Anti-human PD-L1 antibodies and uses thereof

Abstract

The present invention relates to an anti-PD-L1 antibody or an antigen-binding fragment thereof, comprising: a heavy chain variable region sequence comprising three CDRs having a sequence of SEQ ID NO: 2 to 4, or 6 to 8; and a light chain variable region sequence comprising three CDRs having the sequence of SEQ ID NO: 10 to 12, or 14 to 16. The present disclosure also relates to a pharmaceutical composition and a method of detecting the performance of PD-L1 in a sample.

Description

Technical Field

[0001] The present invention relates to novel human antibodies, in particular to human monoclonal antibodies specific for PD-L1 with high affinity. In addition, the present invention relates to the use of such antibodies for the treatment and diagnosis of human diseases. Background Art

[0002] Programmed death protein 1 (PD-1) is an inhibitory member of the CD28 family of receptors, which also includes CD28, CTLA-4, ICOS, and BTLA. PD-1 is expressed on activated B cells, T cells, and myeloid cells (Bennett, Luxenberg et al. 2003, J Immunol 15 January 2003; 170(2):711-718). Its ligand, programmed cell death ligand 1 (PD-L1), is expressed on some tumor cells and is also expressed by activated B cells and T cells, dendritic cells, macrophages, and fibroblasts (Hansen, Du Pasquier et al. 2009, Mol Immunol. 2009 Jan; 46(3):457-72). PD-L1 binds to PD-1 to attenuate the cellular immune response by inducing T cell apoptosis or exhaustion. Blocking the PD-1 / PD-L1 pathway using monoclonal antibodies (anti-PD-1 or PD-L1) is a promising therapeutic approach being explored in studies of many types of human cancers (Sanmamed and Chen 2014, Cancer J. 2014 Jul-Aug; 20(4):256-61). The results of these studies indicate that PD-L1 plays an important role in helping tumors escape the immune system by promoting PD-1 / PD-L1 pathway activation.

[0003] PD-L1 expression has been observed in various solid tumors, including breast cancer, lung cancer, gastric cancer, colorectal cancer, hepatocellular carcinoma, renal cell carcinoma, testicular cancer, and papillary thyroid cancer. In addition, several meta-analyses have shown that PD-L1 overexpression indicates poor prognosis in many cancer types (Wang, Wang et al. 2015, J Intern Med. 2015 Oct; 278(4):369-95; Xu, Xu et al. 2015, Int J Clin Exp Med. 2015 Sep 15; 8(9):14595-603; Zhang, Kang et al. 2015, Medicine 94:e515, Iacovelli, Nole et al. 2016, TargetOncol. 2016; 11:143-148). Therefore, there is a need for preferred antibodies against PD-L1 for the treatment or diagnosis of diseases or conditions mediated by PD-L1. SUMMARY OF THE INVENTION

[0004] In one aspect, the present disclosure relates to an antibody specific for human PD-L1.

[0005] Accordingly, the present disclosure provides an antibody or an antigen-binding fragment thereof of the present disclosure, which comprises a heavy-chain variable region comprising HCDR1, HCDR2, and HCDR3, wherein

[0006] the HCDR1 sequence is GYSITSDYWN (SEQ ID NO: 2), the HCDR2 sequence is YISYTGSTYYNPSLKS (SEQ ID NO: 3), and the HCDR3 sequence is RGEWLSPFAY (SEQ ID NO: 4); or

[0007] the HCDR1 sequence is GYSITSDYWD (SEQ ID NO: 6), the HCDR2 sequence is YISYTGSTYYNPSLRS (SEQ ID NO: 7), and the HCDR3 sequence is RGGWLSPFVY (SEQ ID NO: 8);

[0008] wherein the HCDR sequences are defined according to the method of Kabat nomenclature.

[0009] According to an embodiment of the present disclosure, the complementarity-determining regions (CDRs) in the heavy-chain variable region sequence of the antibody specific for human PD-L1 have the sequences of SEQ ID NO: 2, 3, 4, 6, 7, or 8, as Figures 1A to 1B shown.

[0010] Accordingly, the present disclosure provides an antibody or an antigen-binding fragment thereof of the present disclosure, which comprises a light-chain variable region comprising LCDR1, LCDR2, and LCDR3, wherein

[0011] the LCDR1 sequence is KSSQSLLYSSNQKNSLA (SEQ ID NO: 10), the LCDR2 sequence is WASTRES (SEQ ID NO: 11), and the LCDR3 sequence is QQYYTYPFT (SEQ ID NO: 12); or

[0012] the LCDR1 sequence is KSRQSLLFSSNQKNSLA (SEQ ID NO: 14), the LCDR2 sequence is WASTRES (SEQ ID NO: 15), and the LCDR3 sequence is QQYYTYPFT (SEQ ID NO: 16);

[0013] wherein the LCDR sequences are defined according to the method of Kabat nomenclature.

[0014] According to an embodiment of the present disclosure, the complementarity determining regions in the light chain variable region of an antibody specific for human PD-L1 have the sequences of SEQ ID NO: 10, 11, 12, 14, 15 or 16, as Figures 2A to 2B shown in

[0015] In another aspect, the present disclosure relates to an antibody specific for human PD-L1 or an antigen-binding fragment thereof, which comprises a heavy chain variable region having HCDR1, HCDR2 and HCDR3 and a light chain variable region having LCDR1, LCDR2 and LCDR3, wherein

[0016] the heavy chain variable region, which comprises an HCDR1 having the sequence of SEQ ID NO: 2, an HCDR2 having the sequence of SEQ ID NO: 3 and an HCDR3 having the sequence of SEQ ID NO: 4, and the light chain variable region, which comprises an LCDR1 having the sequence of SEQ ID NO: 10, an LCDR2 having the sequence of SEQ ID NO: 11 and an LCDR3 having the sequence of SEQ ID NO: 12; or

[0017] the heavy chain variable region, which comprises an HCDR1 having the sequence of SEQ ID NO: 6, an HCDR2 having the sequence of SEQ ID NO: 7 and an HCDR3 having the sequence of SEQ ID NO: 8, and the light chain variable region, which comprises an LCDR1 having the sequence of SEQ ID NO: 14, an LCDR2 having the sequence of SEQ ID NO: 15 and an LCDR3 having the sequence of SEQ ID NO: 16.

[0018] In some embodiments of the present disclosure, the antibody is a chimeric, humanized, bispecific or human antibody.

[0019] In some embodiments of the present disclosure, the antibody is multispecific.

[0020] In some embodiments of the present disclosure, the heavy chain variable region of the anti-PD-L1 antibody or its antigen-binding fragment comprises the sequence of SEQ ID NO: 1 or a sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology with the sequence of SEQ ID NO: 1; and the light chain variable region comprises the sequence of SEQ ID NO: 9 or a sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology with the sequence of SEQ ID NO: 9.

[0021] In some embodiments of the present disclosure, the heavy chain variable region of the anti-PD-L1 antibody or its antigen-binding fragment comprises the sequence of SEQ ID NO:5 or a sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology with the sequence of SEQ ID NO:5; and the light chain variable region comprises the sequence of SEQ ID NO:13 or a sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology with the sequence of SEQ ID NO:13.

[0022] In some embodiments of the present disclosure, the heavy chain variable region of the anti-PD-L1 antibody or its antigen-binding fragment comprises the sequence of SEQ ID NO:25, 27 or 28 or a sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology with the sequence of SEQ ID NO:25, 27 or 28, and the light chain variable region comprises the sequence of SEQ ID NO:26, 29 or 30 or a sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology with the sequence of SEQ ID NO:26, 29 or 30.

[0023] In some embodiments of the present disclosure, the heavy chain variable region of the anti-PD-L1 antibody or its antigen-binding fragment comprises the sequence of SEQ ID NO:31 or 35 or a sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology with the sequence of SEQ ID NO:31 or 35, and the light chain variable region comprises the sequence of SEQ ID NO:32, 33 or 34 or a sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology with the sequence of SEQ ID NO:32, 33 or 34.

[0024] In some embodiments of the present disclosure, the anti-PD-L1 antibody or its antigen-binding fragment is a whole antibody, Fab fragment, F(ab')2 fragment or ScFv fragment.

[0025] In some embodiments of the present disclosure, the anti-PD-L1 antibody or its antigen-binding fragment is a fully human antibody.

[0026] In some embodiments of the present disclosure, the anti-PD-L1 antibody or its antigen-binding fragment comprises a heavy chain constant region selected from IgG1, IgG2 or IgG4 isotypes and a light chain constant region selected from κ subtypes or λ isotypes.

[0027] In some embodiments of the present disclosure, the anti-PD-L1 antibody or an antigen-binding fragment thereof forms part of a bispecific or multispecific antibody by binding to a second specific binding domain of a second target. The second specific binding domain of the second target may be, for example, anti-CD3, anti-ICOS, or anti-TIM3, etc.

[0028] In some embodiments of the present disclosure, the anti-PD-L1 antibody or an antigen-binding fragment thereof binds to a therapeutic agent (payload) to form an antibody-drug conjugate (ADC). In some embodiments, the therapeutic agent or payload can be selected for its ability to modulate the function of cells expressing PD-L1 or cells expressing PD-1. Such therapeutic agents or payloads can include, for example, DM1, MMAE, or MMAF.

[0029] In some embodiments of the present disclosure, the antibody or an antigen-binding fragment thereof is expressed on the surface of a cell. The cell can be an immune cell. In one embodiment of the present disclosure, the immune cell is a T cell.

[0030] The present disclosure also provides a vector encoding an antibody or an antigen-binding fragment thereof.

[0031] In some embodiments of the present disclosure, the vector comprises the sequence of SEQ ID NO: 1, 5, 25, 27, 28, 31, or 35 or a sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology to the sequence of SEQ ID NO: 1, 5, 25, 27, 28, 31, or 35; and / or the light chain variable region comprises the sequence of SEQ ID NO: 9, 13, 26, 29, 30, 32, 33, or 34 or a sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology to the sequence of SEQ ID NO: 9, 13, 26, 29, 30, 32, 33, or 34.

[0032] In another aspect, the present disclosure provides a genetically engineered cell expressing an antibody or an antigen-binding fragment thereof or containing the vector. The genetically engineered cell can be an immune cell.

[0033] The present disclosure also provides a method for preparing an antibody or an antigen-binding fragment thereof as disclosed herein, comprising: (a) introducing one or more polynucleotides encoding the antibody or antigen-binding fragment into a host cell; (b) culturing the host cell under conditions conducive to the expression of the one or more polynucleotides; and (c) optionally isolating the antibody or antigen-binding fragment from the host cell and / or the medium in which the host cell is grown.

[0034] The present disclosure provides a pharmaceutical composition comprising an effective amount of an antibody or an antigen-binding fragment thereof or a genetically engineered cell or immune cell and a pharmaceutically acceptable carrier.

[0035] In some embodiments of the present disclosure, the pharmaceutical composition is used to inhibit PD-L1-mediated signaling.

[0036] In some embodiments of the present disclosure, the pharmaceutical composition is used to treat a disease mediated by PD-L1.

[0037] In some embodiments of the present disclosure, the disease mediated by PD-L1 can be cancer. The cancer can include (but is not limited to): lung cancer, breast cancer, prostate cancer, colorectal cancer, gastric cancer, hepatocellular carcinoma, renal cell carcinoma, testicular cancer, melanoma, leukemia or papillary thyroid carcinoma and other advanced solid tumors.

[0038] The present disclosure provides a method for detecting PD-L1 expression, which comprises contacting a sample with an anti-PD-L1 antibody or an antigen-binding fragment thereof described herein.

[0039] Brief Description of the Drawings

[0040] Figures 1A to 1B Displays a composite human heavy chain designed to correspond to a murine anti-human PD-L1 antibody ( Figure 1A , 1G8; Figure 1B , 3C3) variable region sequence.

[0041] Figures 2A to 2B Displays a composite human light chain designed to correspond to a murine anti-human PD-L1 antibody ( Figure 2A , 1G8; Figure 2B , 3C3) variable region sequence.

[0042] Figure 3 Illustrates the binding of an anti-PD-L1 antibody to human PD-L1 using ELISA.

[0043] Figure 4 Displays an anti-PD-L1 antibody bound to HCC827 cells analyzed by flow cytometry.

[0044] Figure 5 Illustrates the ability of various antibodies to induce PD-1 / PD-L1 blockade.

[0045] Figure 6 Displays the in vivo efficacy of anti-PD-L1 mAb treatment in a murine syngeneic MC38 colon cancer model.

[0046] Figure 7Show the sequence analysis for the humanization of the VL and VH sequences of human PD-L1 mAb 3C3 and IMGT. In the first row shown under residue numbering according to the Kabat scheme, the revertant mutation sites are shown underlined.

[0047] Figure 8 Show the sequence analysis for the humanization of the VL and VH sequences of human PD-L1 mAb 1G8 and IMGT. In the first row shown under residue numbering according to the Kabat scheme, the revertant mutation sites are shown underlined.

[0048] Figure 9 Show the expression vectors used to generate the murine-human chimeric and humanized versions of PD-L1 (3C3) mAb. The detailed procedures for purifying the different versions of PD-L1 (3C3) mAb are described in this disclosure.

[0049] Figure 10 Depict the results of using the murine-human chimeric 3C3 MM and humanized 3C3 HuB2Hu0, 3C3 HuB2Hu, and 3C3HuB2Hu2 antibodies to determine the binding affinity of the humanized PD-L1 mAb. The detailed procedures for the expression, purification, and Kd analysis of the chimeric antibodies were carried out as described in this disclosure.

[0050] Figure 11 Depict the results of using the murine-human chimeric 1G8 MM and humanized 1G8 HuHu, 1G8 HuB2Hu0, 1G8HuHu2, 1G8 HuB2Hu, 1G8 HuHu2, and 1G8 HuB2Hu2 antibodies to determine the binding affinity of the humanized PD-L1 mAb. The detailed procedures for the expression, purification, and Kd analysis of the chimeric antibodies were carried out as described in this disclosure.

[0051] Figure 12 Illustrate the ability of various humanized PD-L1 3C3 antibodies to induce PD-1 / PD-L1 blockade.

[0052] Figure 13 Show the in vivo efficacy of treatment with the humanized PD-L1 3C3 antibody in a murine syngeneic MC38 colon cancer model.

[0053] Embodiments

[0054] It should be understood that this disclosure is not limited to the specific materials and methods described herein. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of this disclosure, which will be limited only by the appended claims.

[0055] It must be noted that, unless the context clearly dictates otherwise, as used in this specification and the appended claims, the singular forms "a / an" and "the" include plural referents.

[0056] As used herein, the term "antibody" means any antigen-binding molecule or molecular complex that comprises at least one complementarity determining region (CDR) that specifically binds to or interacts with a particular antigen (PD-L1). The term "antibody" includes immunoglobulin molecules and their multimers (e.g., IgM), which immunoglobulin molecules comprise four polypeptide chains, namely, two heavy (H) chains and two light (L) chains that are interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1, CH2, and CH3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region includes one domain (CL1). The VH and VL regions may be further subdivided into hypervariable regions, known as complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs arranged in the following order from the amino-terminus to the carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In various embodiments of the disclosure, the FRs of the anti-PD-L1 antibody (or its antigen-binding portion) may be identical to human germline sequences or may be naturally or artificially modified. Amino acid consensus sequences can be determined based on the alignment analysis of two or more CDRs.

[0057] As used herein, the term "specifically binds" means that an antibody does not cross-react to a significant extent with other antigenic determinants.

[0058] As used herein, the term "antigenic determinant" refers to the site on an antigen to which an antibody binds.

[0059] As used herein, the term "complementary determining region" (CDR) refers to the discontinuous antigen-combining sites found within the variable regions of heavy and light chain polypeptides. Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat et al., U.S. Dept. of Health and Human Services, "Sequences of proteins of immunological interest" (1991); Chothia et al., J. Mol. Biol. 196:901-917 (1987); and MacCallum et al., J. Mol. Biol. 262:732-745 (1996) have described CDRs, where the definitions include overlapping or subsets of amino acid residues when compared to each other.

[0060] As used herein, the term "monoclonal antibody" is not limited to antibodies produced by hybridoma technology. Monoclonal antibodies are obtained from a single clone by any available or known means in the art, which includes any eukaryotic, prokaryotic, or phage clone.

[0061] As used herein, the term "chimeric" antibody refers to an antibody having variable sequences derived from non-human immunoglobulins and constant regions of human immunoglobulins, typically selected from human immunoglobulin templates.

[0062] A "humanized" form of a non-human antibody is a chimeric immunoglobulin containing very few sequences derived from non-human immunoglobulins. In general, a humanized antibody will contain substantially all of at least one and usually two variable domains, where all or substantially all of the CDR regions correspond to the CDR regions of the non-human immunoglobulin and all or substantially all of the FR regions are FR regions of human immunoglobulin sequences.

[0063] As used herein, the term "composite antibody" refers to an antibody having variable regions that contain germline or non-germline immunoglobulin sequences from two or more non-related variable regions.

[0064] As used herein, the terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and like terms include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex.

[0065] As used in this disclosure, the term "therapeutic agent" means any compound, substance, drug, or active ingredient suitable for administration to a mammal (e.g., a human) that has a therapeutic or pharmacological effect.

[0066] As used herein, the term "immune cell" refers to a cell that plays a role in an immune response. Immune cells are of hematopoietic origin and include lymphocytes, such as B cells and T cells; natural killer cells; myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.

[0067] As used herein, the term "T cell" includes CD4 + T cells and CD8 + T cells. The term T cell also includes T helper type 1 T cells, T helper type 2 T cells, T helper type 17 T cells, and suppressor T cells.

[0068] As used herein, the term "vector" means a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop to which additional DNA fragments can be ligated. Another type of vector is a viral vector, in which an additional DNA segment can be ligated to the viral genome. Some vectors are capable of autonomous replication in the host cells into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can integrate into the genome of the host cell when introduced into the host cell and thereby replicate with the host genome. In addition, some vectors are capable of causing the expression of a gene to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). Generally, expression vectors used in recombinant DNA technology often take the form of plasmids. Since plasmids are the most commonly used form of vectors, the terms "plasmid" and "vector" may be used interchangeably in this specification. However, the present disclosure is intended to include such other forms of expression vectors that perform equivalent functions, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses).

[0069] The term "genetically engineered / genetic engineering" of a cell means the manipulation of genes using genetic material to alter the gene copies and / or the amount of gene expression in the cell. The genetic material can be in the form of DNA or RNA. The genetic material can be transferred into the cell by various means including viral transduction and non-viral transfection. After being genetically engineered, the amount of expression of certain genes in the cell can be permanently or temporarily altered.

[0070] As used in the present disclosure, the term "pharmaceutical composition" means a mixture containing a therapeutic agent administered to a mammal, such as a human, to prevent, treat, or eliminate a specific disease or pathological condition suffered by the mammal.

[0071] As used herein, the term "therapeutically effective amount" or "efficacious amount" refers to the amount of an antibody that, when administered to a mammal or other subject for treating a disease, is sufficient to effect such treatment of the disease.

[0072] As used herein, the terms "treatment / treating" and the like cover any treatment of a disease in a mammal, particularly a human, and include: (a) preventing a disease from occurring in an individual that may be predisposed to the disease but has not yet been diagnosed as having the disease; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing the disease to regress.

[0073] As used interchangeably herein, the terms "individual", "subject", "entity" and "patient" refer to a mammal, including (but not limited to) murine (rat, mouse), non-human primate, human, canine, feline, ungulate (e.g., equine, bovine, ovine, porcine, caprine), etc.

[0074] As used herein, the term "in need of treatment" refers to a judgment made by a caregiver (e.g., in the case of a human, a physician, nurse, nurse practitioner or individual; in the case of an animal (including a non-human mammal), a veterinarian) that an individual needs treatment or would benefit from treatment. This judgment is made based on a variety of factors within the caregiver's area of expertise and includes knowledge that the individual has or will develop a disease condition treatable with the compounds disclosed herein.

[0075] The terms "cancer", "tumor" and the like include pre-cancerous, neoplastic, transformed and cancerous cells, and may refer to solid tumors, or non-solid cancers (see, e.g., Edge et al., AJCC Cancer Staging Manual (7th ed. 2009); Cibas and Ducatman, Cytology: Diagnostic Principles and Clinical Correlates (3rd ed. 2009)). Cancer includes both benign neoplasms and malignant neoplasms (abnormal growths). "Transformation" refers to a spontaneous or induced phenotypic change, e.g., immortalization of cells, morphological changes, abnormal cell growth, reduced contact inhibition and anchorage and / or malignancy (see Freshney, Culture of Animal Cells a Manual of Basic Technique (3rd ed., 1994)). Although transformation can be caused by infection with a transforming virus and incorporation of new genomic DNA or uptake of foreign DNA, it can also occur spontaneously or after exposure to a carcinogen.

[0076] As used herein, the term "sample" encompasses a variety of sample types obtained from an individual, subject or patient and can be used for diagnostic or monitoring assays. The definition encompasses blood and other liquid samples of biological origin; solid tissue samples such as biopsy samples or tissue cultures or cells derived therefrom, and their progeny.

[0077] The present disclosure relates to a novel antibody that is specific for and has high affinity for PD-L1. The anti-PD-L1 antibody or antigen-binding fragment thereof can deliver therapeutic benefits to an individual. The anti-PD-L1 antibody or antigen-binding fragment thereof (which can be human or humanized) of the present disclosure can be used as a therapeutic agent for treating and / or diagnosing various disorders mediated by PD-L1, which are more fully described herein.

[0078] Specifically, the antibody or antigen-binding fragment thereof according to embodiments of the present disclosure is specific for an epitope in human PD-L1 or a fragment thereof.

[0079] The antibody or antigen-binding fragment thereof according to embodiments of the present disclosure can be full-length (e.g., IgG1 or IgG4 antibody), or can comprise only the antigen-binding portion (e.g., Fab, F(ab')2 or scFv fragment), and can be modified as needed to affect functionality.

[0080] The antibody or antigen-binding fragment thereof according to embodiments of the present disclosure is specific for human PD-L1. PD-L1, also known as CD274 or B7 homolog 1, is a 40 kDa type I transmembrane protein that is presumed to play a major role in suppressing the immune system during certain events such as pregnancy, tissue allograft, autoimmune diseases and other disease conditions such as hepatitis. Generally, the immune system reacts to foreign antigens associated with exogenous or endogenous danger signals, which trigger the proliferation of antigen-specific CD8+ T cells and / or CD4+ helper cells. The binding of PD-L1 to PD-1 or B7.1 transmits an inhibitory signal, which reduces the proliferation of these T cells and can also induce apoptosis, which is further mediated by downregulation of the gene Bcl-2.

[0081] Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units that mimic antibody hypervariable regions composed of amino acid residues (e.g., isolated complementarity-determining regions (CDRs), such as CDR3 peptides), or restricted FR3-CDR3-FR4 peptides. Other engineered molecules, such as domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, bispecific antibodies, trispecific antibodies, tetrafunctional antibodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains are also encompassed within the expression "antigen-binding fragment" as used herein.

[0082] Antigen-binding fragments of antibodies typically comprise at least one variable domain. The variable domain can be of any size or amino acid composition and generally will include at least one CDR adjacent to or in-frame with one or more framework sequences. In antigen-binding fragments having a VH domain associated with a VL domain, the VH and VL domains can be positioned relative to each other in any suitable configuration. For example, the variable regions can be dimers and contain VH-VH, VH-VL, or VL-VL dimers. Alternatively, the antigen-binding fragment of an antibody can contain a monomeric VH or VL domain.

[0083] Like whole antibody molecules, antigen-binding fragments can be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies will generally contain at least two different variable domains, where each variable domain is capable of specifically binding to a separate antigen or a different antigenic determinant on the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, can be adapted to the context of the antigen-binding fragments of the antibodies disclosed herein using conventional techniques available in the art.

[0084] In one embodiment of the disclosure, an antibody or its antigen-binding fragment is conjugated to a therapeutic agent. The antibodies disclosed herein can be used as antibody-drug conjugates (ADCs) that can specifically target PD-L1. The conjugate on the ADC can modulate immune cells expressing PD-L1 or cells that interact with cells expressing PD-L1 (e.g., cells expressing PD-1). Such ADCs can use any of the antibodies or their antigen-binding fragments disclosed herein. The drug (payload) conjugated to the antibody (or binding fragment) can be any drug commonly used in ADCs. The methods for conjugation can be those known in the art.

[0085] As applied to polypeptides, the term "homology" means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT with a predefined gap weight, have at least 95% sequence identity, even more preferably at least 98% or 99% sequence identity. According to an embodiment of the present disclosure, the GAP and Best fit programs in the GCG software are used with default parameters to determine sequence homology or sequence identity between closely related polypeptides.

[0086] In an embodiment of the present disclosure, an anti-PD-L1 antibody or an antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three CDR regions, CDRH1 (or HCDR1), CDRH2 (or HCDR2) and CDRH3 (or HCDR3) regions, and the light chain variable region comprises three CDR regions, CDRL1 (or LCDR1), CDRL2 (or LCDR2) and CDRL3 (or CDRL3) regions.

[0087] Reference Figures 1A to 2B , in some embodiments of the present disclosure, the CDRH1 region comprises the amino acid sequence of SEQ ID NO:2, the CDRH2 region comprises the amino acid sequence of SEQ ID NO:3, the CDRH3 region comprises the amino acid sequence of SEQ ID NO:4, the CDRL1 region comprises the amino acid sequence of SEQ ID NO:10, the CDRL2 region comprises the amino acid sequence of SEQ ID NO:11, and the CDRL3 region comprises the amino acid sequence of SEQ ID NO:12.

[0088] In some embodiments of the present disclosure, the CDRH1 region comprises the amino acid sequence of SEQ ID NO:6, the CDRH2 region comprises the amino acid sequence of SEQ ID NO:7, the CDRH3 region comprises the amino acid sequence of SEQ ID NO:8, the CDRL1 region comprises the amino acid sequence of SEQ IDNO:14, the CDRL2 region comprises the amino acid sequence of SEQ ID NO:15, and the CDRL3 region comprises the amino acid sequence of SEQ ID NO:16.

[0089] In some embodiments of the present disclosure, an anti-PD-L1 antibody or an antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ IDNO:1 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:9. In some embodiments of the present disclosure, the heavy chain variable region is encoded by the nucleic acid sequence of SEQ ID NO:17, and the light chain variable region is encoded by the nucleic acid sequence of SEQ IDNO:19.

[0090] In some embodiments of the present disclosure, the anti-PD-L1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:5 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:13. In some embodiments of the present disclosure, the heavy chain variable region is encoded by the nucleic acid sequence of SEQ ID NO:18, and the light chain variable region is encoded by the nucleic acid sequence of SEQ ID NO:20.

[0091] Compared to the corresponding germline sequences from which the antibodies are derived, the anti-PD-L1 antibodies disclosed herein may contain one or more amino acid substitutions, insertions, and / or deletions in the framework and / or CDR regions of the heavy and light chain variable domains. Such mutations can be readily determined by comparing the amino acid sequences disclosed herein with germline sequences obtained from, for example, public antibody sequence databases. The present disclosure includes an antibody and antigen-binding fragment thereof that is derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids in one or more framework regions and / or CDRs are mutated to the corresponding residues of the germline sequence from which the antibody is derived, or to the corresponding residues of another mammalian germline sequence, or to a conservative amino acid substitution of the corresponding germline residue (such sequence variations are collectively referred to herein as "germline mutations"). Starting with the heavy and light chain variable region sequences disclosed herein, one of ordinary skill in the art can readily generate many antibodies and antigen-binding fragments containing one or more individual germline mutations or combinations thereof. In certain embodiments, all of the framework and / or CDR residues within the VH and / or VL domains are mutated back to the residues found in the initial germline sequence from which the antibody was derived. In other embodiments, only certain residues are mutated back to the original germline sequence, such as only the mutated residues found within the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or only the mutated residues found within CDR1, CDR2, or CDR3. In other embodiments, one or more of the framework region and / or CDR residues are mutated to the corresponding residues of a different germline sequence (i.e., a germline sequence different from the germline sequence from which the antibody was originally derived). In addition, the antibodies of the present disclosure can contain any combination of two or more germline mutations within the framework region and / or CDRs, such as where some individual residues are mutated to the corresponding residues of a specific germline sequence while some other residues different from the original germline sequence are maintained or mutated to the corresponding residues of a different germline sequence. Once obtained, the antibodies and antigen-binding fragments containing one or more germline mutations can be readily tested for one or more desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced agonistic or antagonistic biological properties (as appropriate), reduced immunogenicity, etc. The present disclosure encompasses antibodies and antigen-binding fragments obtained in this general manner.

[0092] In some embodiments of the present disclosure, the antibody according to the present disclosure is a humanized antibody. To improve the binding affinity of the humanized antibody according to the present disclosure, some amino acid residues in the human framework region are replaced with the corresponding amino acid residues in a species of CDR (such as a rodent).

[0093] In some embodiments of the present disclosure, the humanized anti-PD-L1 antibody or its antigen-binding fragment comprises a heavy chain variable region having an amino acid sequence containing SEQ ID NO: 25, 27 or 28 and a light chain variable region having an amino acid sequence containing SEQ ID NO: 26, 29 or 30.

[0094] In some embodiments of the present disclosure, the humanized anti-PD-L1 antibody or its antigen-binding fragment comprises a heavy chain variable region having an amino acid sequence containing SEQ ID NO: 31 or 35 and a light chain variable region having an amino acid sequence containing SEQ ID NO: 32, 33 or 34.

[0095] The antibodies of the present disclosure can be monospecific, bispecific or multispecific. Multispecific antibodies can be specific for different antigenic determinants of a target polypeptide or can contain antigen-binding domains specific for more than one target polypeptide. The anti-PD-L1 antibodies of the present disclosure can be linked to another functional molecule (e.g., another peptide or protein) or co-expressed with another functional molecule. For example, the antibody or its fragment can be functionally linked (e.g., by chemical coupling, gene fusion, non-covalent association or other means) to one or more other molecular entities, such as another antibody or antibody fragment, to produce a bispecific or multispecific antibody with a second binding specificity. For example, the present disclosure includes bispecific antibodies in which one arm of the immunoglobulin is specific for PD-L1 or a fragment thereof, and the other arm of the immunoglobulin is specific for a second target or binds to a therapeutic agent.

[0096] In some embodiments of the present disclosure, the antibody or its antigen-binding fragment is in the form of a chimeric antigen receptor.

[0097] The term "chimeric antigen receptor" or alternatively "CAR" refers to a recombinant polypeptide construct comprising at least an extracellular antigen-binding domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as an "intracellular signaling domain") comprising a functional signaling domain derived from a stimulatory molecule as defined below. In some embodiments, the regions in the CAR polypeptide construct are in the same polypeptide chain, such as constituting a chimeric fusion protein. In some embodiments, the domains in the CAR polypeptide construct are not adjacent to each other, such as in different polypeptide chains.

[0098] Genes encoding the heavy and light chains of the antibody of interest can be cloned into cells. For example, genes encoding monoclonal antibodies can be cloned into hybridomas and used to produce recombinant monoclonal antibodies. Gene libraries encoding the heavy and light chains of monoclonal antibodies can also be prepared from hybridomas or plasma cells. Random combination of the heavy and light chain gene products generates a large pool of antibodies with different antigen specificities (see, e.g., Kuby, Immunology (3rd ed. 1997)).

[0099] Examples of methods for making an antibody or antigen-binding fragment include: (a) introducing one or more polynucleotides encoding the antibody or antigen-binding fragment into a host cell; (b) culturing the host cell under conditions conducive to the expression of the one or more polynucleotides; and (c) optionally isolating the antibody or antigen-binding fragment from the host cell and / or the medium in which the host cell is grown.

[0100] Vectors can be used to introduce polynucleotides encoding the antibodies or antigen-binding fragments of the present disclosure into host cells. In one embodiment, one type of vector is a "plasmid", which refers to a circular double-stranded DNA loop that can be ligated to additional DNA fragments. Another type of vector is a viral vector, where the additional DNA segment can be joined to the viral genome. Some vectors are capable of autonomous replication in the host cells into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can integrate into the genome of the host cell upon introduction into the host cell and thereby replicate with the host genome. In addition, some vectors are capable of causing the expression of genes operably linked thereto. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). Generally, expression vectors used in recombinant DNA technology are often in the form of plasmids. Since plasmids are the most commonly used form of vectors, in this specification, "plasmid" and "vector" may be used interchangeably. However, the present disclosure is intended to include such other forms of expression vectors that perform equivalent functions, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses).

[0101] In another aspect, the present disclosure provides genetically engineered cells expressing an antibody or its antigen-binding fragment or containing a vector. The genetically engineered cells can be immune cells or stem cells.

[0102] The present disclosure provides a pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof of the present disclosure, a genetically engineered cell, or an immune cell. The pharmaceutical composition of the present disclosure is formulated with a suitable diluent, carrier, excipient, and other agents that provide improved transfer, delivery, tolerance, and similar properties. The composition can be formulated for a specific use, such as for veterinary use or human medical use. The form of the composition and excipient, diluent, and / or carrier used will depend on the intended use of the antibody and the mode of administration for therapeutic use. Many suitable formulations can be found in the formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa. These formulations include, for example, powders, pastes, ointments, gels, waxes, oils, lipids, vesicles containing lipids (cationic or anionic) (such as LIPOFECTIN.TM., Life Technologies, Carlsbad, Calif.), DNA conjugates, anhydrous absorbent pastes, water-in-oil and oil-in-water emulsions, emulsion carbowaxes (polyethylene glycols of various molecular weights), semisolid gels, and semisolid mixtures containing carbowaxes. See also Powell et al. "Compendium of excipients for parenteral formulations" PDA (1998) J Pharm Sci Technol 52:238-311.

[0103] The dose of the antibody administered to a patient can vary depending on the patient's age and size, the target disease, condition, route of administration, and the like. More preferred doses are typically calculated based on body weight or body surface area. When the antibody of the present disclosure is used to treat a PD-L1-related condition or disease in an adult patient, intravenous administration of the antibody of the present disclosure can be advantageous. Depending on the severity of the condition, the frequency and duration of treatment can be adjusted. The effective dose and schedule for administering the antibody can be determined empirically; for example, the patient's progress can be monitored by periodic evaluation, and the dose can be adjusted accordingly. In addition, interspecies scaling of the dose can be performed using well-known methods in the art (e.g., Mordenti et al., 1991, Pharmaceut. Res. 8:1351).

[0104] A variety of delivery systems are known to those of skill in the art and can be used to administer the pharmaceutical compositions of the present disclosure, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, receptor-mediated endocytosis (see, e.g., Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Introduction methods include (but are not limited to) intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compositions can be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (such as oral mucosa, rectal mucosa, and intestinal mucosa, etc.), and can be administered together with other bioactive agents. Administration can be systemic or local.

[0105] The pharmaceutical compositions of the present disclosure can be delivered subcutaneously or intravenously using standard needles and syringes. Additionally, with respect to subcutaneous delivery, pen-type delivery devices are readily applicable in delivering the pharmaceutical compositions of the present disclosure. Such pen-type delivery devices can be reusable or disposable. Reusable pen-type delivery devices typically utilize replaceable cartridges containing the pharmaceutical composition. After all the pharmaceutical composition in the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen-type delivery device can then be reused. In disposable pen-type delivery devices, there is no replaceable cartridge. In fact, disposable pen-type delivery devices are prefilled with the pharmaceutical composition contained in a reservoir housed within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.

[0106] In certain instances, the pharmaceutical composition can be delivered in a controlled release system. In one embodiment, a pump can be used (see Langer, supra; Sefton 1987 CRC Crit. Ref. Biomed. Eng. 14:201). In another embodiment, polymeric materials can be used; see Controlled Release of Bioactive Agents, Langer and Wise (eds.), 1974, CRC Press, Boca Raton, Fla. In yet another embodiment, the controlled release system can be placed near the target of the composition, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, 1984, in Medical Applications of Controlled Release, supra, Vol. 2, pp. 115-138). Other controlled release systems are discussed in the review by Langer, 1990, Science 249:1527-1533.

[0107] Injectable preparations may include dosage forms for intravenous, subcutaneous, intradermal, and intramuscular injections, drip infusions, etc. These injectable preparations can be prepared by publicly known methods. For example, injectable preparations can be prepared, for example, by dissolving, suspending, or emulsifying the antibodies or their salts described above in a sterile aqueous medium or an oily medium conventionally used for injection. As an aqueous injection medium, there are, for example, physiological saline, isotonic solutions containing glucose and other adjuvants, etc., which can be used in combination with appropriate solubilizers such as alcohols (e.g., ethanol), polyols (e.g., propylene glycol, polyethylene glycol), nonionic surfactants [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], etc. As an oily medium, for example, sesame oil, soybean oil, etc. can be used, which can be used in combination with solubilizers (such as benzyl benzoate, benzyl alcohol, etc.). It is more preferred to fill the injection solution thus prepared into appropriate ampoules.

[0108] The pharmaceutical compositions described above for oral or parenteral use are preferably prepared in dosage forms suitable for unit doses corresponding to the doses of the active ingredients. Such dosage forms in unit doses include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc.

[0109] In some embodiments of the present disclosure, the pharmaceutical composition is for inhibiting PD-L1-mediated signaling.

[0110] In some embodiments of the present disclosure, the pharmaceutical composition is for treating diseases mediated by PD-L1.

[0111] The present disclosure provides a method for detecting the expression of PD-L1, which comprises contacting a sample with the anti-PD-L1 antibody or its antigen-binding fragment described herein.

[0112] The anti-PD-L1 antibody of the present disclosure can also be used for detecting and / or measuring PD-L1-expressing or PD-L1-expressing cells in a sample, for example, for diagnostic purposes. For example, the anti-PD-L1 antibody or its fragment can be used to diagnose conditions or diseases characterized by abnormal expression of PD-L1 (e.g., overexpression, underexpression, lack of expression, etc.). Exemplary diagnostic assays for PD-L1 may include, for example, contacting a sample obtained from a patient with the anti-PD-L1 antibody of the present disclosure, wherein the anti-PD-L1 antibody is labeled with a detectable label or a reporter molecule. Alternatively, an unlabeled anti-PD-L1 antibody can be used in combination with a secondary antibody labeled in a detectable manner itself for diagnostic applications. The detectable label or reporter molecule can be a radioisotope, such as 3 H, 14 C, 32 P, 35 S or 125I; a fluorescent or chemiluminescent moiety such as fluorescein isothiocyanate or rhodamine; or an enzyme such as alkaline phosphatase, β-galactosidase, horseradish peroxidase or luciferase. Specific exemplary assays that can be used to detect or measure PD-L1 in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescence-activated cell sorting (FACS).

[0113] The following examples are provided to assist those skilled in the art in practicing the disclosure.

[0114] Examples

[0115] The antibodies of the disclosure were confirmed to have specific binding to PD-L1 via ELISA. Briefly, PD-L1 was coated on a 96-well ELISA plate (0.1 μg / well). After binding of the anti-PD-L1 antibody, goat anti-mouse IgG conjugated to horseradish peroxidase (HRP) was used as the secondary antibody, and 3,3',5,5'-tetramethylbenzidine (TMB) was used as the substrate to evaluate antibody-PD-L1 binding. OD405 was read to calculate the activity. As shown in Table 1 and Figure 3 as shown, several murine hybridoma anti-human PD-L1 antibodies (mAbs 1G8 and 3C3). All showed specificity and tight binding to PD-L1.

[0116] Table 1. ELISA KD of several murine hybridoma anti-human PD-L1 antibodies

[0117]

[0118] To further verify the utility of the antibodies of the disclosure in cancer treatment, the ability of these antibodies to bind to PD-L1 expressed on cancer cells was evaluated. For example, the binding of anti-PD-L1 antibodies to cells expressing PD-L1 was analyzed by flow cytometry using HCC827 cells (lung adenocarcinoma), which express high levels of PD-L1. Briefly, HCC827 cells (higher PD-L1) were incubated with anti-PD-L1 antibodies for 1 hour and then analyzed by flow cytometry. As shown in Figure 4 as shown, both mAb 1G8 and 3C3 of the disclosure were able to bind to HCC827 cells, indicating that these antibodies can recognize PD-L1 on the surface of cancer cells. Other antibodies of the disclosure also exhibited similar activity. Thus, the antibodies of the disclosure can be used to treat cancer by binding to PD-L1 expressed on cancer cells and thereby inhibiting PD-L1-mediated immunosuppression or exhaustion.

[0119] Although the above experiments tested the binding of the antibodies of the present disclosure to PD-L1 molecules in vitro, such binding was also tested with PD-1 and PD-L1 expressed separately on interacting cells. For example, a PD-1 / PD-L1 blockade assay can use any commercial kit, such as a kit from Promega (Maddison, WI, USA). The Promega PD-1 / PD-L1 Blockade Bioassay is a bioluminescence cell-based assay. The assay kit consists of two genetically engineered cell lines: PD-1 effector cells, which are Jurkat T cells expressing human PD-1 and a luciferase reporter driven by an NFAT response element (NFAT-RE), and PD-L1 aAPC / CHO-K1 cells, which are CHO-K1 cells expressing human PD-L1 and an engineered cell surface protein designed to activate the TCR in an antigen-independent manner.

[0120] When the two cell types are co-cultured, the PD-1 / PD-L1 interaction inhibits TCR signaling and NFAT-RE-mediated luminescence. Addition of the anti-PD-L1 antibody of the present disclosure that blocks the PD-1 / PD-L1 interaction can release the inhibitory signal, causing TCR activation and NFAT-RE-mediated luminescence. The bioluminescence signal can be detected and quantified using a Bio-Glo TM Luciferase Assay System and a standard luminometer, such as those from (Maddison, WI, USA) Discovery System.

[0121] As shown in Table 2 and Figure 5 as indicated, both mAb 1G8 and 3C3 of the present disclosure demonstrated specific and potent activity in blocking the PD-1 / PD-L1 interaction. Other antibodies of the present disclosure also exhibited similar activity. These results confirm that the antibodies of the present disclosure will effectively relieve the immunosuppression mediated by the PD-1 and PD-L1 interaction on interacting cells. Therefore, the antibodies of the present disclosure should be suitable as therapeutic agents for diseases caused by immunosuppression or exhaustion due to PD-1 and / or PD-L1 signaling. Such diseases include various cancers.

[0122] Table 2. PD1 / PD-L1 Blockade Reporter Assay

[0123] 3C3 1G8 <![CDATA[EC 50 > 5.821E-10 6.198E-10

[0124] Some embodiments of the present disclosure relate to methods for treating or alleviating conditions / symptoms of diseases mediated by PD-1 and / or PD-L1 signaling; such diseases may include cancer. To demonstrate the utility of the antibodies of the present disclosure in treating cancer, murine syngeneic models were used. Briefly, B-hPD-1 / hPD-L1 mice were subcutaneously injected in the right anterior abdomen with MC38-hPD-L1 tumor cells (5×10 5 ) suspended in 0.1 mL PBS for tumor development. When the average tumor size reached 75±25 mm 3 , the tumor-bearing animals were randomly selected into seven study groups. Group G1 consisted of 6 mice. Groups G2 to G7 consisted of 8 mice each. Three groups were Mu IgG (5 mg / kg), 3C3 (5 mg / kg), and 1G8 (5 mg / kg). All test articles were administered intraperitoneally to the tumor-bearing mice twice a week for a total of six times. Tumor volume and body weight were measured and recorded twice a week. The study was terminated seven days after the last administration. At the end of this experiment, the tumors were removed from the euthanized animals, weighed, and photographed.

[0125] As shown in Table 3 and Figure 6 , no unplanned animal deaths or obvious clinical signs were found during the study. The body weights of all groups gradually increased during this study, indicating good tolerance of the animals to the test articles. On the 28th day after the start of treatment, the average tumor volume of the Mu IgG (5 mg / kg) group was 2486±447 mm 3 . For the 3C3 (5 mg / kg) treatment group, the average tumor volume was 415±155 mm 3 , with a TGITV of 85.3%. For the 1G8 (5 mg / kg) treatment group, the average tumor volume was 647±216 mm 3 , with a TGITV of 75.8%.

[0126] Table 3. Evaluation of the efficacy of αPD-L1 Ab in the treatment of a subcutaneous MC38-hPD-L1 colon cancer model in humanized B-hPD-1 / hPD-L1 mice

[0127] Mu IgG 3C3 1G8 <![CDATA[TGI 28天 (%)]]> 85.3 75.8

[0128] In this experiment, 3C3 and 1G8 exhibited significant anti-tumor activity at the tested doses and showed no negative impact on animal body weight or induction of any obvious clinical signs. These results clearly demonstrate that the antibodies of the present disclosure will be suitable for clinical use in treating cancers such as lung cancer, breast cancer, prostate cancer, colorectal cancer, etc.

[0129] Mouse monoclonal antibodies can induce strong immunogenicity and anti-drug antibodies in patients. Therefore, humanization of mouse monoclonal antibodies is an essential and critical step for further drug development. By using 3C3 and 1G8 mouse monoclonal antibodies as parental antibodies, the mAb CDR sequences defined based on Kabat are described in Figures 1 and 2 (SEQ ID NO:1 to SEQ ID NO:20).

[0130] For humanized mAb preparation, human germline VL and VH sequences with a higher degree of homology to the framework regions of 3C3 and 1G8 mAbs were identified from the IMGT database (International Immunogenetics Information ) Homology searches can be performed using sequence BLAST or similar methods. The mouse mAb variable region sequences were used as query sequences. These studies identified the human VH germline gene IGHV4-59*01 (SEQ ID NO:21) and VL germline genes IGKV4-1*01 (SEQ ID NO:22), IGKV1-39*01 (SEQ ID NO:23), and IGKV2-29*01 (SEQ ID NO:24) as VH and VL sequences more homologous to the corresponding heavy and light chain framework sequences in the mouse mAbs, respectively.

[0131] Based on the selected human heavy and light chain variable region homologs, anti-PD-L1 antibodies can be constructed by grafting the known CDR sequences from known anti-PD-L1 antibodies (e.g., mAbs 3C3 and 1G8) into the homologous human heavy and light chain variable sequences. As Figure 7 and Figure 8 shown, these light and heavy chain sequence pairs were used as examples for constructing humanized antibodies against human PD-L1.

[0132] CDR grafting onto the framework generates variable domains (VH and VL) from different sources. Such chimeric domains may not have optimal sequences. Therefore, the affinity of the antibody may not be optimal. To improve the binding affinity, some amino acids can be reverted to other species. These key amino acid residues sometimes affect antibody binding in the upper core region and interface region of the antibody (E. Stefan, H. Annemarie and P. Andreas Methods 34 (2004) 184-199). Among the following additional considerations: (i) avoid most of the structurally conserved strands of the Fv b-barrel; (ii) rank surface remodeling sites (mouse amino acids) by relatively high surface accessibility (e.g., greater than 30%); and (iii) classify the commonly reported framework risk sites. Based on the principles described above, six and eight revertant mutation sites were designed on the framework regions of humanized 3C3 Hu-B1 (VH) and 3C3Hu-B2 (VH), respectively ( Figure 7and Table 4). Humanized 1G8 Hu-B2 (VH) was designed with seven back-mutation sites on the framework region ( Figure 8 and Table 5). The resulting heavy chains of mAb 3C3 were HU 3C3 VH as SEQ ID NO:25; HU3C3 VHB2 as SEQ ID NO:27 and HU 3C3 VHB1 as SEQ ID NO:28. The resulting light chains of mAb 3C3 were HU03C3 VL as SEQ ID NO:26; HU 3C3 VL as SEQ ID NO:29 and HU2 3C3 VL as SEQ ID NO:30. The resulting heavy chains of mAb 1G8 were HU 1G8 VHB2 as SEQ ID NO:31 and HU 1G8 VH as SEQ ID NO:35. The resulting light chains of mAb1G8 were HU0 1G8 VL as SEQ ID NO:32; HU 1G8 VL as SEQ ID NO:33 and HU2 1G8 VL as SEQ ID NO:34.

[0133] Table 4: List of framework and back-mutation sites of humanized 3C3.

[0134]

[0135] Table 4 shows the primary sequence alignment of the framework regions of the VH segments of various anti-PD-L1 (3C3) antibodies: murine anti-PD-L1 antibody (M), humanized anti-PD-L1 antibody (Hu), back-mutated humanized anti-PD-L1 antibody (Hu-B1), further improved anti-PD-L1 antibody (Hu-B2). The primary sequence alignment of the framework regions of the VL segments of various anti-PD-L1 antibodies: murine anti-PD-L1 antibody (M), humanized anti-PD-L1 antibody (H), humanized anti-PD-L1 antibody without back-mutation (Hu0), further improved anti-PD-L1 antibody (Hu), and anti-PD-L1 antibody (Hu2).

[0136] Table 5: List of framework and back-mutation sites of humanized 1G8.

[0137]

[0138] Table 5 shows the primary sequence alignment of the framework regions of the VH segments of various anti-PD-L1 (1G8) antibodies: murine anti-PD-L1 antibody (M), humanized anti-PD-L1 antibody (Hu), revertant humanized anti-PD-L1 antibody, and further improved anti-PD-L1 antibody (Hu-B2). The primary sequence alignment of the framework regions of the VL segments of various anti-PD-L1 antibodies: murine anti-PD-L1 antibody (M), humanized anti-PD-L1 antibody (H), non-revertant humanized anti-PD-L1 antibody (Hu0), further improved anti-PD-L1 antibody (Hu), and anti-PD-L1 antibody (Hu2).

[0139] To confirm the affinity changes after humanization of murine antibodies, the variable regions of humanized light chain and humanized heavy chain were directly generated by nucleotide synthesis method. The murine or humanized variable regions were constructed into the human chimeric antibody expression vectors pTCAED heavy chain and pTCAED light chain plasmids ( Figure 9 ), and introduced into host cells to prepare cells expressing recombinant antibodies. FreeStyle TM 293 or Expi 293 cells (manufactured by INVITROGEN TM ) were used as host cells for expression. According to the instruction manual (manufactured by INVITROGEN TM ), the vectors were introduced into host cells by polyethyleneimine (PEI), and about 1.25 μg ratio of antibody expression vectors were introduced into 1x10 6 cells.

[0140] The culture supernatant containing human IgG antibody was prepared by the method described below. The antibody-producing cells were adapted to Free Style TM 293 expression medium (GIBCO TM ). The cells were cultured in a tissue culture flask, and when the viable cell ratio reached 90%, the culture supernatant was collected. The collected supernatant was filtered through 10 μm and 0.2 μm filters (manufactured by Millipore) to remove contaminants. The culture supernatant containing the antibody was affinity purified using Protein A (manufactured by MILLIPORE TM ), PBS as the absorption buffer, and 200 mM glycine buffer (pH 2.5) as the elution buffer. The eluate was adjusted to about pH 6.0 - 7.0 by adding 50 mM Tris buffer (pH 9.0). The prepared antibody solution was replaced with PBS using a dialysis membrane (10,000 MW cut-off value, manufactured by SPECTRUM TM Laboratories), and passed through a membrane filter with a pore size of 0.22 μm (manufactured by MILLIPORE TMManufacture) Filter sterilize to produce purified antibody. Determine the concentration of the purified antibody by measuring the absorbance at 280 nm and converting the measured value based on 1.45 optimal density equal to 1 mg / ml.

[0141] The binding activity of the humanized antibody can be effectively compared by using all combinations of the heavy and light chains expressed by the small-scale antibody. The concentration of the antibody in the culture supernatant is determined by anti-human IgG ELISA. For the PD-L1 ELISA, the plate is coated with 1 μg / ml of PD-L1-hFc and blocked with 5% milk-PBS. The analytical antibody is adjusted to 300 ng / ml, 100 μl / well. The anti-PD-L1 signal is measured by secondary goat anti-human κ HRP IgG 1:4000, and the color is generated by TMB substrate (KPL). The light absorbance value is measured at OD450 - 655 nm by a Bayer Read ELISA reader.

[0142] The binding signals of the 3C3 humanized antibodies HuHu0, HuHu, and HuHu2 to PDL1 are much lower (for comparison, mAb3C3, HuB2Hu0, HuB2Hu, and HuB2Hu2 in the binding ELISA) (Table 6). However, 3C3-HuB2Hu0, 3C3-HuB2Hu, and 3C3-HuB2Hu2 show binding signals more similar to those of the parental mouse pure line. Compared with the sequence of 3C3 Hu (VH), they contain eight beneficial mutations in the heavy chain framework region in 3C3HuB2 (VH) Figure 7 ). It was found that these amino acids had mutated back from the residues in 3C3 Hu (VH) to the corresponding residues in the mouse mAb (i.e., reverse mutations). All of these amino acids are in the heavy chain variable sequence, as Figure 7 shown. The fact that the reverse mutations produce more preferred binders indicates that these residues in the framework region indirectly contribute to the binding to PDL1. It may help to maintain the proper conformation in the CDR region.

[0143] Table 6: Expression levels and PD-L1 binding tests of humanized 3C3. FreeStyle 293 cell cultures grown in format D6. Expression μg / ml

[0144]

[0145]

[0146] Table 6 depicts the results of using chimeric PD-L1 and human PD-L1 mAb 3C3 antibodies to determine the binding affinity of the PD-L1 mAb. The detailed procedures for chimeric antibody expression, purification, and Kd analysis were carried out as described in this disclosure.

[0147] Coat ELISA plates with PDL1-hFc at 1 μg / ml and block with 5% milk-PBS, then add the analytical antibody in the form of 45 nM to 2.7x10 -3 nM dilutions (4-fold dilutions). Analyze by adding goat anti-human KAPPA HRP IgG at 1:4000 and measuring the binding curve, and determine the KD using GraphPad Prism software with a one-site specific binding for non-linear fitting method.

[0148] The binding affinities of the 3C3 humanized antibodies HuB2Hu0, HuB2Hu, and HuB2Hu2 are 1.08x10 -10 M, 9.36x10 -11 M, and 1.01x10 -10 M, respectively. All three of these humanized antibodies exhibit less than 2-fold loss of affinity compared to the parental mouse monoclonal 3C3 ( Figure 10 ). The binding affinities of the 1G8 humanized antibodies HuHu0, HuHu, and HuHu2 are much lower than that of the mouse monoclonal 1G8, being 3.99x10 -9 M, 3.24x10 -9 M, and 9.68x10 -9 M, respectively. However, the binding affinities of the 1G8 humanized antibodies HuB2Hu0, HuB2Hu, and HuB2Hu2 are 2.38x10 -10 M, 2.48x10 -10 M, and 1.08x10 -10 M, respectively. These humanized 1G8 variants exhibit affinities similar to that of the parental mouse monoclonal 1G8 ( Figure 11 ). Compared to the sequence of 1G8 Hu(VH), they contain seven beneficial mutations in the heavy chain framework region in 1G8 HuB2(VH) ( Figure 8 ). The fact that the revertant mutants generate more preferred binders indicates that these residues in the framework region indirectly contribute to the binding to PDL1.

[0149] As shown in Table 7 and Figure 12 , all of the mAbs of the 3C3 humanized antibodies HuB2Hu0, HuB2Hu, and HuB2Hu2 exhibit specific and effective activities in blocking the PD-1 / PD-L1 interaction. Other antibodies of the present disclosure also exhibit similar activities. These results confirm that the antibodies of the present disclosure will effectively relieve the immunosuppression mediated by the PD-1 and PD-L1 interaction on interacting cells. Therefore, the antibodies of the present disclosure should be applicable as therapeutic agents for diseases caused by immunosuppression or exhaustion due to PD-1 and / or PD-L1 signaling. Such diseases include various cancers.

[0150] Table 7.3 Analysis of Blocking Reports of C3 Humanized Antibodies

[0151] αPD-L1 3C3_B2Hu0 αPD-L1 3C3_B2Hu αPD-L1 3C3_B2Hu2 <![CDATA[EC 50 > 4.77E-10 4.41E-10 4.09E-10

[0152] As shown in Table 8 and Figure 13 as indicated, no unplanned animal deaths or obvious clinical signs were found during the study period. The body weights of all groups gradually increased during this study, indicating good tolerance of the animals to the test articles. At the end of this experiment, the mean tumor volume of the IgG group was 2537 ± 300 mm 3 . In the atezolizumab (Atz), αPD-L1 3C3 B2Hu0, αPD-L1 3C3 B2Hu groups, and the Atz group respectively, the mean tumor volume was 1926 ± 436 mm3, among which the TGITV was 24.9%. In the αPD-L1 3C3B2Hu2 group, the mean tumor volume was 999 ± 202 mm 3 , among which the TGITV was 62.7%, 2035 ± 191 mm 3 , among which the TGITV was 20.5%, 1900 ± 344 mm3, among which the TGITV was 26.0%. In this experiment, αPD-L1 3C3 B2Hu0 showed significant anti-tumor activity at a content of 5 mg / kg, and had no negative impact on the body weights of the animals or induced any obvious clinical symptoms.

[0153] Table 8. Evaluation of the Efficacy of αPD-L1 Ab in Subcutaneous Treatment

[0154]

[0155] Although the embodiments of the present disclosure have been illustrated with a limited number of examples, those skilled in the art should understand that other modifications and variations are possible. Therefore, the scope of protection of the present disclosure should be limited only by the appended claims.

Claims

1. An anti-PD-L1 antibody or an antigen-binding fragment thereof, which comprises complementarity-determining regions (CDRs) of a heavy-chain variable region and complementarity-determining regions of a light-chain variable region, wherein the heavy-chain variable region comprises HCDR1 having the sequence of SEQ ID NO:2, HCDR2 having the sequence of SEQ ID NO:3, and HCDR3 having the sequence of SEQ ID NO:4, and the light-chain variable region comprises LCDR1 having the sequence of SEQ ID NO:10, LCDR2 having the sequence of SEQ ID NO:11, and LCDR3 having the sequence of SEQ ID NO:12; or the heavy-chain variable region comprises HCDR1 having the sequence of SEQ ID NO:6, HCDR2 having the sequence of SEQ ID NO:7, and HCDR3 having the sequence of SEQ ID NO:8, and the light-chain variable region comprises LCDR1 having the sequence of SEQ ID NO:14, LCDR2 having the sequence of SEQ ID NO:15, and LCDR3 having the sequence of SEQ ID NO:

16.

2. The anti-PD-L1 antibody or an antigen-binding fragment thereof according to claim 1, wherein the antibody is a chimeric, humanized, bispecific or human antibody.

3. The anti-PD-L1 antibody or an antigen-binding fragment thereof according to claim 1, wherein the heavy-chain variable region comprises the sequence of SEQ ID NO:1, 5, 25, 27, 28, 31 or 35 or a sequence having at least about 95% homology with the sequence of SEQ ID NO:1, 5, 25, 27, 28, 31 or 35; and / or the light-chain variable region comprises the sequence of SEQ ID NO:9, 13, 26, 29, 30, 32, 33 or 34 or a sequence having at least about 95% homology with the sequence of SEQ ID NO:9, 13, 26, 29 or 30, 32, 33 or 34.

4. The anti-PD-L1 antibody or an antigen-binding fragment thereof according to claim 1, wherein the heavy-chain variable region comprises the sequence of SEQ ID NO:1 or a sequence having at least about 95% homology with the sequence, and the light-chain variable region comprises the sequence of SEQ ID NO:9 or a sequence having at least about 95% homology with the sequence; the heavy-chain variable region comprises the sequence of SEQ ID NO:5 or a sequence having at least about 95% homology with the sequence, and the light-chain variable region comprises the sequence of SEQ ID NO:13 or a sequence having at least about 95% homology with the sequence; the heavy-chain variable region comprises the sequence of SEQ ID NO:25, 27 or 28 or a sequence having at least about 95% homology with the sequences, and the light-chain variable region comprises the sequence of SEQ ID NO:26, 29 or 30 or a sequence having at least about 95% homology with the sequences; or The heavy chain variable region comprises the sequence of SEQ ID NO: 31 or 35 or a sequence having at least about 95% homology with such sequences, and the light chain variable region comprises the sequence of SEQ ID NO: 32, 33 or 34 or a sequence having at least about 95% homology with such sequences.

5. The anti-PD-L1 antibody or antigen-binding fragment thereof according to claim 1, which is conjugated to a therapeutic agent.

6. The anti-PD-L1 antibody or antigen-binding fragment thereof according to claim 1, which is covalently linked to a therapeutic agent.

7. The anti-PD-L1 antibody or antigen-binding fragment thereof according to claim 1, which is linked to a second specific binding domain for a second target.

8. The antibody or antigen-binding fragment thereof according to claim 1, which is expressed on the surface of a cell.

9. The antibody or antigen-binding fragment thereof according to claim 8, wherein the cell is an immune cell.

10. The antibody or antigen-binding fragment thereof according to claim 8, wherein the cell is a T cell.

11. A vector encoding the antibody or antigen-binding fragment thereof according to claim 1.

12. The vector according to claim 11, which comprises the sequence of SEQ ID NO: 1, 5, 25, 27, 28, 31 or 35 or a sequence having at least about 95% homology with the sequence of SEQ ID NO: 1, 5, 25, 27, 28, 31 or 35; and / or the light chain variable region comprises the sequence of SEQ ID NO: 9, 13, 26, 29, 30, 32, 33 or 34 or a sequence having at least about 95% homology with the sequence of SEQ ID NO: 9, 13, 26, 29, 30, 32, 33 or 34.

13. A genetically engineered cell expressing the antibody or antigen-binding fragment thereof according to claim 1.

14. A genetically engineered cell containing the vector according to claim 11.

15. The genetically engineered cell according to claim 14, which is an immune cell.

16. The genetically engineered cell according to claim 14, which is a T cell.

17. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, comprising: (a) introducing one or more polynucleotides encoding the antibody or antigen-binding fragment into a host cell; (b) culturing the host cell under conditions conducive to the expression of the one or more polynucleotides; and (c) optionally isolating the antibody or antigen-binding fragment from the host cell and / or the culture medium in which the host cell is grown.

18. A method for inhibiting PD-L1-mediated signaling in an individual in need thereof, comprising administering to the individual an effective amount of a pharmaceutical composition, wherein the pharmaceutical composition comprises the anti-PD-L1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, or the genetically engineered cell according to claim 14 and a pharmaceutically acceptable carrier.

19. A method for treating a PD-L1-mediated disease in an individual in need thereof, comprising administering to the individual an effective amount of a pharmaceutical composition, wherein the pharmaceutical composition comprises an anti-PD-L1 antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 10, or a genetically engineered cell as described in claim 14 and a pharmaceutically acceptable carrier.

20. The method according to claim 19, wherein the disease is cancer.

21. The method according to claim 19, wherein the disease is lung cancer, breast cancer, prostate cancer, colorectal cancer, gastric cancer, hepatocellular carcinoma, renal cell carcinoma, testicular cancer, melanoma, leukemia or papillary thyroid carcinoma.

22. A method for detecting the expression of PD-L1, comprising contacting a sample with an anti-PD-L1 antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 10.

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