDR1 of SEQ ID NO:31; a heavy chain variable region CDR2 of SEQ ID NO:37; a heavy chain variable region CDR3 of SEQ ID NO:43; a light chain variable region CDR1 of SEQ ID NO:13; a light chain variable region CDR2 of SEQ ID NO:19; and a light chain variable region CDR3 of SEQ ID NO:

25.

2. The monoclonal antibody or antigen-binding portion thereof according to claim 1, comprising 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.

3. The monoclonal antibody or antigen-binding portion thereof of claim 1, comprising a heavy chain variable region comprising an 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 shown in SEQ ID NO:

7.

4. The monoclonal antibody or antigen-binding portion thereof according to claim 1, comprising: a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 65 to SEQ ID NO: 67, and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 49 to SEQ ID NO:

51.

5. The monoclonal antibody or antigen-binding portion thereof according to any one of claims 1 to 4, wherein the monoclonal antibody or antigen-binding portion thereof is a Fab fragment, a F(ab')2 fragment, a Fv fragment or a single-chain antibody. The monoclonal antibody according to any one of claims 1 to 4, which is a chimeric antibody.

7. The monoclonal antibody according to any one of claims 1 to 4, which is a humanized antibody.

8. The monoclonal antibody according to any one of claims 1 to 4, which is an immunoglobulin G (IgG), IgM, IgE, IgA or IgD molecule. 9 . The monoclonal antibody according to claim 8 , which is IgG1, IgG2, IgG3 or IgG4.

10. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the affinity (KD) of the antibody or antigen-binding fragment thereof for PD-L1 is 5×10 -8 M to 1×10 -10 M.

11. A pharmaceutical composition comprising the antibody or antigen-binding portion thereof according to any one of claims 1 to 10 and a pharmaceutically acceptable transporter.

12. Use of the pharmaceutical composition according to claim 11 in the preparation of a medicament for treating a cancer associated with PD-L1 overexpression in a subject.

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