Anti-human pd-l1 antibodies and their uses

Abstract

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

Description

Technical Field

[0001] This invention relates to novel human antibodies, and more specifically to human monoclonal antibodies specific for PD-L1, which has a high affinity for it. Furthermore, this invention relates to the use of such antibodies for the treatment and diagnosis of human diseases. Prior Technology

[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 bone marrow 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 also on 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 to attenuate cellular immune responses by inducing T cell apoptosis or exhaustion. Blocking the PD-1 / PD-L1 pathway with monoclonal antibodies (anti-PD-1 or PD-L1) is a promising therapeutic approach being explored in studies of many types of human cancer (Sanmamed and Chen 2014, Cancer J. 2014 Jul-Aug;20(4):256-61). These studies suggest that PD-L1 plays a crucial role in helping tumors escape 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 carcinoma. Furthermore, several meta-analyses have shown that PD-L1 overexpression indicates a 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, Target Oncol. 2016;11:143-148). Therefore, better antibodies targeting PD-L1 are needed to treat or diagnose PD-L1-mediated diseases or conditions. Summary of the Invention

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

[0005] Therefore, the present invention provides an antibody or antigen-binding fragment thereof comprising a heavy chain variable region including 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). These HCDR sequences are defined according to the Kabat nomenclature method.

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

[0007] Therefore, the present invention provides an antibody or antigen-binding fragment thereof comprising a light chain variable region including 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). These LCDR sequences are defined according to the Kabat nomenclature method.

[0008] According to an embodiment of the present invention, the complementarity-determining region in the light chain variable region of an antibody specific to human PD-L1 has a sequence of SEQ ID NO: 10, 11, 12, 14, 15 or 16, as shown in Figures 2A to 2B.

[0009] In another embodiment, the present invention relates to an antibody specific for human PD-L1 or its antigen-binding fragment, comprising a heavy chain variable region having HCDR1, HCDR2, and HCDR3 and a light chain variable region having LCDR1, LCDR2, and LCDR3, 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 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.

[0010] In some embodiments of the present invention, the anti-system is chimeric, humanized, composite, or a human antibody.

[0011] In some embodiments of the present invention, the anti-system is multispecific.

[0012] In some embodiments of the present invention, the heavy chain variable region of the anti-PD-L1 antibody or its antigen-binding fragment contains 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 contains 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.

[0013] In some embodiments of the present invention, the heavy chain variable region of the anti-PD-L1 antibody or its antigen-binding fragment contains 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 contains 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.

[0014] In some embodiments of the present invention, 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.

[0015] In some embodiments of the present invention, the heavy chain variable region of the anti-PD-L1 antibody or its antigen-binding fragment contains 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 contains 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.

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

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

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

[0019] In some embodiments of the present invention, an anti-PD-L1 antibody or its antigen-binding fragment 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.

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

[0021] In some embodiments of the present invention, the antibody or its antigen-binding fragment is expressed on the surface of a cell. The cell may be an immune cell. In one embodiment of the present invention, the immune cell line is a T cell.

[0022] The present invention also provides a vector encoding an antibody or an antigen-binding fragment thereof.

[0023] In some embodiments of the present invention, the vector comprises a 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 a sequence of SEQ ID NO: 1, 5, 25, 27, 28, 31 or 35; and / or the light chain variable region comprises a 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 a sequence of SEQ ID NO: 9, 13, 26, 29, 30, 32, 33 or 34.

[0024] In another embodiment, the present invention provides genetically engineered cells that express antibodies or antigen-binding fragments thereof, or contain vectors. The genetically engineered cells may be immune cells.

[0025] The present invention 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 favorable to the expression of the one or more polynucleotides; and (c) isolating the antibody or antigen-binding fragment from the host cell and / or the culture medium in which the host cell is grown, as appropriate.

[0026] The present invention provides a pharmaceutical composition comprising an effective amount of an antibody or an antigen-binding fragment thereof or genetically engineered cells or immune cells and a pharmaceutically acceptable carrier.

[0027] In some embodiments of the present invention, the pharmaceutical composition is used to inhibit PD-L1-mediated signals.

[0028] In some embodiments of the present invention, the pharmaceutical composition is used to treat PD-L1-mediated diseases.

[0029] In some embodiments of the present invention, PD-L1-mediated diseases may be cancer. Cancer may 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.

[0030] The present invention provides a method for detecting PD-L1 expression, comprising contacting a sample with an anti-PD-L1 antibody or an antigen-binding fragment thereof as described herein. Simple Explanation of the Diagram

[0031] Figures 1A and 1B show the variable region sequences of the complex human heavy chain (Figure 1A, 1G8; Figure 1B, 3C3) designed to correspond to the mouse anti-human PD-L1 antibody.

[0032] Figures 2A and 2B show the variable region sequences of the complex human light chain (Figure 2A, 1G8; Figure 2B, 3C3) designed to correspond to the mouse anti-human PD-L1 antibody.

[0033] Figure 3 illustrates the binding of anti-PD-L1 antibody to human PD-L1 using ELISA.

[0034] Figure 4 shows the anti-PD-L1 antibody bound to HCC827 cells, analyzed by flow cytometry.

[0035] Figure 5 illustrates the ability of various antibodies to induce PD-1 / PD-L1 blockade.

[0036] Figure 6 shows the in vivo efficacy of anti-PD-L1 mAb treatment in a mouse syngeneic MC38 colon cancer model.

[0037] Figure 7 shows the sequence analysis of the humanization of the VL and VH sequences of human PD-L1 mAb 3C3 and IMGT. In the first row, which is shown under the residue number according to the Kabat protocol, reversion mutation sites are underlined.

[0038] Figure 8 shows the sequence analysis of the humanized VL and VH sequences of human PD-L1 mAb 1G8 and IMGT. In the first row, which is displayed under the residue number according to the Kabat protocol, reversion mutation sites are underlined.

[0039] Figure 9 illustrates the expression vectors used to generate humanized versions of mouse-human chimeras and PD-L1 (3C3) mAb. Detailed procedures for purifying different PD-L1 (3C3) mAb versions of the antibody are described in this invention.

[0040] Figure 10 depicts the results of determining the binding affinity of humanized PD-L1 mAb using mouse-human chimeric 3C3 MM and 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 invention.

[0041] Figure 11 depicts the results of determining the binding affinity of humanized PD-L1 mAb using mouse-human chimeric 1G8 MM 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 invention.

[0042] Figure 12 illustrates the ability of various humanized PD-L1 3C3 antibodies to induce PD-1 / PD-L1 blockade.

[0043] Figure 13 shows the in vivo efficacy of humanized PD-L1 3C3 antibody treatment in a mouse syngeneic MC38 colon cancer model. Implementation

[0044] It should be understood that the present invention 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 specific embodiments only and is not intended to limit the scope of the invention, which is limited only by the scope of the appended claims.

[0045] It must be noted that, unless the context clearly specifies otherwise, as used in this specification and the appended claims, the singular forms "a" and "the" include the plural references.

[0046] As used herein, the term "antibody" means any antigen-binding molecule or molecular complex containing at least one complementarity-determining region (CDR) that specifically binds to a particular antigen (PD-L1) or interacts with it. The term "antibody" includes immunoglobulin molecules and their multimers (e.g., IgM), which comprise four polypeptide chains: two heavy (H) chains and two light (L) chains linked by disulfide bonds. 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 includes one domain (CL1). The VH and VL regions can be further subdivided into hypervariable regions, called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments of the present invention, the FRs of the anti-PD-L1 antibody (or its antigen-binding portion) may be identical to the human germline sequence or may be naturally or artificially modified. The common amino acid sequence can be determined based on the side-by-side analysis of two or more CDRs.

[0047] As used in this article, the term "specific binding" means that the antibody does not exhibit significant cross-reactivity with other antigenic determinants.

[0048] As used in this article, the term "antigen determinant" refers to the site at which an antibody binds to an antigen.

[0049] As used herein, the term "complementarity-determining region" (CDR) refers to a discontinuous antigenic combination site found within the variable region of a heavy-chain or light-chain polypeptide. CDRs have been described by Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat et al., USDept. 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), where the definition includes overlap or subsets of amino acid residues when compared with each other.

[0050] As used herein, the term "monoclonal antibody" is not limited to antibodies produced via fusion tumor technology. A monoclonal antibody system is obtained from a single pure line by any available or known means in this technology, including any eukaryotic, prokaryotic, or phage pure line.

[0051] As used herein, the term "chimeric" antibody system refers to antibodies having variable sequences derived from non-human immunoglobulins and constant regions of human immunoglobulins, typically selected from human immunoglobulin templates.

[0052] The "humanized" form of a non-human antibody is a chimeric immunoglobulin containing very few sequences derived from non-human immunoglobulins. Generally, a humanized antibody will contain substantially all of at least one and usually two variable domains, wherein 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 the FR regions of the human immunoglobulin sequence.

[0053] As used herein, the term "composite antibody" refers to an antibody having a variable region containing sequences of germline or non-germinal immunoglobulins derived from two or more unrelated variable regions.

[0054] As used herein, the terms “antigen-binding portion”, “antigen-binding fragment”, and similar terms include any naturally occurring, enzymatically available, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex.

[0055] As used in this invention, the term "therapeutic agent" means any compound, substance, drug, or active ingredient suitable for administration to mammals (e.g., humans) having therapeutic or pharmacological effects.

[0056] As used herein, the term "immune cell" refers to cells that play a role in the immune response. Immune cells are hematopoietic in origin and include lymphocytes, such as B cells and T cells; natural killer cells; and bone marrow cells, such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.

[0057] 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.

[0058] As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is attached. One type of vector is the "plastomer," which refers to a circular double-stranded DNA loop capable of attaching an additional DNA segment. Another type of vector is the viral vector, in which the additional DNA segment attaches to the viral genome. Some vectors can replicate autonomously in the host cell to which they are introduced (e.g., bacterial vectors with a bacterial origin of replication and augmented mammalian vectors). Other vectors (e.g., non-augmented mammalian vectors) can integrate into the host cell's genome upon introduction into the host cell and thereby replicate along with the host genome. Furthermore, some vectors can induce the expression of genes to which they are operatively attached. 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 plastomer form. Because plastomers are the most commonly used form of vector, the terms "plastomer" and "vector" are used interchangeably in this specification. However, the present invention is intended to include other forms of expression vectors that perform equivalent functions, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses).

[0059] In cellular terminology, "genetically engineered" refers to the manipulation of genes using genetic material to alter gene copies and / or gene expression levels within cells. Genetic material can be in the form of DNA or RNA. It can be transferred into cells via various methods, including viral transduction and non-viral transfection. After genetic engineering, the expression levels of certain genes within a cell can be permanently or temporarily altered.

[0060] As used in this invention, the term "medical composition" means a mixture containing a therapeutic agent administered to a mammal, such as a human, to prevent, treat, or eliminate a particular disease or pathological condition afflicting that mammal.

[0061] As used herein, the terms "therapeutic effective dose" or "efficacy dose" refer to the amount of antibody that, when administered to a mammal or other individual for the treatment of a disease, is sufficient to achieve the therapeutic effect on that disease.

[0062] As used herein, the terms “treatment / treating” and similar terms cover any treatment of a disease in mammals (specifically humans), including: (a) preventing the development of a disease in an individual who may be susceptible to the disease but has not yet been diagnosed with it; (b) suppressing the disease, i.e., curbing its development; and (c) alleviating the disease, i.e., causing the disease to subside.

[0063] As used interchangeably in this text, the terms “person,” “individual,” “subject,” and “patient” refer to mammals, including (but not limited to) rats (rat, mouse), non-human primates, humans, canines, felines, ungulates (e.g., equines, bovines, sheep, suidae, goats), etc.

[0064] As used herein, the term "needs treatment" refers to a judgment made by a caregiver (e.g., a physician, nurse, nursing practitioner, or individual in the case of humans; a veterinarian in the case of animals (including non-human mammals)) that an individual needs treatment or will benefit from treatment. This judgment is based on a variety of factors within the caregiver's area of ​​expertise, including the knowledge that the individual is ill or will be ill due to a condition treatable with the compounds of this invention.

[0065] The terms "cancer," "tumor," and similar terms include precancerous, neoplastic, transformative, and cancerous cells, and can refer to solid tumors or non-solid cancers (see, for example, Edge et al., AJCC Cancer Staging Manual (7th edition, 2009); Cibas and Ducatman Cytology: Diagnostic principles and clinical correlates (3rd edition, 2009)). Cancer includes both benign and malignant neoplasia (abnormal growth). "Transformation" refers to spontaneous or induced phenotypic changes, such as cell immortalization, morphological changes, abnormal cell growth, reduced contact inhibition and fixation, and / or malignancy (see Freshney, Culture of Animal Cells a Manual of Basic Technique (3rd edition, 1994)). Although transformation can be caused by infection with a transformed virus and the incorporation of new genomic DNA or the uptake of exogenous DNA, it can also occur spontaneously or after exposure to carcinogens.

[0066] As used herein, the term "sample" encompasses a wide range of sample types obtained from an individual, a person, or a patient and that may be used for diagnostic or monitoring analysis. This definition includes 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.

[0067] This invention relates to a novel antibody that is specific for and has a high affinity for PD-L1. Anti-PD-L1 antibodies or their antigen-binding fragments can deliver therapeutic benefits to individuals. The anti-PD-L1 antibodies or their antigen-binding fragments of this invention (which may be human or humanized) can be used as therapeutic agents for the treatment and / or diagnosis of a variety of PD-L1-mediated conditions, which are more fully described herein.

[0068] Specifically, the antibody or antigen-binding fragment thereof according to embodiments of the present invention is specific for the antigenic determinant of human PD-L1 or its fragment thereof.

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

[0070] The antibodies or antigen-binding fragments thereof according to embodiments of the present invention 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 presumed to play a major role in suppressing the immune system during specific events such as pregnancy, tissue allogeneic transplantation, autoimmune diseases, and other disease conditions such as hepatitis. Normally, the immune system responds to exogenous or endogenous danger signals in response to foreign antigens, triggering 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 inhibitory signals that reduce the proliferation of these T cells and can also induce apoptosis, which is further mediated by the lower regulation of the gene Bcl-2.

[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 of mimicking 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-transplanted antibodies, bifunctional antibodies, trifunctional antibodies, tetrafunctional antibodies, microantibodies, nanoantibodies (e.g., monovalent nanoantibodies, bivalent nanoantibodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also included in the term "antigen-binding fragments" as used herein.

[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 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 region can be a dimer 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.

[0073] Like intact antibody molecules, antigen-binding fragments can be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies will typically contain at least two distinct variable domains, each capable of specifically binding to a different antigenic determinant on an independent antigen or the same antigen. Any type of multispecific antibody, including the exemplary bispecific antibody type disclosed herein, can be adapted to the antigen-binding fragments of the antibodies of this invention using conventional techniques available in this art.

[0074] In one embodiment of the invention, the antibody or its antigen-binding fragment is bound to a therapeutic agent. The antibody of the present invention can be used as an antibody-drug conjugate (ADC) that specifically targets PD-L1. The conjugate on the ADC can modulate immune cells expressing PD-L1 or cells interacting with cells expressing PD-L1 (e.g., cells expressing PD-1). Such ADCs can use any antibody of the present invention or its antigen-binding fragment. The drug (load) bound to the antibody (or binding fragment) can be any drug commonly used in ADCs. The method of binding can be any method known in the art.

[0075] When applied to peptides, the term "homology" means that two peptide sequences have at least 95% sequence similarity when aligned optimally, such as using preset vacancy weights with programs like GAP or BESTFIT, and even better, at least 98% or 99% sequence similarity. According to embodiments of the present invention, the GAP and Bestfit programs in the GCG software are used with preset parameters to determine the sequence homology or sequence similarity between closely related peptides.

[0076] In an embodiment of the present invention, the anti-PD-L1 antibody or its antigen-binding fragment 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.

[0077] Referring to Figures 1A to 2B, in some embodiments of the present invention, the CDRH1 region contains the amino acid sequence of SEQ ID NO: 2, the CDRH2 region contains the amino acid sequence of SEQ ID NO: 3, the CDRH3 region contains the amino acid sequence of SEQ ID NO: 4, the CDRL1 region contains the amino acid sequence of SEQ ID NO: 10, the CDRL2 region contains the amino acid sequence of SEQ ID NO: 11, and the CDRL3 region contains the amino acid sequence of SEQ ID NO: 12.

[0078] In some embodiments of the present invention, the CDRH1 region contains the amino acid sequence of SEQ ID NO: 6, the CDRH2 region contains the amino acid sequence of SEQ ID NO: 7, the CDRH3 region contains the amino acid sequence of SEQ ID NO: 8, the CDRL1 region contains the amino acid sequence of SEQ ID NO: 14, the CDRL2 region contains the amino acid sequence of SEQ ID NO: 15, and the CDRL3 region contains the amino acid sequence of SEQ ID NO: 16.

[0079] In some embodiments of the present invention, the anti-PD-L1 antibody or its antigen-binding fragment 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 invention, 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.

[0080] In some embodiments of the present invention, the anti-PD-L1 antibody or its antigen-binding fragment 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 invention, 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.

[0081] Compared to the corresponding germline sequence that generates the antibody, the anti-PD-L1 antibody 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. These mutations can be readily identified by comparing the amino acid sequences disclosed herein with germline sequences purchased from, for example, public antibody sequence databases. This invention includes an antibody and its antigen-binding fragment derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids in one or more framework regions and / or CDR regions are mutated to corresponding residues of the germline sequence that generates the antibody, or corresponding residues of another mammalian germline sequence, or conserved amino acid substitutions of the corresponding germline residues (such sequence changes are collectively referred to herein as "germline mutations"). Using the heavy and light chain variable region sequences disclosed herein as starting materials, those skilled in the art can readily generate numerous antibody and antigen-binding fragments containing one or more individual germline mutations or combinations thereof. In some embodiments, all framework and / or CDR residues within the VH and / or VL domains are mutated back to residues found in the initial germline sequence of the derived antibody. In other embodiments, only certain residues are mutated back to the original germline sequence, for example, only mutated residues found in the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or only mutated residues found in CDR1, CDR2, or CDR3. In other embodiments, one or more of the framework and / or CDR residues are mutated to corresponding residues of a different germline sequence (i.e., a germline sequence different from the germline sequence from which the antibody was originally derived). Furthermore, the antibody of the present invention may contain any combination of two or more germline mutations in the framework and / or CDR regions, for example, where certain individual residues are mutated to corresponding residues of a specific germline sequence, while certain other residues different from the original germline sequence remain unchanged or are mutated to corresponding residues of 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 desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced antagonistic or activating biological properties (as the case may be), and reduced immunogenicity. This invention encompasses antibodies and antigen-binding fragments obtained in this generally manner.

[0082] In some embodiments of the present invention, the antibody according to the present invention is a humanized antibody. To improve the binding affinity of the humanized antibody according to the present invention, some amino acid residues in the human framework region are replaced with corresponding amino acid residues in a species of CDR (e.g., rodents).

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

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

[0085] The antibodies of this invention can be monospecific, bispecific, or multispecific. Multispecific antibodies may be specific to different antigenic determinants of a single target polypeptide or may contain antigen-binding domains specific to more than one target polypeptide. The anti-PD-L1 antibody of this invention may be linked to another functional molecule (e.g., another peptide or protein) or co-expressed with another functional molecule. For example, the antibody or a fragment thereof may be functionally linked to one or more other molecular entities, such as another antibody or antibody fragment (e.g., by chemical coupling, gene fusion, non-covalent association, or other means), to produce a bispecific or multispecific antibody with a second binding specificity. For example, this invention includes a bispecific antibody wherein one arm of an immunoglobulin is specific to PD-L1 or a fragment thereof, and the other arm of the immunoglobulin is specific to a second target or binds to a therapeutic agent.

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

[0087] 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 located on the same polypeptide chain, for example, constituting a chimeric fusion protein. In some embodiments, the domains in the CAR polypeptide construct are not adjacent to each other, for example, on different polypeptide chains.

[0088] Genes encoding the heavy and light chains of the antibody of interest can be colonized in cells; for example, genes encoding monoclonal antibodies can be colonized in fusion tumors and used to produce recombinant monoclonal antibodies. Gene libraries encoding the heavy and light chains of monoclonal antibodies can also be prepared from fusion tumors or plasma cells. Random combinations of heavy and light chain gene products generate a large pool of antibodies with different antigen specificities (see, for example, Kuby, Immunology (3rd edition, 1997)).

[0089] Examples of methods for manufacturing antibody or antigen-binding fragments 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 favorable to the expression of the one or more polynucleotides; and (c) isolating the antibody or antigen-binding fragment from the host cell and / or the culture medium in which the host cell is grown, as appropriate.

[0090] Vectors can be used to introduce polynucleotides encoding antibody or antigen-binding fragments of the present invention into host cells. In one embodiment, one type of vector is a "plastomer," which refers to a circular double-stranded DNA loop that can be linked to an additional DNA fragment. Another type of vector is a viral vector, in which the additional DNA segment can bind to a viral genome. Some vectors are capable of autonomous replication in the host cell in which they are introduced (e.g., bacterial vectors with a bacterial origin of replication and augmented mammalian vectors). Other vectors (e.g., non-augmented mammalian vectors) can integrate into the host cell's genome upon introduction into the host cell and thereby replicate together with the host genome. Furthermore, some vectors can induce the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors"). Generally, expression vectors used in recombinant DNA technologies are often in plastomer form. Because plastomers are the most commonly used form of vector, the terms "plastomer" and "vector" are used interchangeably in this specification. However, the present invention is intended to include other forms of expression vectors that perform equivalent functions, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses).

[0091] In another embodiment, the present invention provides genetically engineered cells that express antibodies or antigen-binding fragments thereof or contain vectors. These genetically engineered cells may be immune cells or stem cells.

[0092] This invention provides pharmaceutical compositions comprising the antibody of the present invention or an antigen-binding fragment thereof, genetically engineered cells, or immune cells. The pharmaceutical compositions of the present invention are formulated with suitable diluents, carriers, excipients, and other agents that provide improved transfer, delivery, tolerability, and similar properties. The compositions may be formulated for specific uses, such as for veterinary or human pharmaceutical purposes. The form of the compositions and excipients, diluents, and / or carriers used will depend on the intended use of the antibody and the mode of administration for therapeutic purposes. Many suitable formulations can be found in all formularies known to pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa. Such formulations include, for example, powders, pastes, ointments, gels, waxes, oils, lipids, lipid-containing vesicles (such as LIPOFECTIN.TM., Life Technologies, Carlsbad, Calif.), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsion carbon waxes (polyethylene glycol of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbon waxes. See also Powell et al., "Compendium of excipients for parenteral formulations," PDA (1998), J Pharm Sci Technol 52:238-311.

[0093] The dosage of the antibody administered to a patient can vary depending on the patient's age and body size, target disease, symptoms, route of administration, and similar factors. A preferred dosage is typically calculated based on body weight or body surface area. Intravenous administration of the antibody of the present invention may be advantageous when used to treat adult patients with PD-L1-related conditions or diseases. The frequency and duration of treatment can be adjusted depending on the severity of the symptoms. The effective dosage and duration of antibody administration can be determined empirically; for example, patient progression can be monitored through periodic assessments, and the dosage adjusted accordingly. Furthermore, interspecies ratio adjustments of the dosage can be made using methods well-known in the art (e.g., Mordenti et al., 1991, Pharmaceut. Res. 8:1351).

[0094] Various delivery systems are known to us and can be used to administer the pharmaceutical compositions of the present invention, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, and receptor-mediated endolysin (see, for example, Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Methods of administration 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, by absorption through the epithelial or mucosal lining of the skin (e.g., oral mucosa, rectal mucosa, and intestinal mucosa), and can be administered together with other bioactive agents. Administration can be systemic or local.

[0095] The pharmaceutical compositions of this invention can be delivered subcutaneously or intravenously using standard needles and syringes. Additionally, regarding subcutaneous delivery, pen-type delivery devices are readily applicable for delivering the pharmaceutical compositions of this invention. Such pen-type delivery devices can be reusable or disposable. Reusable pen-type delivery devices typically utilize a replaceable sleeve containing the pharmaceutical composition. After all the pharmaceutical composition has been dispensed into the sleeve and the sleeve is empty, the empty sleeve can be easily discarded and replaced with a new sleeve containing the pharmaceutical composition. The pen-type delivery device can then be reused. In disposable pen-type delivery devices, there is no replaceable sleeve. In practice, disposable pen-type delivery devices are pre-filled with the pharmaceutical composition contained in a reservoir within the device. Once the pharmaceutical composition in the reservoir is emptied, the entire device is discarded.

[0096] In some cases, the pharmaceutical composition can be delivered using a controlled-release system. In one embodiment, a pump can be used (see Langer, above; 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, the controlled-release system can be placed near the target of the composition, thus requiring only a portion of the systemic dose (see, for example, Goodson, 1984, Medical Applications of Controlled Release, above, Vol. 2, pp. 115-138). Other controlled-release systems are discussed in the review in Langer, 1990, Science 249: 1527-1533.

[0097] Injectable formulations may include dosage forms for intravenous, subcutaneous, intradermal, and intramuscular injection, infusion, etc. These injectable formulations can be prepared by methods known to the public. For example, injectable formulations can be prepared, for instance, by dissolving, suspending, or emulsifying the antibodies or salts thereof described above in a sterile aqueous or oily medium known for injection. As an aqueous medium for injection, there are, for example, physiological saline, isotonic solutions containing glucose and other adjuvants, which can be used in combination with suitable solubilizers, such as alcohols (e.g., ethanol), polyols (e.g., propylene glycol, polyethylene glycol), nonionic surfactants [e.g., polysorbate 80, HCO-50 (a polyoxyethylene (50 mol) adduct of hydrogenated castor oil)]. Oily media, such as sesame oil or soybean oil, can be used in combination with solubilizers (e.g., methyl benzoate, benzyl alcohol, etc.). Preferably, the injection solution prepared thereby is filled into a suitable ampoule.

[0098] The pharmaceutical compositions described above for oral or non-enteral use should preferably be formulated in unit doses suitable for containing the active ingredient. Such unit dose dosage forms include, for example, tablets, pills, capsules, ampoules, suppositories, etc.

[0099] In some embodiments of the present invention, the pharmaceutical composition is used to inhibit PD-L1-mediated signals.

[0100] In some embodiments of the present invention, the pharmaceutical composition is used to treat PD-L1-mediated diseases.

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

[0102] The anti-PD-L1 antibody of this invention can also be used to detect and / or measure PD-L1 expression or PD-L1-containing cells in a sample, for example, for diagnostic purposes. For instance, the anti-PD-L1 antibody or a fragment thereof can be used to diagnose conditions or diseases characterized by abnormal PD-L1 expression (e.g., overexpression, underexpression, lack of expression, etc.). Exemplary diagnostic analyses of PD-L1 may include, for example, contacting a sample obtained from a patient with the anti-PD-L1 antibody of this invention, wherein the anti-PD-L1 antibody is detectably labeled or a reporter molecular marker. Alternatively, unlabeled anti-PD-L1 antibodies can be combined with self-labeled secondary antibodies in a detectably labeled manner for diagnostic applications. Detectable markers or reporter molecules can be radioactive isotopes, such as 3H, 14C, 32P, 35S, or 125I; fluorescent or chemiluminescent components, such as luciferin isothiocyanate or rhodamine; or enzymes, such as alkaline phosphatase, β-galactosidase, horseradish peroxidase, or luciferase. Specific illustrative analyses that can be used to detect or measure PD-L1 in samples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS).

[0103] The following examples are provided to help those skilled in this art to implement the present invention. Example

[0104] The antibodies of this invention have been confirmed to specifically bind to PD-L1 via ELISA. In short, PD-L1 was coated onto a 96-well ELISA plate (0.1 µg / well). After binding the anti-PD-L1 antibody, goat anti-mouse IgG conjugated with horseradish peroxidase (HRP) was used as a secondary antibody, and 3,3',5,5'-tetramethylbenzidine (TMB) was used as the recipient to assess antibody-PD-L1 binding. OD405 was read to calculate activity. As shown in Table 1 and Figure 3, several mouse fusion tumor anti-human PD-L1 antibodies (mAbs 1G8 and 3C3) were analyzed. All showed specificity and tight binding to PD-L1. Table 1. ELISA KD of several mouse fusion tumor anti-human PD-L1 antibodies Pure concentration (mg / ml) volume endotoxin (EU / mg) ELISA KD Luc reports EC50 (M) 1G8 4.66 5.0 <5 4.35×10⁻¹¹ 6.2×10-10 3C3 5.42 8.5 <5 3.56×10⁻¹¹ 5.8×10-10

[0105] To further verify the efficacy of the antibodies of this invention 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 exhibit high levels of PD-L1. In short, HCC827 cells (with high PD-L1 levels) were incubated with anti-PD-L1 antibodies for 1 hour, followed by analysis using flow cytometry. As shown in Figure 4, both mAbs 1G8 and 3C3 of this invention bound to HCC827 cells, indicating that these antibodies can recognize PD-L1 on the surface of cancer cells. Other antibodies of this invention also exhibited similar activity. Therefore, the antibodies of this invention can be used to treat cancer by binding to PD-L1 expressed on cancer cells, thereby inhibiting PD-L1-mediated immunosuppression or depletion.

[0106] Although the above experiments test the binding of the antibody of the present invention to the in vivo PD-L1 molecule, such binding is also tested using PD-1 and PD-L1 expressed on interacting cells, respectively. For example, the PD-1 / PD-L1 blocking assay can use any commercial kit, such as those from Promega (Maddison, WI, USA). The Promega PD-1 / PD-L1 blocking 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 driven by NFAT response elements (NFAT-RE) expressing human PD-1 and luciferase reporter proteins, and PD-L1 aAPC / CHO-K1 cells, which are CHO-K1 cells designed to activate the TCR in an antigen-independent manner, expressing human PD-L1 and engineered cell surface proteins.

[0107] When two cell types are co-cultured, the interaction between PD-1 and PD-L1 inhibits TCR signaling and NFAT-RE-mediated luminescence. Adding the anti-PD-L1 antibody of this invention, which blocks the PD-1 / PD-L1 interaction, releases an inhibitory signal, inducing TCR activation and NFAT-RE-mediated luminescence. The bioluminescent signal can be detected and quantified using the Bio-Glo™ luciferase analysis system and standard photometers, such as the GloMax® discovery system from PROMEGA® (Maddison, WI, USA).

[0108] As shown in Table 2 and Figure 5, mAbs 1G8 and 3C3 of the present invention both exhibit specific and effective 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 can effectively alleviate immunosuppression mediated by PD-1 and PD-L1 interaction on interacting cells. Therefore, the antibodies of the present invention should be suitable as therapeutic agents for diseases caused by immunosuppression or depletion due to PD-1 and / or PD-L1 signaling. These diseases include various cancers. Table 2. Analysis of reported PD1 / PD-L1 blocking [] [3C3] [1G8] [EC, 50 , ] 5.821E-10 6.198E-10

[0109] Some embodiments of this invention 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 efficacy of the antibody of this invention in treating cancer, a murine syngeneic model was used. Briefly, B-hPD-1 / hPD-L1 mice were subcutaneously injected in the right anterior abdomen with MC38-hPD-L1 tumor cells (5 × 10⁵) suspended in 0.1 mL PBS to promote tumor development. When the average tumor size reached 75 ± 25 mm³, tumor-bearing animals were randomly assigned to seven study groups. Group G1 consisted of 6 mice. Groups G2 through G7 consisted of 8 mice. The three groups were Mu IgG (5 mg / kg), 3C3 (5 mg / kg), and 1G8 (5 mg / kg). All test items were administered intraperitoneally to tumor-bearing mice twice weekly for a total of six weeks. Tumor volume and body weight were measured and recorded twice weekly. The study was terminated seven days after the last administration. At the end of the experiment, the tumors were removed from the euthanized animals, and they were weighed and photographed.

[0110] As shown in Table 3 and Figure 6, no unplanned animal deaths or obvious clinical signs were observed during the study period. Body weight gradually increased in all groups during the study, indicating good tolerance of the test items to the animals. At day 28 after treatment initiation, the mean tumor volume in the Mu IgG (5 mg / kg) group was 2486 ± 447 mm³. In the 3C3 (5 mg / kg) treatment group, the mean tumor volume was 415 ± 155 mm³, with a TGITV of 85.3%. In the 1G8 (5 mg / kg) treatment group, the mean tumor volume was 647 ± 216 mm³, with a TGITV of 75.8%. Table 3. Efficacy evaluation of αPD-L1 Ab in subcutaneous MC38-hPD-L1 colon cancer model treatment in humanized B-hPD-1 / hPD-L1 mice Mu IgG 3C3 1G8 TGI 28 days (%) 85.3 75.8

[0111] In this experiment, 3C3 and 1G8 exhibited significant antitumor activity at the tested doses, without negatively impacting animal weight or inducing any obvious clinical signs. These results clearly demonstrate that the antibodies of this invention are suitable for clinical use in treating cancers such as lung cancer, breast cancer, prostate cancer, and colorectal cancer.

[0112] Mouse monoclonal antibodies can induce strong immunogenicity and anti-drug antibodies in patients. Therefore, humanization of mouse monoclonal antibodies is a necessary and critical step for further drug development. Using 3C3 and 1G8 mouse monoclonal antibodies as parental antibodies, the mAb CDR sequences based on the Kabat definition are described in Figures 1 and 2 (SEQ ID NO: 1 to SEQ ID NO: 20).

[0113] For the preparation of humanized mAbs, human germline VL and VH sequences with higher homology to the 3C3 and 1G8 mAb framework regions were identified from the IMGT database (International Immunogenetic Information System®). Homology searches were performed using sequence BLAST or similar methods. Mouse mAb variable region sequences were used as lookup sequences. These studies identified the human VH germline gene IGHV4-59*01 (SEQ ID NO: 21) and the 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 mouse mAbs.

[0114] Based on selected human heavy and light chain variable region homologs, anti-PD-L1 antibodies can be constructed by grafting known CDR sequences from known anti-PD-L1 antibodies (e.g., mAb 3C3 and 1G8) into homologous human heavy and light chain variable sequences. Examples of these light and heavy chain sequence pairs used for constructing humanized antibodies against human PD-L1 are shown in Figures 7 and 8.

[0115] CDR transplantation onto the scaffold generates variable domains (VH and VL) from different sources. These chimeric domains may not have optimal sequences. Therefore, antibody affinity may not be optimal. To improve 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 interfacial regions of the antibody (E. Stefan, H. Annemarie and P. Andreas Methods 34 (2004) 184-199). Among the additional considerations are: (i) avoiding most structurally conserved strands of the Fv b-tube; (ii) grading surface remodeling sites (mouse amino acids) by relatively high surface accessibility (e.g., greater than 30%); and (iii) classifying commonly reported scaffold risk sites. Based on the principles described above, six and eight reversion mutation sites were designed in the framework region for humanized 3C3 Hu-B1 (VH) and 3C3 Hu-B2 (VH), respectively (Figure 7 and Table 4). Humanized 1G8 Hu-B2 (VH) was designed with seven reversion mutation sites in the framework region (Figure 8 and Table 5). The heavy chains obtained from 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 obtained from 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 chain obtained from mAb 1G8 is HU 1G8 VHB2 as SEQ ID NO: 31 and HU 1G8 VH as SEQ ID NO: 35. The light chain obtained from mAb 1G8 is 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. Table 4: List of humanized 3C3 framework and reversion mutation sites. 3C3 heavy chain name Framework Reverse mutation reply M mice All mice Hu lGH4-59*01F 16(E16Q) 1 Hu-B1 1,16,48,67,71,78 6 Hu-B2 1,16,39,47,48,67,71,78 8 3C3 Light Chain Hu0 IGKV4-1*01 No-repair mutation 0 Hu IGKV1-39*01 No-repair mutation 0 Hu2 IGKV2-29*01 No-repair mutation 0

[0116] Table 4 shows the primary sequence alignments of the VH region of various anti-PD-L1 (3C3) antibodies: mouse anti-PD-L1 antibody (M), humanized anti-PD-L1 antibody (Hu), reversion mutant humanized anti-PD-L1 antibody (Hu-B1), and further improved anti-PD-L1 antibody (Hu-B2). The primary sequence alignments of the VL fragment of various anti-PD-L1 antibodies are also shown: mouse anti-PD-L1 antibody (M), humanized anti-PD-L1 antibody (H), non-reversion mutant humanized anti-PD-L1 antibody (Hu0), further improved anti-PD-L1 antibody (Hu), and anti-PD-L1 antibody (Hu2). Table 5: List of humanized 1G8 framework and reversion mutation sites. 1G8 heavy chain name Framework Reverse mutation reply M mice All mice Hu IGH4-59*01F 16(E16Q) 1 Hu-B2 1,16,39,47,67,71,78 7 1G8 light chain Hu0 lGKV4-1*01 No-repair mutation 0 Hu IGKV1-39*01 No-repair mutation 0 Hu2 IGKV2-29*01 No-repair mutation 0

[0117] Table 5 shows the primary sequence alignments of the VH region of various anti-PD-L1 (1G8) antibodies: mouse anti-PD-L1 antibody (M), humanized anti-PD-L1 antibody (Hu), reversion mutant humanized anti-PD-L1 antibody, and further improved anti-PD-L1 antibody (Hu-B2). The primary sequence alignments of the VL fragment of various anti-PD-L1 antibodies are also shown: mouse anti-PD-L1 antibody (M), humanized anti-PD-L1 antibody (H), non-reversion mutant humanized anti-PD-L1 antibody (Hu0), further improved anti-PD-L1 antibody (Hu), and anti-PD-L1 antibody (Hu2).

[0118] To confirm the changes in affinity after humanization of mouse antibodies, variable regions of the humanized light and heavy chains were directly generated using nucleotide synthesis methods. The mouse or humanized variable regions were constructed into the pTCAED heavy and light chain plastids of the human chimeric antibody expression vector (Figure 9) and introduced into host cells to prepare cells expressing the recombinant antibody. FreeStyle™ 293 or Expi 293 cells (manufactured by INVITROGEN™) were used as host cells for expression. Following the instruction manual (manufactured by INVITROGEN™), the vector was introduced into the host cells using polyethyleneimine (PEI), with approximately 1.25 μg of the antibody expression vector introduced into 1 x 10⁶ cells.

[0119] Culture supernatants containing human IgG antibodies were prepared using the method described below. Antibody-producing cells were acclimated to Free Style™ 293 expression medium (GIBCO™). Cells were cultured in tissue culture flasks, and the culture supernatant was collected when the viable cell percentage reached 90%. The collected supernatant was filtered through 10 µm and 0.2 µm filters (manufactured by Millipore) to remove contaminants. The antibody-containing culture supernatant was purified for affinity using protein A (manufactured by Millipore™), PBS as an absorption buffer, and 200 mM glycine buffer (pH 2.5) as a dissociation buffer. The dissolved fraction was adjusted to approximately 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 cutoff, manufactured by SPECTRUM™ Laboratories) and sterilized by filtration through a 0.22 μm membrane filter (manufactured by MILLIPORE™) to produce purified antibody. The concentration of purified antibody was determined by measuring absorbance at 280 nm and based on a conversion measure of 1.45, which is equivalent to 1 mg / ml of optimal density.

[0120] The binding activity of humanized antibodies can be effectively compared by using small-scale antibody expression of all combinations of heavy and light chains. The antibody concentration in the culture supernatant was determined by anti-human IgG ELISA. For PD-L1 ELISA, the pans were coated with PD-L1-hFc 1 μg / ml and blocked with 5% milk-PBS. The analytical antibody was adjusted to 300 ng / ml, 100 μl / well. The anti-PD-L1 signal was measured by secondary goat anti-human κ HRP IgG 1:4000, and the color was generated by TMB receptor (KPL). Absorbance was measured at OD 450-655 nm using a Bayer Reid ELISA reader.

[0121] The 3C3 humanized antibodies HuHu0, HuHu, and HuHu2 showed significantly lower binding signals for PDL1 (for comparison, mAbs 3C3, HuB2Hu0, HuB2Hu, and HuB2Hu2 in binding ELISA) (Table 6). However, 3C3-HuB2Hu0, 3C3-HuB2Hu, and 3C3-HuB2Hu2 exhibited binding signals more similar to their parental mouse purelines. Compared to the sequence of 3C3 Hu (VH), 3C3 HuB2 (VH) contained eight beneficial mutations in the heavy chain framework region (Figure 7). These amino acids were found to have been mutated back from residues in 3C3 Hu (VH) to their corresponding residues in the mouse mAbs (i.e., reversion mutations). All of these amino acids are in the variable sequence of the heavy chain, as shown in Figure 7. The fact that reversion mutations produce better binders suggests that these residues in the framework region indirectly contribute to binding to PDL1. It may help maintain the proper configuration in the CDR region. Table 6: Humanized 3C3 expression level and PD-L1 binding assay in free-form 293 cell cultures cultured in D6. Performance μg / ml Light chain Heavy chain M Hu0 Hu Hu2 M 2.80 4.99 1.65 3.66 Hu 2.46 8.61 1.02 2.31 Hu-B1 6.16 15.31 9.21 16.68 Hu-B2 4.90 12.36 4.52 6.73 300 ng / ml PDL1 binding OD450-650 nM Light chain Heavy chain M Hu0 Hu Hu2 M 1.524 1.577 1.851 1.548 Hu 0.25 0.15 0.148 0.081 Hu-B1 0.61 0.539 0.455 0.349 Hu-B2 1.204 1.128 1.352 1.053

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

[0123] PDL1-hFc 1ug / ml was spread onto ELISA plates and blocked with 5% milk-PBS, followed by the addition of 45 nM of the analytical antibody to a 2.7 x 10⁻³ nM dilution (4×). Analysis was performed by adding goat anti-human KAPPA HRP IgG 1:4000 and measuring the binding curve. KD was determined using GraphPad Prism software with site-specific binding for a non-linear fitting method.

[0124] The binding affinities of the 3C3 humanized antibodies HuB2Hu0, HuB2Hu, and HuB2Hu2 were 1.08 x 10⁻¹⁰ M, 9.36 x 10⁻¹¹ M, and 1.01 x 10⁻¹⁰ M, respectively. All three humanized antibodies showed less affinity loss than the parental pure mouse 3C3 (Figure 10). The binding affinities of the 1G8 humanized antibodies HuHu0, HuHu, and HuHu2 were significantly lower than those of the pure mouse 1G8, at 3.99 x 10⁻⁹ M, 3.24 x 10⁻⁹ M, and 9.68 x 10⁻⁹ M, respectively. However, the binding affinities of the humanized 1G8 antibodies HuB2Hu0, HuB2Hu, and HuB2Hu2 were 2.38 x 10⁻¹⁰ M, 2.48 x 10⁻¹⁰ M, and 1.08 x 10⁻¹⁰ M, respectively. These humanized 1G8 variants exhibited similar affinities to the parental pure mouse 1G8 (Figure 11). Compared to the 1G8 Hu (VH) sequence, 1G8 HuB2 (VH) contained seven beneficial mutations in the heavy chain framework region (Figure 8). The fact that the reversion mutations produced better binders suggests that these residues in the framework region indirectly contribute to binding to PDL1.

[0125] As shown in Table 7 and Figure 12, the mAbs of the 3C3 humanized antibodies HuB2Hu0, HuB2Hu, and HuB2Hu2 all exhibit specific and effective 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 can effectively alleviate immunosuppression mediated by PD-1 and PD-L1 interaction on interacting cells. Therefore, the antibodies of the present invention are suitable as therapeutic agents for diseases caused by immunosuppression or depletion due to PD-1 and / or PD-L1 signaling. These diseases include various cancers. Table 7. Analysis of reports on 3C3 humanized antibody blocking αPD-L1 3C3_B2Hu0 αPD-L1 3C3_B2Hu αPD-L1 3C3_B2Hu2 EC50 4.77E-10 4.41E-10 4.09E-10

[0126] As shown in Table 8 and Figure 13, no unplanned animal deaths or obvious clinical signs were observed during the study. Body weight gradually increased in all groups during the study, indicating good tolerance of the test materials to the animals. At the end of the experiment, the mean tumor volume in the IgG group was 2537 ± 300 mm3. In the atezolizumab (Atz), αPD-L1 3C3 B2Hu0, and αPD-L1 3C3 B2Hu2 groups, the mean tumor volume was 1926 ± 436 mm3, with a TGITV of 24.9%. In the αPD-L1 3C3 B2Hu2 group, the mean tumor volume was 999 ± 202 mm3, with a TGITV of 62.7%; in the αPD-L1 3C3 B2Hu2 group, the mean tumor volume was 2035 ± 191 mm3, with a TGITV of 20.5%; and in the αPD-L1 3C3 B2Hu2 group, the mean tumor volume was 1900 ± 344 mm3, with a TGITV of 26.0%. In this experiment, αPD-L1 3C3 B2Hu0 at a concentration of 5 mg / kg showed significant antitumor activity without negatively affecting animal weight or inducing any obvious clinical symptoms. Hu IgG Atz αPD-L1 3C3_B2Hu0 αPD-L1 3C3_B2Hu αPD-L1 3C3_B2Hu2 TGI 28 days (%) 24.9 62.7 20.5 26.0 Table 8. Evaluation of the effectiveness of αPD-L1 Ab in subcutaneous treatment

[0127] Although embodiments of the present invention have been described with a limited number of examples, those skilled in the art will understand that other modifications and variations are possible. Therefore, the scope of protection of the present invention should be limited only to the scope of the appended patent applications.

[0128] TW202547859A_114117762_SEQL.xml

Claims

1. An anti-PD-L1 antibody or an antigen-binding fragment thereof, comprising a complementarity-determining region (CDR) of a heavy chain variable region and a complementarity-determining region 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.

2. The anti-PD-L1 antibody or its antigen-binding fragment as claimed in claim 1, wherein the antibody is a chimeric, humanized, complex, or human antibody.

3. The anti-PD-L1 antibody or its antigen-binding fragment as claimed in claim 1, wherein the heavy chain variable region comprises the sequence of SEQ ID NO: 1, 31 or 35 or a sequence having at least about 95% homology with the sequence of SEQ ID NO: 1, 31 or 35; and / or the light chain variable region comprises the sequence of SEQ ID NO: 9, 32, 33 or 34 or a sequence having at least about 95% homology with the sequence of SEQ ID NO: 9, 32, 33 or 34.

4. The anti-PD-L1 antibody or its antigen-binding fragment as claimed in 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 thereto, 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 thereto; 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 the sequences 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 with the sequences thereto.

5. The anti-PD-L1 antibody or its antigen-binding fragment, as requested in item 1, which binds to the therapeutic agent.

6. The anti-PD-L1 antibody or its antigen-binding fragment, as requested in item 1, is covalently linked to a therapeutic agent.

7. The anti-PD-L1 antibody or its antigen-binding fragment as claimed in claim 1, which is linked to a second specific binding domain of a second target.

8. The antibody or antigen-binding fragment thereof, as claimed in claim 1, is expressed on the surface of a cell.

9. The antibody or antigen-binding fragment thereof as claimed in claim 8, wherein the cell line is an immune cell.

10. The antibody or antigen-binding fragment thereof as requested in claim 8, wherein the cell is a T cell.

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

12. The vector of claim 11, wherein the antibody or its antigen-binding fragment comprises the sequence of SEQ ID NO: 1, 31 or 35 or a sequence having at least about 95% homology with the sequence of SEQ ID NO: 1, 31 or 35; and / or the light chain variable region comprises the sequence of SEQ ID NO: 9, 32, 33 or 34 or a sequence having at least about 95% homology with the sequence of SEQ ID NO: 9, 32, 33 or 34.

13. A genetically engineered cell that exhibits the antibody or antigen-binding fragment thereof of claim 1.

14. A genetically engineered cell containing the vector as claimed in claim 11.

15. The genetically engineered cells in claim 14 are immune cells.

16. The genetically engineered cells, as described in claim 14, are T cells.

17. A method for preparing an antibody or an antigen-binding fragment thereof as claimed in 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; and (b) culturing the host cell under conditions favorable to the expression of the one or more polynucleotides.

18. The method of claim 17, further comprising step (c) isolating the antibody or antigen-binding fragment from the host cell and / or the culture medium in which the host cell is grown.

19. Use of a pharmaceutical composition for preparing a medicament for inhibiting PD-L1-mediated signaling in an individual of need, wherein the pharmaceutical composition comprises an anti-PD-L1 antibody or an antigen-binding fragment thereof as claimed in any one of claims 1 to 10, or a genetically engineered cell and a medically acceptable carrier as claimed in claim 14.

20. Use of a pharmaceutical composition for preparing a medicament for treating a PD-L1-mediated disease in an individual in need, wherein the pharmaceutical composition comprises an anti-PD-L1 antibody or an antigen-binding fragment thereof as claimed in any one of claims 1 to 10, or a genetically engineered cell and a medically acceptable carrier as claimed in claim 14, wherein the disease is cancer.

21. As claimed in claim 20, 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 contacting a sample with an anti-PD-L1 antibody or an antigen-binding fragment thereof as claimed in any one of claims 1 to 10.

Images