Anti-human PD-L1 antibodies and their uses

Abstract

The present disclosure relates to anti-PD-L1 antibodies or antigen-binding fragments thereof, comprising a heavy chain variable region sequence comprising three CDRs having the sequences of SEQ ID NOs: 2 to 4, or SEQ ID NOs: 6 to 8, and a light chain variable region sequence comprising three CDRs having the sequences of SEQ ID NOs: 10 to 12, or SEQ ID NOs: 14 to 16. The disclosure also relates to pharmaceutical compositions and methods for detecting PD-L1 expression in a sample.

Description

[Technical Field]

[0001] [Field of the Invention] This disclosure relates to novel human antibodies, particularly human monoclonal antibodies that are specific for and have high affinity to PD-L1. Further, this disclosure relates to the use of such antibodies in the treatment and diagnosis of human disease.

[0002] [Background of the invention] The PD-1 (Programmed Death 1) protein is an inhibitory member of the CD28 receptor family, 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. 2003 Jan. 15;170(2):711-718). Its ligand, PD-L1 (programmed cell death-ligand 1), is expressed on some tumor cells, activated B 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 and attenuates cellular immune responses by inducing apoptosis or exhaustion of T cells. Blockade of the PD-1 / PD-L1 pathway with monoclonal antibodies (directed against PD-1 or PD-L1) is a promising therapeutic approach being investigated in studies of many types of human cancers (Sanmamed and Chen 2014, Cancer J. 2014 July-August;20(4):256-61). Results of these studies suggest that PD-L1 plays an important role in helping tumors evade the immune system by promoting activation of the PD-1 / PD-L1 pathway.

[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. Furthermore, several meta-analyses have shown that overexpression of PD-L1 is associated with 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;8(9):14595-603; Zhang, Kang et al., 2015, Medicine 94:e515; Iacovelli, Nole et al., 2016, Target Oncol. 2016;11:143-148). Therefore, there is a need for better antibodies against PD-L1 for the treatment or diagnosis of diseases or conditions mediated by PD-L1.

[0004] [Summary of the Invention]

[0004] In one aspect, the present disclosure relates to antibodies that are specific for human PD-Ll.

[0005]

[0005] Accordingly, the present disclosure provides an antibody or antigen-binding fragment thereof of the present invention, comprising a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, wherein 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 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); Here, HCDR sequences are defined according to the Kabat nomenclature.

[0006] According to an embodiment of the present disclosure, the complementarity determining regions (CDRs) in the heavy chain variable region sequence of an antibody specific for human PD-L1 have the sequence of SEQ ID NO: 2, 3, 4, 6, 7, or 8 shown in Figures 1A-1B.

[0007]

[0007] Accordingly, the present disclosure provides an antibody or antigen-binding fragment thereof of the present invention, comprising a light chain variable region comprising LCDR1, LCDR2, and LCDR3, wherein 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 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); Here, LCDR sequences are defined according to the Kabat nomenclature.

[0008] According to some embodiments of the present disclosure, the complementarity determining regions in the light chain variable region of the antibody specific for human PD-L1 have the sequence of SEQ ID NO: 10, 11, 12, 14, 15, or 16 shown in Figures 2A-2B.

[0009] In another aspect, the disclosure provides an antibody, or antigen-binding fragment thereof, specific for human PD-L1, comprising a heavy chain variable region having HCDR1, HCDR2, and HCDR3, and a light chain variable region having LCDR1, LCDR2, and LCDR3; the heavy chain variable region 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 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 the heavy chain variable region 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 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, or an antigen-binding fragment thereof, specific for human PD-L1.

[0010]

[0010] In some embodiments of the present disclosure, the antibody is a chimeric antibody, a humanized antibody, a composite antibody, or a human antibody.

[0011]

[0011] In some embodiments of the present disclosure, the antibody is multispecific.

[0012] In some embodiments of the present disclosure, the anti-PD-L1 antibody or antigen-binding fragment thereof has a heavy chain variable region comprising 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 to the sequence of SEQ ID NO:1; and a light chain variable region comprising 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 to the sequence of SEQ ID NO:9.

[0013] In some embodiments of the present disclosure, the anti-PD-L1 antibody or antigen-binding fragment thereof has a heavy chain variable region comprising 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 to the sequence of SEQ ID NO:5; and a light chain variable region comprising 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 to the sequence of SEQ ID NO:13.

[0014] In some embodiments of the present disclosure, the anti-PD-L1 antibody or antigen-binding fragment thereof has a heavy chain variable region comprising 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 to the sequence of SEQ ID NO:25, 27, or 28; and a light chain variable region comprising 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 to the sequence of SEQ ID NO:26, 29, or 30.

[0015] In some embodiments of the present disclosure, the anti-PD-L1 antibody or antigen-binding fragment thereof has a heavy chain variable region comprising 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 to the sequence of SEQ ID NO: 31 or 35; and a light chain variable region comprising 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 to the sequence of SEQ ID NO: 32, 33 or 34.

[0016] In some embodiments of the present disclosure, the anti-PD-L1 antibody or antigen-binding fragment thereof is a full-length antibody, a Fab fragment, a F(ab')2 fragment, or an ScFv fragment.

[0017] In some embodiments of the present disclosure, the anti-PD-L1 antibody or antigen-binding fragment thereof is a fully human antibody.

[0018] In some embodiments of the present disclosure, the anti-PD-L1 antibody or antigen-binding fragment thereof comprises a heavy chain constant region selected from an IgG1, IgG2, or IgG4 isoform, and a light chain constant region selected from a κ subtype or a λ isoform.

[0019] In some embodiments of the present disclosure, the anti-PD-L1 antibody, or antigen-binding fragment thereof, is conjugated to a second specific binding domain for a second target to form part of a bispecific or multispecific antibody, such as anti-CD3, anti-ICOS, or anti-TIM3.

[0020] In some embodiments of the present disclosure, the anti-PD-L1 antibody or antigen-binding fragment thereof is conjugated to a therapeutic agent (payload) to form an antibody-drug conjugate (ADC). In some embodiments, the therapeutic agent or payload may be selected for its ability to modulate the function of PD-L1-expressing cells or PD-1-expressing cells. Such therapeutic agents or payloads may include, for example, DM1, MMAE, or MMAF.

[0021] In some embodiments of the present disclosure, the antibody or 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.

[0022]

[0022] The present disclosure also provides a vector encoding the above-described antibody or antigen-binding fragment thereof.

[0023]

[0023] 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.

[0024]

[0024] In another aspect, the present disclosure provides a genetically engineered cell that expresses the above-described antibody or antigen-binding fragment thereof or contains the above-described vector. The genetically engineered cell can be an immune cell.

[0025]

[0025] The present disclosure also provides a method for producing an antibody or antigen-binding fragment thereof disclosed herein, comprising the steps of: (a) introducing one or more polynucleotides encoding the antibody or antigen-binding fragment into a host cell; (b) culturing the host cell under conditions favorable for 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.

[0026]

[0026] The present disclosure provides a pharmaceutical composition comprising an effective amount of the above-described antibody or antigen-binding fragment thereof, or genetically engineered cell or immune cell, and a pharmaceutically acceptable carrier.

[0027]

[0027] In some embodiments of the present disclosure, the pharmaceutical composition is for use in inhibiting PD-L1-mediated signaling.

[0028] In some embodiments of the present disclosure, the pharmaceutical composition is for use in the treatment of a disease mediated by PD-L1.

[0029] In some embodiments of the present disclosure, the PD-L1-mediated disease can be cancer, which 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 cancer and other advanced solid tumors.

[0030]

[0030] The present disclosure provides a method for detecting PD-L1 expression, comprising contacting a sample with an anti-PD-L1 antibody or antigen-binding fragment thereof described herein. [Brief explanation of the drawings]

[0031] [Figure 1A] Figure 1 shows composite human heavy chain (Figure 1A, 1G8; Figure 1B, 3C3) variable region sequences designed to correspond to the variable region sequences of murine anti-human PD-L1 antibodies. [Figure 1B] Figure 1 shows composite human heavy chain (Figure 1A, 1G8; Figure 1B, 3C3) variable region sequences designed to correspond to the variable region sequences of murine anti-human PD-L1 antibodies. [Figure 2A] Figure 2 shows composite human light chain (Figure 2A, 1G8; Figure 2B, 3C3) variable region sequences designed to correspond to the variable region sequences of mouse anti-human PD-1 antibodies. [Figure 2B] Figure 2 shows composite human light chain (Figure 2A, 1G8; Figure 2B, 3C3) variable region sequences designed to correspond to the variable region sequences of mouse anti-human PD-1 antibodies. [Figure 3] Figure 1 shows the binding of anti-PD-L1 antibodies to human PD-L1 using ELISA. [Figure 4] Figure 1 shows the binding of anti-PD-L1 antibodies to HCC827 cells as assayed by flow cytometry. [Figure 5] FIG. 1 shows the ability of various antibodies to induce PD-1 / PD-L1 blockade. [Figure 6] Figure 1 shows the in vivo efficacy of anti-PD-L1 mAb treatment in a mouse syngeneic MC38 colon cancer model. [Figure 7-1] Figure 1 shows the sequence analysis performed for the humanization of the VL and VH sequences of hum PD-L1 mAbs 3C3 and IMGT. The first line shows residue numbering according to the Kabat scheme, with backmutation sites underlined. [Figure 7-2] Figure 1 shows the sequence analysis performed for the humanization of the VL and VH sequences of hum PD-L1 mAbs 3C3 and IMGT. The first line shows residue numbering according to the Kabat scheme, with backmutation sites underlined. [Figure 8-1]Figure 1 shows the sequence analysis performed for the humanization of the VL and VH sequences of hum PD-L1 mAbs 1G8 and IMGT. The first line shows residue numbering according to the Kabat scheme, with backmutation sites underlined. [Figure 8-2] Figure 1 shows the sequence analysis performed for the humanization of the VL and VH sequences of hum PD-L1 mAbs 1G8 and IMGT. The first line shows residue numbering according to the Kabat scheme, with backmutation sites underlined. [Figure 9-1] Figure 1 shows the expression vectors for generating mouse-human chimeric and humanized versions of PD-L1(3C3) mAb.Detailed procedures for antibody purification of the various PD-L1(3C3) mAb versions are described in this disclosure. [Figure 9-2] Figure 1 shows the expression vectors for generating mouse-human chimeric and humanized versions of PD-L1(3C3) mAb.Detailed procedures for antibody purification of the various PD-L1(3C3) mAb versions are described in this disclosure. [Figure 9-3] Figure 1 shows the expression vectors for generating mouse-human chimeric and humanized versions of PD-L1(3C3) mAb.Detailed procedures for antibody purification of the various PD-L1(3C3) mAb versions are described in this disclosure. [Figure 10] Figure 1 shows the binding affinity of humanized PD-L1 mAbs determined using the mouse-human chimeric 3C3 MM antibody, and the humanized 3C3 HuB2Hu0, 3C3 HuB2Hu, and 3C3 HuB2Hu2 antibodies. Detailed procedures for chimeric antibody expression, purification, and Kd analysis were performed as described in this disclosure. [Figure 11] Figure 1 shows the binding affinity of humanized PD-L1 mAbs determined using mouse-human chimeric 1G8 MM antibody, and humanized 1G8 HuHu, 1G8 HuB2Hu0, 1G8 HuHu2, 1G8 HuB2Hu, 1G8 HuHu2 and 1G8 HuB2Hu2 antibodies. Detailed procedures for chimeric antibody expression, purification and Kd analysis were performed as described in this disclosure. [Figure 12]Figure 1 shows the ability of various humanized PD-L1 3C3 antibodies to induce PD-1 / PD-L1 blockade. [Figure 13] Figure 1 shows the in vivo efficacy of humanized PD-L1 3C3 antibody treatment in a mouse syngeneic MC38 colon cancer model.

[0032] [Detailed Description of the Invention]

[0044] It is understood that this invention is not limited to the particular materials and methods described herein. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments and is not intended to limit the scope of the invention, which will be limited only by the appended claims.

[0033]

[0045] Please note that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise.

[0034]

[0046] The term "antibody," as used herein, refers to any antigen-binding molecule or molecular complex containing 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, including four polypeptide chains—two heavy (H) chains and two light (L) chains—interconnected by disulfide bonds, as well as multimers thereof (e.g., IgM). Each heavy chain contains a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region contains three domains: CH1, CH2, and CH3. Each light chain contains a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region contains one domain (CL1). The VH and VL regions can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs), which are separated by more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In various embodiments of the present disclosure, the FRs of an anti-PD-L1 antibody (or antigen-binding portion thereof) may be identical to human germline sequences or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on a side-by-side analysis of two or more CDRs.

[0035]

[0047] As used herein, the term "specifically binds" means that the antibody does not cross-react to any significant extent with other epitopes.

[0036]

[0048] As used herein, the term "epitope" refers to a site on an antigen to which an antibody binds.

[0037]

[0049] As used herein, the term "complementarity-determining region" (CDR) refers to the noncontiguous antigen-binding sites found in the variable regions of both heavy and light chain polypeptides. CDRs are described in Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat et al., US 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), and the definition includes overlapping or subsets of amino acid residues when compared with each other.

[0038]

[0050] The term "monoclonal antibody," as used herein, is not limited to antibodies produced through hybridoma technology. Monoclonal antibodies may be obtained from a single clone, including any eukaryotic, prokaryotic, or phage clone, by any means available or known in the art.

[0039]

[0051] The term "chimeric" antibody, as used herein, refers to an antibody having variable sequences derived from a non-human immunoglobulin and a human immunoglobulin constant region, which is generally chosen from a human immunoglobulin template.

[0040]

[0052] "Humanized" forms of non-human antibodies are chimeric immunoglobulins that contain minimal sequence derived from non-human immunoglobulin. Generally, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence.

[0041]

[0053] As used herein, the term "composite antibody" refers to an antibody having a variable region that comprises germline or non-germline immunoglobulin sequences from two or more unrelated variable regions.

[0042]

[0054] The terms "antigen-binding portion" of an antibody and "antigen-binding fragment" of an antibody, etc., as used herein, include a polypeptide or glycoprotein that specifically binds to an antigen to form a complex, whether said polypeptide or glycoprotein is naturally occurring, enzymatically obtainable, synthetic, or genetically engineered.

[0043]

[0055] As used herein, the term "therapeutic agent" means any compound, substance, drug, medication or active ingredient having a therapeutic or pharmacological effect that is suitable for administration to a mammal, e.g., a human.

[0044]

[0056] As used herein, the term "immune cell" refers to a cell that plays a role in the 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.

[0045]

[0057] As used herein, the term "T cells" refers to CD4 + T cells and CD8 + Includes T cells. The term T cells also includes T helper type 1 T cells, T helper type 2 T cells, T helper type 17 T cells and suppressor T cells.

[0046]

[0058] As used herein, the term "vector" is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid molecule to which it has been linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell 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 be integrated into the genome of a host cell upon introduction into the host cell and replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. As used herein, "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include other forms of expression vectors, such as viral vectors (eg, replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

[0047]

[0059] The terms "genetically engineered" or "genetic engineering" of a cell refer to the use of genetic material to manipulate genes to change the gene copies and / or gene expression levels within the cell. Genetic material can be in the form of DNA or RNA. Genetic material can be transferred to cells by a variety of means, including viral transduction and non-viral transfection. Genetic engineering can permanently or temporarily change the expression levels of certain genes within a cell.

[0048]

[0060] As used herein, the term "pharmaceutical composition" means a mixture containing a therapeutic agent that is administered to a mammal, e.g., a human, to prevent, treat, or eliminate a particular disease or pathological condition from which the mammal is afflicted.

[0049]

[0061] As used herein, the terms "therapeutically effective amount" or "effective amount" refer 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 for the disease.

[0050]

[0062] As used herein, the terms "treatment" and "treating" and the like encompass any treatment of disease in a mammal, particularly a human, and include (a) preventing the disease from occurring in a subject who may be predisposed to the disease but has not yet been diagnosed as having the disease; (b) inhibiting the disease, i.e., halting the progression of the disease; and (c) relieving the disease, i.e., causing regression of the disease.

[0051]

[0063] The terms "individual," "subject," "host," and "patient," used interchangeably herein, refer to mammals, including but not limited to murines (rats, mice), non-human primates, humans, dogs, cats, ungulates (e.g., horses, cattle, sheep, pigs, goats), and the like.

[0052]

[0064] As used herein, the term "in need of treatment" refers to a determination made by a treating provider (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 a subject needs or will benefit from treatment. This determination is made based on a variety of factors within the provider's experience, including knowledge that the subject is ill or will become ill as a result of a condition treatable by the compounds of the present disclosure.

[0053]

[0065] Terms such as "cancer" and "tumor" include precancerous cells, neoplastic cells, transformed cells, and cancerous cells and can 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 and malignant neoplasms (abnormal growths). "Transformation" refers to spontaneous or induced phenotypic changes, such as cellular immortalization, morphological changes, abnormal cell proliferation, contact inhibition and loss of anchorage, and / or malignant tumors (see Freshney, Culture of Animal Cells: A Manual of Basic Technique (3rd ed., 1994)). Transformation can result from infection with a transforming virus and integration of de novo genomic DNA or uptake of exogenous DNA, but can also occur spontaneously or after exposure to carcinogens.

[0054]

[0066] As used herein, the term "sample" encompasses a variety of sample types obtained from an individual, subject, or patient and can be used in a diagnostic or monitoring assay. The definition includes blood and other liquid samples of biological origin, solid tissue samples such as biopsy specimens or tissue cultures, or cells derived therefrom and their progeny.

[0055]

[0067] The present invention relates to novel antibodies that are specific for and have high affinity to PD-L1. The anti-PD-L1 antibodies, or antigen-binding fragments thereof, can provide therapeutic benefits to subjects. The anti-PD-L1 antibodies, or antigen-binding fragments thereof, according to the present disclosure may be human or humanized and can be used as therapeutic agents for treating and / or diagnosing various disorders mediated by PD-L1, as described in more detail herein.

[0056]

[0068] In particular, the antibodies or antigen-binding fragments thereof according to embodiments of the present disclosure are specific for an epitope on human PD-L1 or a fragment thereof.

[0057]

[0069] Antibodies or antigen-binding fragments thereof according to embodiments of the present disclosure may be full-length (e.g., IgG1 or IgG4 antibodies) or may comprise only the antigen-binding portion (e.g., Fab, F(ab')2, or scFv fragments), and may be modified, if necessary, to affect functionality.

[0058]

[0070] The antibodies or antigen-binding fragments thereof according to embodiments of the present disclosure are specific for human PD-L1. PD-L1, also known as CD274 or B7 homolog 1, is a 40 kDa type 1 transmembrane protein that is suspected to play an important role in suppressing the immune system during certain events, such as pregnancy, tissue allotransplantation, autoimmune diseases, and other disease states such as hepatitis. Normally, the immune system responds to foreign antigens associated with exogenous or endogenous danger signals, resulting in the proliferation of antigen-specific CD8+ T cells and / or CD4+ helper cells. Binding of PD-L1 to PD-1 or B7.1 delivers an inhibitory signal that reduces the proliferation of these T cells and can induce apoptosis, further mediated by downregulation of the gene Bcl-2.

[0059]

[0071] 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 consisting of amino acid residues mimicking the hypervariable regions of an antibody (e.g., isolated complementarity-determining regions (CDRs) such as CDR3 peptides), or constrained FR3-CDR3-FR4 peptides. As used herein, the phrase "antigen-binding fragment" also encompasses other engineered molecules, such as domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), SMIPs (small modular immunopharmaceuticals), and shark variable IgNAR domains.

[0060]

[0072] Antigen-binding fragments of antibodies typically contain at least one variable domain. The variable domain can be of any size or amino acid composition and generally contains at least one CDR, which is adjacent to or in frame with one or more framework sequences. In antigen-binding fragments having a VH domain accompanied by a VL domain, the VH and VL domains can be positioned relative to each other in any suitable configuration. For example, the variable region can be dimeric, including VH-VH, VH-VL, or VL-VL dimers. Alternatively, the antigen-binding fragment of an antibody can contain a monomeric VH or VL domain.

[0061]

[0073] Like full-length antibody molecules, antigen-binding fragments can be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies typically comprise at least two different variable domains, each capable of specifically binding to a separate antigen or to a different epitope on the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, can be adapted for use in connection with the antigen-binding fragments of antibodies of the invention using routine techniques available in the art.

[0062]

[0074] In one embodiment of the present disclosure, the antibody or antigen-binding fragment thereof is conjugated to a therapeutic agent. The antibody of the present disclosure can be used as an antibody-drug conjugate (ADC) that can specifically target PD-L1. The conjugate on the ADC can regulate immune cells that express PD-L1 or cells that interact with cells that express PD-L1 (e.g., PD-1-expressing cells). These ADCs can use any antibody of the present invention or an antigen-binding fragment thereof. The drug (payload) conjugated to the antibody (or binding fragment) can be any drug commonly used in ADCs. The conjugation method can be any method known in the art.

[0063]

[0075] The term "homologous," as applied to polypeptides, means that two peptide sequences share at least 95% sequence identity, and even more preferably at least 98% or 99% sequence identity, when optimally aligned, such as with the programs GAP or BESTFIT, using default gap weights. According to an embodiment of the invention, sequence homology or sequence identity between closely related polypeptides was determined using the Gap and Bestfit programs of the GCG software using default parameters.

[0064]

[0076] In embodiments of the present disclosure, the anti-PD-L1 antibody or 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 LCDR3) regions.

[0065]

[0077] Referring to Figures 1A-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.

[0066]

[0078] 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 ID NO: 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.

[0067]

[0079] 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: 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 ID NO: 19.

[0068]

[0080] 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.

[0069]

[0081] The anti-PD-L1 antibodies disclosed herein may contain at least one amino acid substitution, insertion, and / or deletion in the framework and / or CDR regions of the heavy and light chain variable domains, relative to the corresponding germline sequence from which the antibody is derived. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available, for example, from public antibody sequence databases. The present disclosure includes antibodies, and antigen-binding fragments thereof, derived from any of the amino acid sequences disclosed herein, in which one or more amino acids in one or more framework and / or CDR regions have been mutated to the corresponding residue(s) in the germline sequence from which the antibody is derived, to the corresponding residue(s) in another mammalian germline sequence, or to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are collectively referred to herein as "germline mutations"). Starting with the heavy and light chain variable region sequences disclosed herein, one of skill in the art can readily generate numerous antibodies and antigen-binding fragments containing at least one individual germline mutation or a combination thereof. In certain embodiments, all of the framework and / or CDR residues in the VH and / or VL domains are backmutated to the residue found in the original germline sequence from which the antibody was derived. In other embodiments, only certain residues are backmutated to the original germline sequence, for example, only the mutated residues found in the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or only the mutated residues found in CDR1, CDR2, or CDR3. In other embodiments, one or more of the framework and / or CDR residues are mutated to the corresponding residue(s) in a different germline sequence (i.e., a different germline sequence from the germline sequence from which the antibody was originally derived).Furthermore, antibodies of the present disclosure can contain any combination of two or more germline mutations in the framework and / or CDR regions, exemplified by those in which certain individual residues are mutated to the corresponding residue in a particular germline sequence, while certain other residues that differ from the original germline sequence are either maintained or mutated to the corresponding residue in a different germline sequence. Once obtained, antibodies and antigen-binding fragments containing one or more germline mutations can be readily tested for one or more desirable properties, such as improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as the case may be), reduced immunogenicity, etc. Antibodies and antigen-binding fragments obtained by this general means are encompassed by the present disclosure.

[0070]

[0082] 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 regions are replaced with corresponding amino acid residues in various CDRs (e.g., rodent).

[0071]

[0083] In some embodiments of the present disclosure, the humanized anti-PD-L1 antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 25, 27, or 28, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 26, 29, or 30.

[0072]

[0084] In some embodiments of the present disclosure, the humanized anti-PD-L1 antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 31 or 35, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 32, 33, or 34.

[0073]

[0085] Antibodies of the present disclosure may be monospecific, bispecific, or multispecific. Multispecific antibodies may be specific for different epitopes of a single target polypeptide or may contain antigen-binding domains specific for more than one target polypeptide. The anti-PD-L1 antibodies of the present disclosure may be linked to or co-expressed with another functional molecule, e.g., another peptide or protein. For example, an antibody or fragment thereof may be operatively linked (e.g., by chemical coupling, genetic fusion, or non-covalent association) to one or more other molecular entities, e.g., another antibody or antibody fragment, to generate 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 is conjugated to a therapeutic agent.

[0074]

[0086] In some embodiments of the present disclosure, the antibody or antigen-binding fragment thereof is in the form of a chimeric antigen receptor.

[0075]

[0087] The term "chimeric antigen receptor" or "CAR" refers to a recombinant polypeptide construct that includes at least an extracellular antigen-binding domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as an "intracellular signaling domain") that includes a functional signaling domain derived from a stimulatory molecule, as defined below. In some embodiments, the domains in a CAR polypeptide construct are on the same polypeptide chain, e.g., comprising a chimeric fusion protein. In some embodiments, the domains in a CAR polypeptide construct are not contiguous with each other, e.g., are on different polypeptide chains.

[0076]

[0088] Genes encoding the heavy and light chains of an antibody of interest can be cloned from cells; for example, genes encoding a monoclonal antibody can be cloned from a hybridoma and used to generate recombinant monoclonal antibodies. Gene libraries encoding the heavy and light chains of monoclonal antibodies can also be generated from hybridomas or plasma cells. Random combinations of heavy and light chain gene products create a large pool of antibodies with different antigen specificities (see, e.g., Kuby, Immunology (3rd ed. 1997)).

[0077]

[0089] An example method for producing the antibody or antigen-binding fragment thereof includes: (a) introducing one or more polynucleotides encoding the antibody or antigen-binding fragment into a host cell; (b) culturing the host cell under conditions favoring 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.

[0078]

[0090] Vectors can be used to introduce polynucleotides encoding the antibodies or antigen-binding fragments of the invention into host cells. In one embodiment, one type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell 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 be integrated into the genome of a host cell upon introduction into the host cell and replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. As used herein, "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (eg, replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

[0079]

[0091] In another aspect, the present disclosure provides a genetically engineered cell that expresses the above-described antibody or antigen-binding fragment thereof or contains the above-described vector. The genetically engineered cell can be an immune cell or a stem cell.

[0080]

[0092] The present disclosure provides pharmaceutical compositions comprising the antibodies or antigen-binding fragments thereof, genetically engineered cells, or immune cells of the present disclosure. The pharmaceutical compositions of the present disclosure are formulated with appropriate diluents, carriers, excipients, and other agents that improve uptake, delivery, and tolerability. The compositions may be formulated for a particular use, for example, for veterinary use or for human pharmaceutical use. The form of the composition and the excipients, diluents, and / or carriers used will depend on the intended use of the antibody, as well as the therapeutic use and mode of administration. Many suitable formulations can be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., which is common knowledge to all pharmaceutical chemists. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic)-containing vesicles (e.g., LIPOFECTIN™, Life Technologies, Carlsbad, Calif.), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsion carbowaxes (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al., "Compendium of excipients for parenteral formulations," PDA (1998) J Pharm Sci Technol 52:238-311.

[0081]

[0093] The dose of an antibody administered to a patient may vary depending on the patient's age and size, the target disease, condition, and route of administration. A preferred dose is usually 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, it may be advantageous to administer the antibody intravenously. The frequency and duration of treatment may be adjusted depending on the severity of the condition. Effective dosages and schedules for administering the antibody may be determined empirically; for example, patients may be monitored by periodic evaluation, and the dose adjusted accordingly. Furthermore, scaling of administration between species may be performed using methods well known in the art (e.g., Mordenti et al., 1991, Pharmaceut. Res. 8:1351).

[0082]

[0094] Various delivery systems, such as liposomes, microparticles, encapsulation in microcapsules, recombinant cells capable of expressing mutant viruses, and receptor-mediated endocytosis, are known and can be used to administer the pharmaceutical compositions of the present disclosure (see, e.g., Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Methods of introduction 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, such as by infusion or bolus injection, absorption through epithelial or mucocutaneous linings (e.g., oral, rectal, and intestinal mucosa), and can be administered together with other biologically active agents. Administration can be systemic or local.

[0083]

[0095] The pharmaceutical compositions of the present disclosure can be delivered subcutaneously or intravenously using a standard needle and syringe. Furthermore, for subcutaneous delivery, pen delivery devices are readily adapted to deliver the pharmaceutical compositions of the present disclosure. Such pen delivery devices may be reusable or disposable. Reusable pen delivery devices generally utilize a replaceable cartridge containing the pharmaceutical composition. Once all of the pharmaceutical composition in the cartridge has been administered and the cartridge is emptied, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device can then be reused. In disposable pen delivery devices, there is no replaceable cartridge. Instead, disposable pen delivery devices are pre-filled with the pharmaceutical composition held in a reservoir within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.

[0084]

[0096] In certain circumstances, pharmaceutical compositions can be delivered in controlled-release systems. In one embodiment, a pump can be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201). In another embodiment, a polymeric material can be used; see Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Fla. In yet another embodiment, a controlled-release system is placed in proximity to the target of the composition, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, 1984, supra, Medical Applications of Controlled Release, Vol. 2, pp. 115-138). Other controlled-release systems are discussed in the review by Langer (1990, Science 249:1527-1533).

[0085]

[0097] Injectable preparations may include dosage forms for intravenous, subcutaneous, intradermal, and intramuscular injections, drip infusions, and the like. These injectable preparations can be prepared by known methods. For example, injectable preparations can be prepared by, for example, dissolving, suspending, or emulsifying the above-mentioned antibody or its salt in a sterile aqueous or oily medium commonly used for injections. Aqueous media for injection include, for example, isotonic solutions containing saline, glucose, and other auxiliary agents, which can be used in combination with appropriate solubilizers such as alcohols (e.g., ethanol), polyalcohols (e.g., propylene glycol, polyethylene glycol), nonionic surfactants [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], and the like. Oily media include, for example, sesame oil and soybean oil, which can be used in combination with solubilizers such as benzyl benzoate, benzyl alcohol, and the like. The injectable solutions prepared in this manner are preferably filled into appropriate ampoules.

[0086]

[0098] The above-mentioned pharmaceutical compositions for oral or parenteral use are conveniently prepared in a dosage form of a unit dose appropriate for the dose of the active ingredient, such as tablets, pills, capsules, injections (ampoules), suppositories, etc.

[0087]

[0099] In some embodiments of the present disclosure, the pharmaceutical composition is for use in inhibiting PD-L1-mediated signaling.

[0088]

[0100] In some embodiments of the present disclosure, the pharmaceutical composition is for use in the treatment of a disease mediated by PD-L1.

[0089]

[0101] The present disclosure provides methods for detecting PD-L1 expression comprising contacting a sample with an anti-PD-L1 antibody, or antigen-binding fragment thereof, described herein.

[0090]

[0102] The anti-PD-L1 antibodies of the present disclosure can also be used to detect and / or measure PD-L1 or PD-L1-expressing cells in a sample, e.g., for diagnostic purposes. For example, anti-PD-L1 antibodies, or fragments thereof, can be used to diagnose conditions or diseases characterized by aberrant expression of PD-L1 (e.g., overexpression, underexpression, lack of expression, etc.). An exemplary diagnostic assay for PD-L1 may involve, for example, contacting a sample obtained from a patient with an anti-PD-L1 antibody of the present disclosure, where the anti-PD-L1 antibody has been labeled with a detectable label or reporter molecule. Alternatively, an unlabeled anti-PD-L1 antibody may be used for diagnostic purposes in combination with a secondary antibody that is itself detectably labeled. The detectable label or reporter molecule may be: a radioisotope, e.g., 3 H, 14 C. 32 P, 35 S, or 125 a fluorescent or chemiluminescent moiety, such as fluorescein isothiocyanate or rhodamine; or an enzyme, such as alkaline phosphatase, beta-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).

[0091]

[0103] The following examples are provided to aid those of ordinary skill in the art in practicing the present disclosure.

[0092] [Example]

[0104] The antibodies of the present disclosure have been confirmed to specifically bind to PD-L1 by ELISA. Briefly, PD-L1 was coated onto a 96-well ELISA plate (0.1 μg / well). After binding of the anti-PD-L1 antibodies, antibody-PD-L1 binding was assessed using goat anti-mouse IgG conjugated with horseradish peroxidase (HRP) as the secondary antibody and 3,3',5,5'-tetramethylbenzidine (TMB) as the substrate. OD405 readings were taken to calculate activity. As shown in Table 1 and Figure 3, several mouse hybridoma anti-human PD-L1 antibodies (mAbs 1G8 and 3C3) all exhibited specific and strong binding to PD-L1.

[0093] [Table 1]

[0094]

[0105] To further validate the usefulness of the antibodies of the present invention in cancer treatment, the antibodies were evaluated for their ability to bind to PD-L1 expressed on cancer cells. For example, the binding of anti-PD-L1 antibodies to PD-L1-expressing cells was assessed by flow cytometry using HCC827 cells (lung adenocarcinoma) that express high levels of PD-L1. Briefly, HCC827 cells (PD-L1 high) were incubated with anti-PD-L1 antibodies for 1 hour and then analyzed using flow cytometry. As shown in Figure 4, mAbs 1G8 and 3C3 of the present invention were all able to bind to HCC827 cells, indicating that these antibodies can recognize PD-L1 on the surface of cancer cells. Other antibodies of the present invention also exhibit similar activity. Therefore, the antibodies of the present invention can be used to treat cancer by inhibiting PD-L1-mediated immune suppression or exhaustion by binding to PD-L1 expressed on cancer cells.

[0095]

[0106] While the above experiments tested the binding of antibodies of the invention to PD-L1 molecules in vitro, such binding was also tested for PD-1 and PD-L1 expressed on interacting cells. For example, the PD-1 / PD-L1 blockade assay can use any commercially available kit, such as a kit from Promega (Maddison, WI, USA). The Promega PD-1 / PD-L1 blockade bioassay is a bioluminescent 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 the 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 cognate TCR in an antigen-independent manner.

[0096]

[0107] When the two cell types are co-cultured, PD-1 / PD-L1 interaction suppresses TCR signaling and NFAT-RE-mediated luminescence. Addition of an anti-PD-L1 antibody of the invention that blocks the PD-1 / PD-L1 interaction can relieve the inhibitory signal, leading to TCR activation and NFAT-RE-mediated luminescence. The bioluminescent signal can be detected and quantified using the Bio-Glo™ Luciferase Assay System and a standard luminometer, such as the GloMax® Discover System from Promega® (Maddison, WI, USA).

[0097]

[0108] As shown in Table 2 and Figure 5, the mAbs 1G8 and 3C3 of the present invention all exhibit specific and potent activity in blocking PD-1 / PD-L1 interaction. Other antibodies of the present invention also exhibit similar activity. These results confirm that the antibodies of the present invention may be effective in alleviating immune suppression mediated by the interaction between PD-1 and PD-L1 on interacting cells. Therefore, the antibodies of the present invention are believed to be useful as therapeutic agents for diseases resulting from immune suppression or exhaustion due to PD-1 and / or PD-L1 signaling. Such diseases include various cancers.

[0098] [Table 2]

[0099]

[0109] Some embodiments of the present invention relate to methods for treating or alleviating the pathology / 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 invention in treating cancer, a mouse syngeneic model was used. Briefly, B-hPD-1 / hPD-L1 mice were inoculated with MC38-hPD-L1 tumor cells (5 x 10) suspended in 0.1 mL PBS. 5 ) were injected subcutaneously into the right anterior flank and tumors were allowed to develop. Tumor-bearing animals had a mean tumor size of 75 ± 25 mm. 3 When the mice reached 18 days of age, they were randomly enrolled into seven study groups. Group G1 consisted of six mice. Groups G2 to G7 consisted of eight mice. The three groups were Mu IgG (5 mg / kg), 3C3 (5 mg / kg), and 1G8 (5 mg / kg). All test articles were intraperitoneally administered to tumor-bearing mice twice weekly for a total of six doses. Tumor volume and body weight were measured and recorded twice weekly. The study was terminated 7 days after the last dose. At the end of the experiment, tumors were removed from the animals in a comfortable environment, weighed, and photographed.

[0100]

[0110] As shown in Table 3 and Figure 6, no unscheduled deaths or obvious clinical signs were observed during the study. Body weights in all groups gradually increased during the study, indicating that the test product was well tolerated by the animals. On day 28 after the start of treatment, the Mu IgG (5 mg / kg) group had a mean tumor volume of 2486 ± 447 mm 3 The mean tumor volume in the 3C3 (5 mg / kg) treatment group was 415 ± 155 mm 3 The mean tumor volume in the 1G8 (5 mg / kg) treated group was 647 ± 216 mm 3 and TGITV was 75.8%.

[0101] [Table 3]

[0102]

[0111] In this experiment, 3C3 and 1G8 showed significant antitumor activity at the tested dosages without adversely affecting the body weight of the individuals or inducing obvious clinical symptoms. These results clearly demonstrate that the antibodies of the present invention are useful in clinical applications for treating cancers, such as lung cancer, breast cancer, prostate cancer, and colon cancer.

[0103]

[0112] Mouse monoclonal antibodies have the potential to induce strong immunogenicity and anti-drug antibodies in patients. Therefore, humanization of mouse monoclonal antibodies is an essential and important step for further drug development. Using the 3C3 and 1G8 mouse monoclonal antibodies as parent antibodies, the mAb CDR sequences based on the Kabat definition are listed in Figures 1 and 2 (SEQ ID NO: 1 to SEQ ID NO: 20).

[0104]

[0113] For humanized mAb preparation, human germline VL and VH sequences with a high degree of homology to the 3C3 and 1G8 mAb framework regions were identified from the IMGT database (International Immunogenetics Information System®). Homology searches can be performed by sequence BLAST or similar methods. 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 the VL germline gene IGKV4-1 * 01 (SEQ ID NO: 22), IGKV1-39 * 01 (SEQ ID NO: 23) and IGKV2-29 * 01 (SEQ ID NO: 24) was identified as the VH and VL sequences more homologous to the corresponding heavy and light chain framework sequences in the murine mAb, respectively.

[0105]

[0114] Based on the selected human heavy and light chain variable region homologs, anti-PDL1 antibodies can be constructed by grafting known CDR sequences from known anti-PDL1 antibodies (e.g., mAbs 3C3 and 1G8) onto homologous human heavy and light chain variable sequences. As shown in Figures 7 and 8, these light and heavy chain sequence pairs are used as examples for constructing humanized antibodies against human PDL1.

[0106]

[0115] The grafting of CDRs onto the framework results in 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 binding affinity, some amino acids can be backmutated to those of other species. These important amino acid residues are often located in the upper core region and interface of the antibody and can affect antibody binding (E. Stefan, H. Annemarie, and P. Andreas, Methods 34 (2004) 184-199). Additional considerations include: (i) avoiding the most structurally conserved strands of the Fv β-barrel; (ii) ranking resurfacing sites (murine amino acids) by their relative surface accessibility (e.g., greater than 30%); and (iii) categorizing commonly reported risk sites of the framework. According to the above principles, six and eight backmutation sites were designed in the framework regions of humanized 3C3 Hu-B1 (VH) and 3C3 Hu-B2 (VH), respectively (Figure 7 and Table 4). Seven backmutation sites were designed in the framework regions of humanized 1G8 Hu-B2 (VH) (Figure 8 and Table 5). The heavy chains of the resulting mAb 3C3 are HU 3C3 VH as SEQ ID NO: 25; HU 3C3 VHB2 as SEQ ID NO: 27; and HU 3C3 VHB1 as SEQ ID NO: 28. The light chains of the resulting mAb 3C3 are HU0 3C3 VL as SEQ ID NO: 26; HU 3C3 VL as SEQ ID NO: 29; and HU2 3C3 VL as SEQ ID NO: 30. The heavy chains of the resulting mAb 1G8 are HU 1G8 VHB2 as SEQ ID NO: 31; and HU 1G8 VH as SEQ ID NO: 35. The resulting light chains of mAb 1G8 are 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.

[0107] [Table 4]

[0108]

[0116] 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), humanized anti-PD-L1 antibody with back mutations (Hu-B1), and further improved anti-PD-L1 antibody (Hu-B2). Table 4 shows 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 mutations (Hu0), further improved anti-PD-L1 antibody (Hu), and anti-PD-L1 antibody (Hu2).

[0109] [Table 5]

[0110]

[0117] 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), humanized anti-PD-L1 antibody with back mutations, and further improved anti-PD-L1 antibody (Hu-B2). Table 5 shows 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 mutations (Hu0), further improved anti-PD-L1 antibody (Hu), and anti-PD-L1 antibody (Hu2).

[0111]

[0118] To confirm the change in affinity after humanization of a mouse antibody, the variable regions of the humanized light and heavy chains were directly produced by nucleotide synthesis. The mouse or humanized variable regions were constructed into the human chimeric antibody expression vectors pTCAED heavy and pTCAED light plasmids (Figure 9), which were then introduced into host cells to prepare recombinant antibody-expressing cells. FreeStyle™ 293 or Expi 293 cells (manufactured by INVITROGEN™) were used as host cells for expression. The above vectors were prepared according to the manufacturer's instructions (manufactured by INVITROGEN™) by dissolving approximately 1.25 micrograms of antibody expression vector in 1 x 10 cells. 6 The vector was introduced into the host cells by polyethylimine (PEI) at the rate of introduction into the cells.

[0112]

[0119] Culture supernatant containing human IgG antibodies was prepared as follows. Antibody-producing cells were conditioned with Freestyle™ 293 Expression Medium (GIBCO™). The cells were cultured in tissue culture flasks, and the culture supernatant was harvested when cell viability reached 90%. The harvested supernatant was filtered through 10-micrometer and 0.2-micrometer filters (manufactured by Millipore) to remove contaminants. Affinity purification of the antibody-containing culture supernatant was performed using Protein A (manufactured by Millipore™), PBS as the adsorption buffer, and 200 mM glycine buffer (pH 2.5) as the elution buffer. The pH of the eluted fraction was adjusted to approximately 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 cutoff, manufactured by SPECTRUM™ Laboratories) and sterilized by filtration through a membrane filter (manufactured by Millipore™) with a pore size of 0.22 micrometers to obtain purified antibody. The concentration of the purified antibody was determined by measuring absorbance at 280 nm and converting the measured value based on an optimal density of 1.45 (equivalent to 1 mg / ml).

[0113]

[0120] The binding activity of humanized antibodies was efficiently compared across all combinations of heavy and light chains using mini-scale antibody expression. Antibody concentrations in culture supernatants were determined by anti-human IgG ELISA. For PDL1 ELISA, plates were coated with 1 μg / ml PDL1-hFc and blocked with 5% milk in PBS. The assay antibody was adjusted to 300 ng / ml, 100 μl per well. The anti-PDL1 signal was measured with secondary goat anti-human kappa HRP IgG 1:4000 and developed with TMB substrate (KPL). Absorbance values ​​were measured at OD 450-655 nm using a Bio-Rad ELISA reader.

[0114]

[0121] The 3C3 humanized antibodies, HuHu0, HuHu, and HuHu2, exhibited significantly lower binding signals to PDL1 (for comparison, mAbs 3C3, HuB2Hu0, HuB2Hu, and HuB2Hu2 in the binding ELISA) (Table 6). However, 3C3-HuB2Hu0, 3C3-HuB2Hu, and 3C3-HuB2Hu2 exhibited binding signals similar to those of the parental mouse clone. Compared with the sequence of 3C3 Hu(VH), the sequence of 3C3 HuB2(VH) contains eight beneficial mutations in the heavy chain framework region (Figure 7). These amino acids are found to be backmutated (i.e., backmutations) from residues in 3C3 Hu(VH) to the corresponding residues in the mouse mAb. All of these amino acids are present in the heavy chain variable sequence, as shown in Figure 7. The fact that the backmutations generated better binding antibodies suggests that these residues in the framework regions indirectly contribute to binding to PDL1. These backmutations likely contribute to maintaining the proper conformation of the CDR regions.

[0115] [Table 6]

[0116]

[0122] Table 6 shows the results of determining the binding affinity of PD-L1 mAbs using chimeric PD-L1 and hum PD-L1 mAb 3C3 antibodies. Detailed procedures for chimeric antibody expression, purification, and Kd analysis were performed as described in this disclosure.

[0117]

[0123] ELISA plates were coated with 1 μg / ml PDL1-hFc and blocked with 5% milk in PBS. Afterwards, the assay antibodies were added at 45 nM to 2.7 × 10 -3 The assay was run by adding goat anti-human kappa HRP IgG 1:4000, and binding curves and KDs were determined using GraphPad Prism software with a one-site specific binding for nonlinear fitting.

[0118]

[0124] The binding affinities of the 3C3 humanized antibodies HuB2Hu0, HuB2Hu, and HuB2Hu2 were 1.08 × 10 -10 M, 9.36 x 10 -11 M, and 1.01 x 10 -10 All three of these humanized antibodies demonstrate a greater than 2-fold loss in affinity compared to the parental murine clone 3C3 (Figure 10). The binding affinities of the 1G8 humanized antibodies HuHu0, HuHu, and HuHu2 are 3.99 x 10 -9 M, 3.24 x 10 -9 M and 9.68 x 10 -9 M, which is much lower than that of the mouse clone 1G8. However, the binding affinities of the 1G8 humanized antibodies HuB2Hu0, HuB2Hu, and HuB2Hu2 are 2.38 × 10 -10 M, 2.48 x 10 -10 M and 1.08 x 10 -10These humanized 1G8 variants exhibited similar affinities to the parental murine clone 1G8 (Figure 11). Compared with the sequence of 1G8 Hu(VH), the sequence of 1G8 HuB2(VH) contains seven beneficial mutations in the heavy chain framework region (Figure 8). The fact that backmutations generated better-binding antibodies suggests that these residues in the framework region indirectly contribute to binding to PDL1.

[0119]

[0125] As shown in Table 7 and Figure 12, the 3C3 humanized antibody mAbs HuB2Hu0, HuB2Hu, and HuB2Hu2 all exhibit specific and potent activity in blocking PD-1 / PD-L1 interaction. Other antibodies of the present invention also exhibit similar activity. These results confirm that the antibodies of the present invention may be effective in alleviating immune suppression mediated by the interaction between PD-1 and PD-L1 on interacting cells. Therefore, the antibodies of the present invention are believed to be useful as therapeutic agents for diseases resulting from immune suppression or exhaustion due to PD-1 and / or PD-L1 signaling. Such diseases include various cancers.

[0120] [Table 7]

[0121]

[0126] As shown in Table 8 and Figure 13, no unscheduled animal deaths or overt clinical signs were observed during the study. Body weights in all groups gradually increased during the study, indicating that the test article was well tolerated by the animals. At the end of the experiment, the mean tumor volume in the IgG group was 2537 ± 300 mm. 3 In the atezolizumab (Atz) group, the mean tumor volume was 999 ± 202 mm for αPD-L1 3C3 B2Hu0, αPD-L1 3C3 B2Hu, and αPD-L1 3C3 B2Hu2, respectively. 3 The TGITV was 62.7%, and the mean tumor volume was 2035 ± 191 mm 3In the study, αPD-L1 3C3 B2Hu0 showed significant antitumor activity at a level of 5 mg / kg without adversely affecting animal body weight or inducing overt clinical signs.

[0122] [Table 8]

[0127] While embodiments of the present invention have been described with a limited number of examples, those skilled in the art will appreciate that other modifications and variations are possible. Accordingly, the scope of protection of the present invention may be limited only by the appended claims.

Claims

1. An anti-PD-L1 antibody or its antigen-binding fragment comprising a complementarity-determining region (CDR) of the heavy chain variable region and a complementarity-determining region of the light chain variable region, The heavy chain variable region includes 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 includes 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; or An anti-PD-L1 antibody or its antigen-binding fragment, 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.

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

3. The heavy chain variable region includes the sequence of sequence numbers 5, 25, 27, 28, 1, 31, or 35, or a sequence having at least about 95% homology to the sequence of sequence numbers 5, 25, 27, 28, 1, 31, or 35; and / or The anti-PD-L1 antibody or antigen-binding fragment thereof according to claim 1, wherein the light chain variable region includes the sequence of SEQ ID NOs: 13, 26, 29, 30, 9, 32, 33, or 34, or a sequence having at least about 95% homology to the sequence of SEQ ID NOs: 13, 26, 29, 30, 9, 32, 33, or 34.

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

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

6. An 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. An antibody or antigen-binding fragment thereof, expressed on the surface of a cell, as described in claim 1.

9. The antibody or antigen-binding fragment thereof according to claim 8, wherein the cells are immune cells.

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

11. A vector encoding an antibody or an antigen-binding fragment thereof as described in claim 1.

12. The heavy chain variable region includes the sequence of SEQ ID NOs. 5, 25, 27, 28, 1, 31, or 35, or a sequence having at least about 95% homology to the sequence of SEQ ID NOs. 5, 25, 27, 28, 1, 31, or 35; and / or The vector according to claim 11, wherein the light chain variable region includes the sequence of sequence numbers 13, 26, 29, 30, 9, 32, 33, or 34, or a sequence having at least about 95% homology to the sequence of sequence numbers 13, 26, 29, 30, 9, 32, 33, or 34.

13. Genetically modified cells expressing the antibody or antigen-binding fragment thereof as described in claim 1.

14. Genetically engineered cells comprising the vector according to claim 11.

15. A genetically modified cell according to claim 14, which is an immune cell.

16. A genetically modified cell according to claim 14, which is a T cell.

17. A method for producing an antibody or an 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 favorable to the expression of 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 cultured.

18. A method for inhibiting PD-L1-mediated signaling in a subject requiring such inhibition, comprising the step of administering an effective amount of a pharmaceutical composition to the subject, 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 modified cell as described in claim 14, and a pharmaceutically acceptable carrier.

19. A method for treating a disease mediated by PD-L1 in a subject requiring such treatment, comprising the step of administering an effective amount of a pharmaceutical composition to the subject, 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 modified 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, stomach cancer, hepatocellular carcinoma, renal cell carcinoma, testicular cancer, melanoma, leukemia, or papillary thyroid carcinoma.

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

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