CN115181180A

CN115181180A - Antibody against human programmed death ligand-1 (PD-L1) and application thereof

Info

Publication number: CN115181180A
Application number: CN202210218622.6A
Authority: CN
Inventors: 刘杨; 邓婧; 卢士强; 曹卓晓; 唐任宏; 任晋生
Original assignee: Shandong Simcere Bio Pharmaceutical Co ltd; Jiangsu Simcere Pharmaceutical Co Ltd
Current assignee: Shandong Simcere Bio Pharmaceutical Co ltd
Priority date: 2021-03-03
Filing date: 2022-03-03
Publication date: 2022-10-14

Abstract

The present invention discloses antibodies or antigen-binding fragments that specifically bind to human programmed death ligand-1 (PD-L1), which antibodies or antigen-binding fragments are capable of enhancing T cell function, up-regulating T cell-mediated immune responses and for use in the treatment of diseases associated with aberrant PD-L1 expression and/or T cell function, such as tumors.

Description

Antibody against human programmed death ligand-1 (PD-L1) and application thereof

Technical Field

The present invention relates to antibodies against human programmed death ligand-1 (PD-L1) or antigen binding fragments thereof, nucleic acids encoding, expression vectors and expression cells, methods of preparation, pharmaceutical compositions, and their use for enhancing T cell function, up-regulating T cell mediated immune responses, and for treating diseases associated with aberrant PD-L1 expression and T cell function, such as tumors.

Background

Immunotherapy is one of the most rapidly developing and promising research fields in tumor therapy, and the use of immune checkpoint inhibitors, such as PD-1/PD-L1 monoclonal antibody and CTLA-4 monoclonal antibody, is a revolutionary therapy for tumor immunotherapy, greatly improving the survival of malignant patients.

T cell-mediated immune responses are tightly regulated by costimulatory and cosuppressive mechanisms, maintaining an optimal balance between antigenic immune responses and maintenance of self-tolerance. This balance is involved by a variety of activating and inhibitory proteins. Inhibitory proteins, also known as immune checkpoint like proteins, modulate the activation and effector functions of killer T Cells (CTLs) to maintain self-tolerance. The immune checkpoint inhibitory protein plays a key role in the regulation pathway of tumors, and an important immune checkpoint protein PD-1 can transmit an immunosuppressive signal after being combined with a ligand PD-L1 thereof, so that the activity of T cells is reduced. Meanwhile, tumor cells can also inhibit the activation and proliferation of T cells by expressing PD-L1 on the cell surface, thereby escaping the attack and killing of CTL. The function of T cells can be partially restored by using the PD-1 or PD-L1 monoclonal antibody to prevent the combination and interaction of PD-1/PD-L1, thereby enhancing the capacity of killing tumor cells.

In 2011, the first immunodetection point inhibitor, namely, the ipipril monoclonal antibody, which is an anti-CTLA-4 monoclonal antibody, becomes a tumor immunotherapy successfully used for treating melanoma, and many patients treated so far have a 5-year survival time better than that of the traditional treatment method. Then, the FDA approved 3 PD-1 mabs and 3 PD-L1 mabs in sequence, and successfully used for immunotherapy of more than ten kinds of tumors except melanoma, and became a first-line treatment for various cancers, such as non-small cell lung cancer (NSCLC), renal Cell Carcinoma (RCC), and bladder or urothelial cancer. China has approved 2 imported PD-1 antibodies and 4 homemade PD-1 antibodies to market so far, but has not yet homemade PD-L1 antibody to obtain an approval, and in view of the difference of PD-L1 antibody and PD-1 antibody treatment mechanism and current clinical test combined medicine and applicable indications, the research and development of a new PD-L1 monoclonal antibody and a PD-L1-based double antibody still have great social and economic significance.

Disclosure of Invention

The invention provides chimeric or humanized antibodies or antigen-binding fragments, multispecific antigen-binding molecules, chimeric antigen receptors, immune effector cells, nucleic acid fragments, vectors, cells, compositions, methods of preparation, pharmaceutical uses, and methods of treatment of disease that specifically bind to PD-L1.

In some embodiments, the heavy chain variable region and the light chain variable region of an isolated antibody or antigen-binding fragment that specifically binds human programmed death ligand-1 (PD-L1) have the sequences set forth in SEQ ID NO 3 and SEQ ID NO 4, respectively, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity compared to the sequences set forth in SEQ ID NO 3 and SEQ ID NO 4.

In a preferred embodiment, the antibody or antigen-binding sheet of the present inventionA segment that binds human programmed death ligand-1 (PD-L1) with a dissociation constant (KD) of no more than 2.1 x 10 ^-9 M, dissociation constant (KD) for binding to cynomolgus monkey programmed death ligand-1 (PD-L1) is not more than 1.2 x 10 ^-9 M。

In a preferred embodiment, the antibody or antigen-binding fragment of the invention is chimeric or humanized or fully human.

In a preferred embodiment, the antibody or antigen-binding fragment thereof of the invention comprises the sequence of a constant region of any one of human or murine antibodies IgG1, igG2, igG3, igG4, igA, igM, igE or IgD; preferably comprising the sequence of the constant region of a human or murine antibody IgG1, igG2, igG3, or IgG 4; or a sequence of a constant region of a human or murine antibody IgG1, igG2, igG3, or IgG4 carrying a mutation.

In a preferred embodiment, the antigen binding fragment of the invention is selected from one or more of F (ab) 2, fab', fab, fv, scFv, diabody, nanobody, and antibody minimal recognition unit.

In a preferred embodiment, the antibody or antigen-binding fragment thereof of the invention competitively binds PD-L1 with an antibody selected from number 156 and has the following properties:

1) Specifically bind to PD-L1 recombinant protein and cells expressing PD-L1;

2) Blocking the binding of PD-L1 to PD-1 protein;

3) Inhibiting the binding of PD-1 to cell surface expressed PD-L1;

4) Enhancing T cell activity; or/and

5) Inhibiting tumor growth.

In a preferred embodiment, the antibody or antigen-binding fragment thereof of the invention is further conjugated to a therapeutic agent or tracer; preferably, the therapeutic agent is selected from a radioisotope, a chemotherapeutic agent or an immunomodulatory agent and the tracer is selected from a radiocontrast agent, a paramagnetic ion, a metal, a fluorescent label, a chemiluminescent label, an ultrasound contrast agent or a photosensitizer.

In a preferred embodiment, the present invention also provides a multispecific antigen-binding molecule; preferably, the multispecific antigen-binding molecule comprises a first antigen-binding moiety comprising the antibody or antigen-binding fragment of any one of the above, and a second antigen-binding moiety that specifically binds to an antigen other than PD-L1 or binds to a different epitope of PD-L1 than the first antigen-binding moiety;

preferably, the additional antigen is selected from PD-1, TNFR2, CTLA-4, LAG-3, CD28, CD122, 4-1BB, TIM3, OX-40, OX40L, CD40L, LIGHT, ICOS, ICOSL, GITR, GITRL, TIGIT, CD27, VISTA, B7H3, B7H4, HEVM, BTLA, CD47, or CD73;

preferably, the multispecific antibody is "bispecific", "trispecific" or "tetraspecific".

In a preferred embodiment, the present invention provides a Chimeric Antigen Receptor (CAR); preferably, the chimeric antigen receptor comprises at least an extracellular antigen-binding domain comprising the PD-L1 antibody or antigen-binding fragment of any one of the above, a transmembrane domain, and an intracellular signaling domain.

In a preferred embodiment, the present invention provides an immune effector cell; preferably, said immune effector cell comprises said chimeric antigen receptor described above or a nucleic acid fragment comprising said chimeric antigen receptor described above;

preferably, the immune effector cell is selected from a T cell, an NK cell, an NKT cell, a monocyte, a macrophage, a dendritic cell, or a mast cell; the T cell may be selected from an inflammatory T cell, a cytotoxic T cell, a regulatory T cell (Treg), or a helper T cell;

preferably, the immune effector cells are allogeneic immune effector cells or autologous immune cells.

In a preferred embodiment, the present invention provides an isolated nucleic acid molecule encoding a nanobody, an antigen-binding fragment, or any combination thereof, as described in any of the above, a multispecific antigen-binding molecule as described above, or a chimeric antigen receptor as described above.

In some embodiments, the present invention provides an expression vector comprising the isolated nucleic acid molecule of the present invention described above.

In some embodiments, the invention provides a host cell comprising the isolated nucleic acid molecule or expression vector of the invention described above.

In a preferred embodiment, the host cell is a eukaryotic cell or a prokaryotic cell; more preferably, the host cell is derived from a mammalian cell, a yeast cell, an insect cell, E.coli and/or Bacillus subtilis; more preferably, the host cell is selected from Chinese hamster ovary Cells (CHO).

In some embodiments, the invention provides a method of producing an antibody or antigen-binding fragment or multispecific antigen-binding molecule, culturing a host cell of the invention described above under suitable conditions, and isolating the antibody or antigen-binding fragment or multispecific antigen-binding molecule.

In some embodiments, the invention provides a method of making an immune effector cell, introducing into the immune effector cell a nucleic acid fragment of the CAR described above, preferably the method further comprises priming the immune effector cell to express the CAR described above.

In some embodiments, the invention provides a pharmaceutical composition comprising an antibody or antigen-binding fragment of the invention described above, a multispecific antigen-binding molecule of the invention described above, a chimeric antigen receptor of the invention described above, an immune effector cell of the invention described above, an isolated nucleic acid molecule of the invention described above, an expression vector of the invention described above, a cell of the invention described above, or a product (e.g., an antibody or antigen-binding fragment) made by a method of the invention described above, and a pharmaceutically acceptable carrier.

In a preferred embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier (carrier), diluent or adjuvant; more preferably, the pharmaceutical composition further comprises an additional antineoplastic agent.

In some embodiments, the present invention provides a method of preventing and/or treating a disease associated with aberrant PD-L1 expression and/or T cell function, comprising administering to a patient in need thereof an antibody or antigen-binding fragment of the invention described above, a multispecific antigen-binding molecule of the invention described above, a chimeric antigen receptor of the invention described above, an immune effector cell of the invention described above, an isolated nucleic acid molecule of the invention described above, an expression vector of the invention described above, a cell of the invention described above, a product made by a method of the invention described above, or a pharmaceutical composition of the invention described above; the disease is preferably a tumor;

preferably, the tumor is selected from lymphoma, leukemia, melanoma, glioma, breast cancer, lung cancer, bone cancer, ovarian cancer, bladder cancer, kidney cancer, liver cancer, testicular cancer, salivary gland cancer, thyroid cancer, thymus cancer, epithelial cancer, pancreatic cancer, colon cancer, rectal cancer, hematological malignancies, head and neck cancer, glioma, stomach cancer, nasopharyngeal cancer, laryngeal cancer, cervical cancer, uterine corpus cancer, and osteosarcoma.

In some embodiments, the present invention provides the use of the antibody or antigen-binding fragment of the invention described above, the multispecific antigen-binding molecule of the invention described above, the chimeric antigen receptor of the invention described above, the immune effector cell of the invention described above, the isolated nucleic acid molecule of the invention described above, the expression vector of the invention described above, the cell of the invention described above, the product (e.g., antibody and antigen-binding fragment) produced by the method of the invention described above, or the pharmaceutical composition of the invention described above, in the manufacture of a medicament for the prevention and/or treatment of a disease associated with aberrant PD-L1 expression and/or T cell dysfunction, preferably a tumor;

In some embodiments, the invention provides the antibody or antigen-binding fragment of the invention described above, the multispecific antigen-binding molecule of the invention described above, the chimeric antigen receptor of the invention described above, the immune effector cell of the invention described above, the isolated nucleic acid molecule of the invention described above, the expression vector of the invention described above, the cell of the invention described above, a product (e.g., an antibody and antigen-binding fragment) of the invention prepared by the method described above, or the pharmaceutical composition of the invention described above for use in preventing and/or treating a disease associated with aberrant PD-L1 expression and/or T-cell dysfunction; the disease is preferably a tumor;

In some embodiments, the invention provides a kit comprising an antibody or antigen-binding fragment of the invention described above, a multispecific antigen-binding molecule of the invention described above, a chimeric antigen receptor of the invention described above, an immune effector cell of the invention described above, an isolated nucleic acid molecule of the invention described above, an expression vector of the invention described above, a cell of the invention described above, or a product (e.g., an antibody or antigen-binding fragment) made by a method of the invention described above, or a pharmaceutical composition of the invention described above, and instructions for use.

Definition and description of terms

As used herein, the term "antibody" (Ab) refers to immunoglobulin molecules that specifically bind or are immunoreactive with an antigen of interest, including polyclonal, monoclonal, genetically engineered, and other modified forms of antibodies (including, but not limited to, chimeric, humanized, fully human, heteroconjugate antibodies (e.g., bispecific, trispecific, and tetraspecific antibodies, diabodies, triabodies, and tetrabodies, antibody conjugates), and antigen-binding fragments of antibodies (including, for example, fab ', F (Ab ') 2, fab, fv, rgigg, and scFv fragments). Furthermore, unless otherwise specified, the term "monoclonal antibody" (mAb) is intended to include intact antibody molecules capable of specifically binding a target protein as well as incomplete antibody fragments (e.g., fab and F (Ab ') 2 fragments that lack the Fc fragment of an intact antibody (cleared more rapidly from the animal circulation), and therefore lack Fc-mediated effector function (effector function) (see Wahl et al, j.nucl.24: 316,1983; the contents of which are incorporated herein).

The "antibody" herein may be derived from any animal, including but not limited to humans and non-human animals which may be selected from primates, mammals, rodents and vertebrates, such as camelids, llamas, ostriches, alpacas, sheep, rabbits, mice, rats or chondroiidaes (e.g. shark).

The term "monospecific" herein refers to having one or more binding sites, wherein each binding site binds to the same epitope of the same antigen.

The term "multispecific" herein refers to having at least two antigen binding sites, each of which binds to a different epitope of the same antigen or to a different epitope of a different antigen. Thus, terms such as "bispecific," "trispecific," "tetraspecific," and the like refer to the number of different epitopes that an antibody/antigen binding molecule can bind.

"full-length antibody," "intact antibody," and "intact antibody" are used interchangeably herein and refer to antibodies that have a structure that is substantially similar to the structure of a native antibody.

As used herein, the term "antigen-binding fragment" refers to one or more antibody fragments that retain the ability to specifically bind to a target antigen. The antigen binding function of an antibody may be performed by a fragment of a full-length antibody. The antibody fragment may be a Fab, F (ab') 2, scFv, SMIP, diabody, triabody, affibody, nanobody, aptamer, or domain antibody. Examples of binding fragments encompassing the term "antigen-binding fragment" of an antibody include, but are not limited to: (i) Fab fragment, a monovalent fragment consisting of VL, VH, CL and CHl domains; (ii) A F (ab) 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bond at the hinge region; (iii) an Fd fragment consisting of VH and CHl domains; (iv) (ii) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (V) a dAb comprising VH and VL domains; (vi) dAb fragments consisting of VH domains (Ward et al, nature 341 544-546, 1989); (vii) a dAb consisting of a VH or VL domain; (viii) an isolated Complementarity Determining Region (CDR); and (ix) a combination of two or more isolated CDRs, which may optionally be joined by a synthetic linker. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, these two domains can be joined using recombinant methods by a linker that enables them to be made into a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see, e.g., bird et al, science 242, 423-426,1988 and Huston et al, proc.natl.acad.sci.usa 85-5879-5883, 1988). These antibody fragments can be obtained using conventional techniques known to those skilled in the art, and these fragments are screened for use in the same manner as intact antibodies. Antigen-binding fragments may be produced by recombinant DNA techniques, enzymatic or chemical cleavage of intact immunoglobulins, or in some embodiments by chemical peptide synthesis procedures known in the art.

As used herein, the term "PD-L1" refers to programmed death ligand-1, also known as CD279 (cluster of differentiation 279), an important immunosuppressive molecule. The PD-L1 is preferably human PD-L1.

As used herein, the terms "anti-programmed death ligand-1 antibody," "anti-PD-L1 antibody," "anti-PD-L1 antibody portion," and/or "anti-PD-L1 antibody fragment," and the like, refer to any protein-or peptide-containing molecule comprising at least a portion of an immunoglobulin molecule capable of specifically binding PD-L1 (such as, but not limited to, at least one Complementarity Determining Region (CDR) of a heavy chain or light chain or a ligand-binding portion thereof, a heavy chain or light chain variable region, a heavy chain or light chain constant region, a framework region, or any portion thereof). The PD-L1 antibody also includes an antibody-like protein scaffold (e.g., the tenth fibronectin type III domain (10 Fn 3)) that contains BC, DE, and FG structural loops similar in structure and solvent accessibility to the antibody CDRs. The tertiary structure of the 10Fn3 domain is similar to that of the IgG heavy chain variable region, and the skilled person can graft, for example, the CDRs of the PD-L1 monoclonal antibody onto a fibronectin scaffold by replacing residues of the BC, DE and FG loops of 10Fn3 with residues from the CDR-H1, CDR-H2 or CDR-H3 regions of the PD-L1 monoclonal antibody.

The term "bispecific antibody" as used herein refers to an antibody, typically a human or humanized antibody, having monoclonal binding specificity for at least two different antigens. In the present invention, one of the binding specificities may be detected against an epitope of PD-L1, and the other may be detected against another epitope of PD-L1 or any other antigen than PD-L1, for example, against a cell surface protein, a receptor subunit, a tissue-specific antigen, a virus-derived protein, a virus-encoded envelope protein, a bacterium-derived protein or a bacterium surface protein, etc.

As used herein, the term "chimeric" antibody refers to an antibody having the variable sequences of an immunoglobulin derived from one source organism (e.g., rat or mouse) and the constant regions of an immunoglobulin derived from a different organism (e.g., human). Methods for producing chimeric antibodies are known in the art. See, e.g., morrison,1985, science 229 (4719): 1202-7; oi et al, 1986, bio Techniques 4; gillies et al, 1985J Immunol Methods 125; the above is incorporated by reference.

As used herein, the term "complementarity determining region" (CDR) refers to a hypervariable region found in both the light chain and heavy chain variable domains. The more highly conserved portions of the variable domains are called Framework Regions (FR). As understood in the art, the amino acid positions representing hypervariable regions of an antibody can vary according to the context and various definitions known in the art. Some positions within a variable domain may be considered to be hybrid hypervariable positions in that these positions may be considered to be within a hypervariable region under one set of criteria (such as IMGT or KABAT) but outside a hypervariable region under a different set of criteria (such as KABAT or IMGT). One or more of these positions may also be found in extended hypervariable regions. The invention includes antibodies comprising modifications in these hybrid hypervariable positions. The variable domains of native heavy and light chains each comprise four framework regions, largely in a sheet configuration, connected by three CDRs (CDR 1, CDR2 and CDR 3) that form loops connecting, and in some cases forming part of, the sheet structure. The CDRs in each chain are held tightly together by the FR regions in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and contribute to the formation of the antigen binding site of the antibody with CDRs from other antibody chains (see Kabat et al, sequences of Protein of immunological Interest, national Institute of Health, bethesda, md.1987; which is incorporated herein by reference).

As used herein, the term "monoclonal antibody" refers to an antibody derived from a single clone (including any eukaryotic, prokaryotic, or phage clone) and is not limited to the method of production of the antibody.

As used herein, the term "VH" refers to the variable region of the immunoglobulin heavy chain of an antibody (including the heavy chain of Fv, scFv or Fab). The term "VL" refers to the variable region of an immunoglobulin light chain (including the light chain of an Fv, scFv, dsFv, or Fab).

The term "heavy chain constant region" herein refers to the carboxy-terminal portion of an antibody heavy chain that is not directly involved in binding of the antibody to an antigen, but exhibits effector functions, such as interaction with an Fc receptor, which has a more conserved amino acid sequence relative to the variable domain of the antibody. The "heavy chain constant region" comprises at least one of: a CH1 domain, a hinge region, a CH2 domain, a CH3 domain, or a variant or fragment thereof. The "heavy chain constant region" includes a "full-length heavy chain constant region" having a structure substantially similar to a natural antibody constant region, and a "heavy chain constant region fragment" including only a portion of the full-length heavy chain constant region. Illustratively, a typical "full-length antibody heavy chain constant region" consists of a CH1 domain-hinge region-CH 2 domain-CH 3 domain; when the antibody is IgE, it further comprises a CH4 domain; when the antibody is a heavy chain antibody, it does not include a CH1 domain. Exemplary "heavy chain constant region fragments" may be selected from the CH1, fc or CH3 domains.

The term "light chain constant region" herein refers to the carboxy-terminal portion of an antibody light chain that is not directly involved in binding of the antibody to an antigen, which light chain constant region may be selected from a constant kappa domain or a constant lambda domain.

The term "Fc" herein refers to the carboxy-terminal portion of an antibody that is papain hydrolysis of intact antibodies, typically comprising the CH3 and CH2 domains of the antibody. Fc regions include, for example, native sequence Fc regions, recombinant Fc regions, and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain may vary slightly, the Fc region of a human IgG heavy chain is generally defined as extending from the amino acid residue at position Cys226 or from Pro230 to its carboxy terminus. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) may be removed, for example, during production or purification of the antibody, or by recombinant engineering of the nucleic acid encoding the heavy chain of the antibody, and thus, the Fc region may or may not include Lys447.

The term "humanized antibody" herein refers to a non-human antibody that has been genetically engineered to have an amino acid sequence modified to increase homology to the sequence of a human antibody. Generally, all or part of the CDR regions of a humanized antibody are derived from a non-human antibody (donor antibody), and all or part of the non-CDR regions (e.g., variable region FRs and/or constant regions) are derived from a human immunoglobulin (acceptor antibody). Humanized antibodies typically retain or partially retain the desired properties of the donor antibody, including, but not limited to, antigen specificity, affinity, reactivity, the ability to increase the activity of immune cells, the ability to enhance an immune response, and the like.

As used herein, the term "percent (%) sequence identity" refers to the percentage of amino acid (or nucleotide) residues of a candidate sequence that are identical to the amino acid (or nucleotide) residues of a reference sequence, after aligning the sequences and introducing gaps, if necessary, for maximum percent sequence identity (e.g., gaps can be introduced in one or both of the candidate and reference sequences for optimal alignment, and non-homologous sequences can be disregarded for comparison purposes). Alignment can be achieved in a variety of ways well known to those skilled in the art for the purpose of determining percent sequence identity, for example using publicly available computer software such as BLAST, ALIGN or Megalign (DNASTAIi) software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm that requires maximum alignment over the full length of the sequences being compared. For example, a reference sequence aligned for comparison to a candidate sequence can show that the candidate sequence exhibits from 50% to 100% sequence identity over the full length of the candidate sequence or over a selected portion of contiguous amino acid (or nucleotide) residues of the candidate sequence. The length of a candidate sequence aligned for comparison purposes can be, e.g., at least 30% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) of the length of a reference sequence. When a position in the candidate sequence is occupied by the same amino acid (or nucleotide) residue as the corresponding position in the reference sequence, then the molecules are identical at that position.

The term "Kabat numbering system" herein generally refers to the immunoglobulin alignment and numbering system proposed by Elvin a.

As used herein, the term "specific binding" refers to a binding reaction that determines the presence of an antigen in a heterogeneous population of proteins and other biomolecules that are specifically recognized, for example, by antibodies or antigen-binding fragments thereof. An antibody or antigen-binding fragment thereof that specifically binds to an antigen will bind to the antigen with a KD of less than 100 nM. For example, an antibody or antigen-binding fragment thereof that specifically binds to an antigen will bind to the antigen with a KD of up to 100nM ((e.g., between 1pM and 100 nM.) an antibody or antigen-binding fragment thereof that does not exhibit specific binding to a particular antigen or epitope thereof will exhibit a KD of greater than 100nM (e.g., greater than 500nM, 1 μ M, 100 μ M, 500 μ M, or 1 mM) for the particular antigen or epitope thereof.

The term "antibody conjugate" as used herein refers to a conjugate/conjugate formed by an antibody molecule chemically bonded to another molecule, either directly or through a linking linker. Such as an antibody-drug conjugate (ADC) in which the drug molecule is the other molecule.

The term "Chimeric Antigen Receptor (CAR)" herein refers to a recombinant protein comprising at least (1) an extracellular antigen-binding domain, e.g., a variable heavy or light chain of an antibody, (2) a transmembrane domain that anchors the CAR into an immune effector cell, and (3) an intracellular signaling domain. In certain embodiments, the extracellular antigen-binding domain of the CAR comprises an scFv. The scFv can be derived from the variable region of a fusion antibody. Alternatively or additionally, the scFv may be derived from Fab's (rather than antibodies, e.g. from a Fab library). In certain embodiments, the scFv is fused to a transmembrane domain, and then to an intracellular signaling domain.

The term "nucleic acid" herein includes any compound and/or substance comprising a polymer of nucleotides. Each nucleotide consists of a base, in particular a purine or pyrimidine base (i.e. cytosine (C), guanine (G), adenine (a), thymine (T) or uracil (U)), a sugar (i.e. deoxyribose or ribose) and a phosphate group. Typically, a nucleic acid molecule is described by the sequence of bases, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule. The sequence of bases is usually indicated as 5 'to 3'. In this context, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA), including for example complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), in particular messenger RNA (mRNA), synthetic forms of DNA or RNA, and mixed polymers comprising two or more of these molecules. The nucleic acid molecule may be linear or circular. In addition, the term nucleic acid molecule includes both sense and antisense strands, as well as single-and double-stranded forms. Furthermore, the nucleic acid molecules described herein may contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases having derivatized sugar or phosphate backbone linkages or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules suitable as vectors for direct expression of the antibodies of the invention in vitro and/or in vivo, e.g., in a host or patient. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors may be unmodified or modified. For example, mRNA can be chemically modified to enhance the stability of the RNA vector and/or expression of the encoded molecule so that the mRNA can be injected into a subject to produce antibodies in vivo (see, e.g., stadler et al, nature Medicine 2017, published online 2017, 12.6.12.doi: 10.1038/nm 4356 or EP 2 101 823 B1).

The term "pharmaceutical composition" herein refers to a formulation that is present in a form that allows for the biological activity of the active ingredient contained therein to be effective and that does not contain additional ingredients that have unacceptable toxicity to the subject to which the pharmaceutical composition is administered.

As used herein, the terms "subject," "subject," and "patient" refer to an organism that receives treatment for a particular disease or condition (such as cancer or an infectious disease) as described herein. Examples of subjects and patients include mammals, such as humans, primates, pigs, goats, rabbits, hamsters, cats, dogs, guinea pigs, members of the family bovidae (e.g., cattle, bison, buffalo, elk, and yak, etc.), sheep and horses, etc., that are being treated for a disease or disorder (e.g., a cell proliferative disorder, such as cancer or an infectious disease).

As used herein, the term "treatment" refers to surgery or drug treatment (therapeutic or therapeutic treatment) with the purpose of preventing, slowing (reducing) the progression of an undesirable physiological change or pathology, such as a cell proliferative disorder (e.g., cancer or infectious disease), in a subject being treated. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or complete), whether detectable or undetectable. Subjects in need of treatment include subjects already suffering from a condition or disease as well as subjects susceptible to a condition or disease or subjects for whom prevention of a condition or disease is intended. When terms such as slow, moderate, weak, moderate, etc. are referred to, their meanings also include elimination, disappearance, nonoccurrence, etc.

The term "effective amount" as used herein refers to an amount of a therapeutic agent that is effective to prevent or ameliorate a condition of a disease or the progression of the disease when administered to a cell, tissue or subject alone or in combination with another therapeutic agent. An "effective amount" also refers to an amount of a compound that is sufficient to alleviate symptoms, e.g., to treat, cure, prevent or alleviate the associated medical condition, or to increase the rate at which such condition is treated, cured, prevented or alleviated. When the active ingredient is administered alone to an individual, a therapeutically effective dose refers to the ingredient alone. When a combination is used, a therapeutically effective dose refers to the combined amounts of the active ingredients that produce the therapeutic effect, whether administered in combination, sequentially or simultaneously.

The term "cancer" herein refers to or describes a physiological condition in mammals that is typically characterized by unregulated cell growth. Included in this definition are benign and malignant cancers.

The term "tumor" as used herein refers to all neoplastic (neoplastic) cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer" and "tumor" are not mutually exclusive when referred to herein.

Advantageous effects

Compared with the prior art, the technical scheme of the invention has at least one of the following beneficial effects:

1. compared with a murine antibody, the humanized antibody disclosed by the invention has the capacity of combining PD-L1, ensures that the molecular stability and the biological function meet the requirements, reduces the immunogenicity, and is favorable for reducing the immunological rejection risk of a human subject in use.

2. The humanized antibody disclosed by the invention shows good binding capacity with human PD-L1 and/or monkey PD-L1, and is favorable for improving the treatment effect and/or developing preclinical animal experiments.

3. The humanized antibody disclosed by the invention can effectively block the combination of PD-1 and PD-L1 protein, enhance the function of T cells, up-regulate T cell mediated immune response and is beneficial to treating diseases related to PD-L1 expression abnormality and/or T cell function abnormality.

Drawings

Unless defined otherwise herein, scientific and technical terms used in connection with the present invention shall have the meanings that are understood by those of ordinary skill in the art.

FIG. 1 shows the SEC-HPLC analysis result of the purity of the purified humanized PD-L1 antibody.

FIG. 2 shows the result of blocking the binding between PD-L1 and PD-1 by the humanized PD-L1 antibody, in which Avelumab is the positive control.

FIG. 3 is a FACS assay of the binding capacity of humanized PD-L1 antibody to PD-L1 at cellular level, with a positive control of Avelumab.

FIG. 4 is a Jurkat-PD-1/CHO-PD-L1-NFAT system testing the blocking ability of the humanized PD-L1 antibody against PD-L1/PD-1, wherein the positive control is Avelumab.

FIG. 5 shows the result of IFN-. Gamma.secretion by humanized anti-PD-L1 antibody in mixed lymphocyte reaction, in which negative control was anti-Hel antibody and positive control was Avelumab.

FIG. 6 shows the in vivo inhibition of the growth of A375 human melanoma by the humanized PD-L1 antibody.

FIG. 7 shows the results of monitoring the in vivo efficacy of the humanized PD-L1 antibody in evaluating body weight.

Detailed Description

The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The examples are exemplary only and do not limit the scope of the invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and substitutions are intended to be within the scope of the invention.

Example 1 humanization of antibodies

Firstly, the method of classical "CDRs transplantation" is adopted to perform antibody humanization, i.e., the humanized antibody is formed by selecting the humanized antibody with the highest homology by sequence selection to provide antibody Framework Regions (FRs), and transplanting antigen binding fragment Complementarity Determining Regions (CDRs) of the target antibody based on the Kabat naming method to the former. Secondly, to better maintain antibody activity and affinity, analysis was performed by MOE software based on antibody structure modeling: 1) Selecting amino acid residues of which the framework region of the antibody is positioned on a VH-VL interface, close to or has direct interaction with CDRs and the like for carrying out back mutation, wherein the amino acid residues are more important for keeping the CDRs in a more conformational state; 2) In consideration of immunogenicity, the amino acids embedded in the protein are selected as much as possible for carrying out back mutation; 3) Energy reducing mutations are preferred for the molecule in view of antibody stability and expression level. Humanized antibodies with affinity, antibody characterization and activity function equivalent or better to murine PD-L1 antibody were screened by testing the affinity of humanized antibodies containing different mutations for human PD-L1 and binding to cells expressing PD-L1 on the surface.

The amino acid information of the heavy-light chain variable region of the preferred candidate antibody molecule 156-BM12 after the murine PD-L1 antibody PDL1-156 is humanized is shown in the following table 1.

TABLE 1 detailed sequence information of heavy and light chain variable regions of murine and humanized anti-PD-L1 antibodies

Example 2 humanized antibody expression and purification

2.1 expression of humanized antibodies

One day before transfection, expicCHO-S cells (Thermo fisher, A29127) were cultured at 2.5X 10 ⁶ -4×10 ⁶ cells/mL density was inoculated in fresh ExpicHO Expression medium (Yinxi Weiji, A29100-01) and cultured overnight in a shaker. On the day of transfection, overnight cultured ExpicHO-S cell suspension was counted, with a cell viability > 95% and a density at 7X 10 ⁶ -10×10 ⁶ And viable cells/mL. Taking the desired cell suspension, diluting with ExpicCHO Expression medium (Yinxi Weiji, A29100-01) to 6X 10 ⁶ cells/mL, placed on a shaker for use. Diluting the prepared humanized antibody expression plasmid into a culture medium, gently shaking the centrifuge tube to mix the humanized antibody expression plasmid and the culture medium uniformly, adding the humanized antibody expression plasmid into OptiPROTMSFM-DNA diluent (England WeiJie foundation, 12309-019), gently shaking the centrifuge tube to mix the humanized antibody expression plasmid and the DNA diluent uniformly, and standing the mixture at room temperature for 1 to 5mins. The plasmid compound is slowly dripped into the cell suspension to be transfected, and the shaking flask is rotated in the dripping process. After transfection, cells were placed on a shaker overnight. Cells on the first day after transfection were supplemented with ExpifactamineTMCHO Enhancer (Endor) and ExpicCHOTMFeed (Endor, A29129) at 0.6% of the cell volume, while gently shaking the flask and transferring the cells to a shaker for 4 days. The transfected cells were supplemented with ExpichotMFeed (Endor Weiji, A29129) at 16% of the cell volume on day 5 after transfection, and the flasks were gently shaken during the addition. The culture medium was collected 12 days after transfection, and centrifuged for 10mins to harvest the supernatant.

2.2 purification of humanized antibodies

The cell culture supernatant collected in example 2.1 was centrifuged at high speed and passed through a 0.45+0.22 μm filter for the first purification step by affinity chromatography. The chromatography medium was protein A packing Mbaselect Sure (GE, 17543803) interacting with Fc, and the equilibration buffer was PBS (2.5 g/L Na) ₂ HPO ₄ ·12H ₂ O，0.408g/L NaH ₂ PO ₄ 8.76g/L NaCl, pH 7.2), after balancing 4 times of the column volume, the cell supernatant is combined, and the flow rate is controlled so that the retention time of the sample on the column is more than or equal to 5min. After the end of the loading, the column was washed with PBS (pH 7.2) until the column was emptyTo a280 uv absorption down to baseline. Then 2 column volumes were washed with 20mM PB +1M NaCl (pH 6.0). The column was then washed with PBS (pH 7.2) until the A280 UV absorbance and conductance reached baseline. Finally, the column was washed with an elution buffer of 20mM citric acid (pH 3.4), and the elution peak was collected from the A280 UV absorption peak, and the collected elution sample was neutralized with 1M Tris-HCl (pH 9.0) to neutrality.

Ultrafiltering and concentrating the neutralized eluate, performing size exclusion chromatography with 20mM citric acid (pH 5.5) as buffer, GE HiLoad Superdex 26/600 200pg (GE, 28989336) as column, controlling flow rate at 2.6ml/min, and loading volume less than 10ml, and combining the target protein peaks according to A280 ultraviolet absorption. The collected protein is shown in figure 1, and the purity of the protein is more than 95% by SEC-HPLC, so that the protein can be used for subsequent research.

Example 3 KD assay for antibody binding to human and cynomolgus PD-L1 recombinant proteins

The binding affinity of the PD-L1 antibody to human and cynomolgus monkey PD-L1-His proteins was determined using Biacore T200 (GE Healthcare). Anti-human IgG Fc (Genway, cat. GWB-20A 705) was immobilized on a CM5 chip (GE Healthcare, cat. BR-1005-30) at 25 ℃. Anti-human Fc (Genway, cat. GWB-20A 705) was diluted to 20. Mu.g/mL with Acetate pH5.0 (GE Healthcare, BR-1003-51). Immobilization was performed using the Amine method in Immobilization method. Alternatively, detection was carried out using a commercial Protein A (GE Healthcare, cat.29127556) chip. The affinity of the antibody to the antigen was determined by multi-cycle kinetics at 25 ℃ by capturing the antibody to be detected on a fixed CM5 chip, injecting recombinant human PD-L1-His (Novoprotein, cat. 315) and cynomolgus PD-L1-His protein (Sino Biological, cat. 90251-C08H) in each cycle, and regenerating with Glycine pH1.5 (Hu test, cat. 62011516). The mobile phase was HBS-EP + Buffer (GE Healthcare, cat. BR-1006-69), the flow rate was 30. Mu.L/min, and the binding time was 300 seconds. The regeneration flow rate was 30. Mu.L/min for 30 seconds. Apply Biacore T200 Evaluation Software (version 3.0) with 1:1 binding model, analysis of experimental data, fitting of the equilibrium dissociation constant KD of the antibody antigen, determination of the association rate constant ka and dissociation rate constant KD.

From the results, it was found that the tested PD-L1 humanized antibodies showed nM or higher affinity for binding of human PD-L1 recombinant protein to cynomolgus monkey PD-L1 recombinant protein, as detailed in table 2 below.

TABLE 2 binding affinity KD assay results for humanized PD-L1 antibody Biacore

Antibody numbering	Human PD-L1 (M)	Cynomolgus monkey PD-L1 (M)
			156-BM12	2.04E-09	1.17E-09

Example 4 IC50 assay of antibodies blocking the interaction of PD-L1 and PD-1

The IC50 of the anti-PD-L1 antibody blocking the binding of the PD-L1 protein to the PD-1 protein was determined by a competitive ELISA method. Human PD-L1 recombinant protein (Nano Biological, cat.10084-H05H) was diluted with carbonate buffer and added to a 96-well microplate at a final concentration of 1. Mu.g/ml. Blocking with 3% BSA in PBS, adding a gradiently diluted anti-PD-L1 antibody (40 nM to 0.02 nM) and human PD-1-His recombinant protein (Sino Biological, cat.10377-H08H) for co-incubation, adding HRP-labeled anti-His tag antibody (MBL, cat.D291-7), TMB (Thermo, cat.34029) for color development, and reading OD values (dual wavelength 450nM to 630 nM) after 1M sulfuric acid termination. The competitive binding curve of the test antibody can be drawn by corresponding the antibody concentration and the OD value, and the IC50 value is calculated. FIG. 2 shows the competitive binding curve of anti-PD-L1 antibodies to human PD-L1 recombinant protein. The results show that the tested 156-BM12 antibodies were effective in blocking the interaction of human PD-L1 protein with human PD-1 protein with an IC50 of 1.489nM and a positive control Avelumab (Pfizer, lot: AU 020322) of 1.263nM.

Example 5FACS determination of the EC50 of PD-L1 antibody on cell surface PD-L1 binding

Mixing gradient concentration antibody to be detected (antibody concentration: 10000ng/ml-0.1 ng/ml) with CHO-PD-L1 cell (Nanjing Yongshan bioscience Co., ltd., 10) ⁵ One/well), incubated at 4 ℃ for 30min. After the incubation is finished, 1. The results in FIG. 3 show that the EC50 of the 156-BM12 antibody is 104.9ng/ml, which is similar to Avelumab (EC 50 of 72 ng/ml), the positive control in this experiment. This assay quantitatively demonstrates the ability of the 156-BM12 antibody to dose-dependent binding of PD-L1 target on the cell surface. Mffold = experimental/no-drug control MFI value.

Example 6PD-1/PD-L1-NFAT reporter Gene testing anti-PD-L1 antibodies inhibit PD-1

The antagonism of the PD-L1 antibody against the PD-1/PD-L1 protein interaction and its signaling pathway was compared using the Jurkat cell line stably transfected with PD-1 (GenScript, cat.00612) and the CHO cell line stably transfected with PD-L1 (GenScript, cat.M00613). When the inhibition of the signal pathway is suppressed, the expression of the NFAT-controlled luminescence reporter gene is enhanced and the luminescence signal value is increased. The blocking effect of the antibody on PD-L1 is strong and weak through the strong and weak (relative light units, RLU) reaction of luminescence reading values.

The CHO cell strain of the stable PD-L1 is planted on a white bottom plate with 96 holes, 40000 cells in each hole and 100 mul/hole, and is put back into the incubator for overnight; and taking out the pore plate, absorbing the culture medium, adding the cell strain of the stably-transformed PD-1 and the PD-L1 antibody to be detected for co-incubation, wherein the sample adding amount of each pore of the PD-1 cell is 16000 per pore, the antibody is subjected to gradient dilution, each dosage is 3 multiple pores, the incubation volume is 100 mu L/pore, the incubation time is 6 hours, when the incubation is finished, taking out the pore plate, adding a luminescence detection reagent with the same volume (100 mu L), and reading. And 4-parameter analysis is carried out by Graphpad according to the detection value to make a regression curve, so as to obtain the EC50 value of each antibody. FIG. 4 shows that the EC50 value of the 156-BM12 antibody tested (340.8 ng/ml) is similar to that of the positive control antibody Avelumab (291 ng/ml). This assay quantitatively demonstrated that the 156-BM12 antibody was specific for cell surface PD-1: the inhibition of T cell activity by PD-L1 interaction exhibits dose-dependent repression ability, thereby dose-dependently potentiating the activity of reporter genes in Jurkat cells.

Example 7ELISA detection of IFN-. Gamma.secreted by T cells in a Mixed lymphocyte reaction

PD-L1 mAb was assayed for enhancement of T cell activity by Mixed Lymphocyte Reaction (MLR). Isolation of CD14 from Peripheral Blood Mononuclear Cells (PBMC) of healthy human donors 1 ⁺ Monocytes were induced in vitro to differentiate into Dendritic Cells (DCs) using recombinant human granulocyte-macrophage colony stimulating factor (GM-CSF, peprotech, cat.300-03) and recombinant human interleukin-4 (rhIL-4, peprotech, cat.200-04), LPS (Sigma, cat: L4516) was added to stimulate mature DCs on day 6 of culture, and the DC cells from donor 1 and CD4 enriched from PBMC from healthy donor 2 on day 7 ⁺ T cell mixed co-culture, DC: CD4 ⁺ The ratio of the number of T cells was 1. After 4 days, cell culture supernatants were collected and assayed for IFN-. Gamma.content by ELISA. As shown in FIG. 5, 156-BM12 and the positive control antibody Aveluab significantly enhanced CD4 in MLR experiments compared with the anti-Hel monoclonal antibody negative control group ⁺ The capacity of the T cells to secrete IFN-gamma is reduced, and the activity of increasing the secretion of IFN-gamma is also reduced along with the reduction of the drug concentration of the PD-L1 antibody. This result indicates that 156-BM12 enhances T cell function and is dose dependent. (T-test,. P)<0.05，**P<0.01，***P<0.001，****P<0.0001.)

Example 8HuPBMC humanized mice in vivo efficacy assessment

A375 cells (Beinai biology, BNCC 100266) at 5X 10 ⁶ 0.1mL of the cells were inoculated into female 5-6 week-old NPG mice (North)Kyvontoda biotechnology limited) on the right side. After A375 cell inoculation, hu PBMC cells (ALLCELLs, PB 005F-C) were plated at 5X 10 ⁶ The tumor is injected into mice in a tail vein with a concentration of 0.2mL until the tumor grows to about 100mm ³ 24 random groups of 8 in each group were selected according to tumor volume, and the groups were 3 groups: vehicle (PBS), tecntriq (30 mg/kg; lotNO. HK65567, roche), 156-BM12 (30 mg/kg). All groups were administered by intraperitoneal injection 3 times a week, 5 times a week, and 2 days after the last administration. Mice body weight and tumor volume were measured 3 times per week during administration and observation, and the measured values were recorded to calculate tumor volume (major diameter. Times. Minor diameter) ² /2) and growth inhibition ratio TGI _TV (%) = (1- (Tn/T0)/(Vn/V0)) × 100%, where Tn represents the mean tumor volume at the experimental end of the treatment group; t0 represents the mean tumor volume at the starting point of treatment; vn represents the mean tumor volume at the end of the vehicle group; v0 represents mean volume of starting point tumor in vehicle group. On day 11 of group administration, the positive drug Tecentriq group and the PD-L1 antibody 156-BM12 group had significant and similar inhibitory effects on tumor volume as compared to the control group, and had statistical differences (P)<0.05). See table 6, table 3 for details.

TABLE 3 Effect of test Agents on tumor volume in A375 cell-transplanted HuPBMC NPG mice

Note: a: mean ± standard error;

b: tumor volumes in the administered groups were statistically compared to tumor volumes in the Vehicle control groups at day 11 of the divided administration, two-way ANOVA analysis, P <0.05, P <0.01, P <0.001, P <0.0001.

The experimental animals had good activity and eating status during the administration period, and the body weight showed an overall rising trend, which indicates that 156-BM12 has higher safety, and the details are shown in FIG. 7 and Table 4.

TABLE 4 Effect of test Agents on body weight in A375 cell transplantation HuPBMC NPG mice

Note: a: mean ± standard error;

the final result shows that the humanized PD-L1 antibody 156-BM12 has obvious inhibition effect on the growth of subcutaneous A375 tumor transplanted tumor and has higher safety. Both TGI levels were comparable compared to the positive control antibody Tecentriq.

Claims

1. An isolated antibody or antigen-binding fragment that specifically binds human programmed death ligand-1 (PD-L1), wherein the heavy chain variable region and the light chain variable region of the antibody or antigen-binding fragment have the sequences shown in SEQ ID NO 3 and SEQ ID NO 4, respectively, or a sequence that has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity compared to the sequences shown in SEQ ID NO 3 and SEQ ID NO 4.

2. The antibody or antigen-binding fragment of claim 1, which binds human programmed death ligand-1 (PD-L1) with a dissociation constant (KD) of no greater than 2.1 x 10 ^-9 M, dissociation constant (KD) for binding to cynomolgus monkey programmed death ligand-1 (PD-L1) is not more than 1.2 x 10 ^-9 M。

3. The antibody or antigen-binding fragment of claim 1 or 2, wherein the antibody or antigen-binding fragment is:

(1) A chimeric antibody or fragment thereof;

(2) A humanized antibody or fragment thereof;

(3) A fully human antibody or fragment thereof.

4. The antibody or antigen-binding fragment of any one of claims 1 to 3, characterized in that the antibody comprises the sequence of a constant region of any one of human or murine antibodies IgG1, igG2, igG3, igG4, igA, igM, igE, or IgD; preferably comprising the sequence of the constant region of a human or murine antibody IgG1, igG2, igG3 or IgG 4.

5. The antibody or antigen-binding fragment of any one of claims 1 to 4, characterized in that the antigen-binding fragment is selected from one or more of F (ab) 2, fab', fab, fv, scFv, diabody.

6. An antibody or antigen-binding fragment that competitively binds to PD-L1 or an epitope thereof with the antibody or antigen-binding fragment of any one of claims 1-5 and has the following properties:

1) Specifically bind to PD-L1 recombinant protein and cells expressing PD-L1;

2) Blocking the binding of PD-L1 to PD-1 protein;

3) Inhibiting the binding of PD-1 to cell surface expressed PD-L1;

4) Enhancing T cell activity; or/and

5) Inhibiting tumor growth.

7. The antibody or antigen-binding fragment of any one of claims 1 to 6, wherein the antibody or antigen-binding fragment is further conjugated to a therapeutic agent or tracer; preferably, the therapeutic agent is selected from a radioisotope, a chemotherapeutic agent or an immunomodulatory agent and the tracer is selected from a radiocontrast agent, a paramagnetic ion, a metal, a fluorescent label, a chemiluminescent label, an ultrasound contrast agent or a photosensitizer.

8. A multispecific antigen-binding molecule, comprising a first antigen-binding moiety comprising the antibody or antigen-binding fragment of any one of claims 1 to 7, and a second antigen-binding moiety that specifically binds to an antigen other than PD-L1 or binds to a different PD-L1 epitope than the first antigen-binding moiety;

9. A Chimeric Antigen Receptor (CAR) comprising at least an extracellular antigen-binding domain comprising the PD-L1 antibody or antigen-binding fragment of any one of claims 1-7, a transmembrane domain, and an intracellular signaling domain.

10. An immune effector cell comprising the chimeric antigen receptor of claim 9 or a nucleic acid fragment encoding the chimeric antigen receptor of claim 9;

11. An isolated nucleic acid molecule encoding the antibody, antigen-binding fragment, or any combination thereof of any one of claims 1-7, the multispecific antigen-binding molecule of claim 8, or the chimeric antigen receptor of claim 9.

12. An expression vector comprising the isolated nucleic acid molecule of claim 11.

13. An isolated host cell comprising the isolated nucleic acid molecule of claim 11, or the expression vector of claim 12; preferably, the host cell is a eukaryotic cell or a prokaryotic cell; more preferably, the host cell is derived from a mammalian cell, a yeast cell, an insect cell, E.coli and/or Bacillus subtilis; more preferably, the host cell is selected from Chinese hamster ovary Cells (CHO).

14. A method of producing the antibody or antigen-binding fragment according to any one of claims 1 to 7 or the multispecific antigen-binding molecule according to claim 8, comprising culturing the host cell according to claim 13 under suitable conditions and isolating the antibody or antigen-binding fragment or multispecific antigen-binding molecule.

15. A method of making the immune effector cell of claim 10, comprising introducing a nucleic acid fragment encoding the CAR of claim 9 into the immune effector cell, optionally further comprising priming the immune effector cell to express the CAR of claim 9.

16. A pharmaceutical composition comprising the antibody or antigen-binding fragment of any one of claims 1 to 7, the multispecific antigen-binding molecule of claim 8, the chimeric antigen receptor of claim 9, the immune effector cell of claim 10, the isolated nucleic acid molecule of claim 11, the expression vector of claim 12, the cell of claim 13, or a product made by the method of claim 14 or 15; preferably, the composition further comprises a pharmaceutically acceptable carrier (carrier), diluent or adjuvant; preferably, the pharmaceutical composition further comprises an additional antineoplastic agent.

17. Use of the antibody or antigen-binding fragment of any one of claims 1 to 7, the multispecific antigen-binding molecule of claim 8, the chimeric antigen receptor of claim 9, the immune effector cell of claim 10, the isolated nucleic acid molecule of claim 11, the expression vector of claim 12, the cell of claim 13, the product made by the process of claim 14 or 15, or the pharmaceutical composition of claim 16 in the manufacture of a medicament for the prevention and/or treatment of a disease associated with aberrant PD-L1 expression and/or T cell function, preferably a tumor;

preferably, the tumor is selected from lymphoma, leukemia, melanoma, glioma, breast cancer, lung cancer, bone cancer, ovarian cancer, bladder cancer, kidney cancer, liver cancer, testicular cancer, salivary gland cancer, thyroid cancer, thymus cancer, epithelial cancer, pancreatic cancer, colon cancer, rectal cancer, hematological malignancies, head and neck cancer, glioma, stomach cancer, nasopharyngeal cancer, laryngeal cancer, cervical cancer, uterine body cancer, and osteosarcoma.

18. A method of preventing and/or treating a disease associated with aberrant PD-L1 expression and/or T cell function, comprising administering to a patient in need thereof the antibody or antigen-binding fragment of any one of claims 1-7, the multispecific antigen-binding molecule of claim 8, the chimeric antigen receptor of claim 9, the immune effector cell of claim 10, the isolated nucleic acid molecule of claim 11, the expression vector of claim 12, a product made by the method of claim 14 or 15, or the pharmaceutical composition of claim 16; the disease is preferably a tumor;

19. The antibody or antigen-binding fragment of any one of claims 1 to 7, the multispecific antigen-binding molecule of claim 8, the chimeric antigen receptor of claim 9, the immune effector cell of claim 10, the isolated nucleic acid molecule of claim 11, the expression vector of claim 12, the cell of claim 13, the product made by the process of claim 14 or 15, or the pharmaceutical composition of claim 16, for use in and/or treating a tumor;

20. A kit comprising the antibody or antigen-binding fragment thereof of any one of claims 1-7, the multispecific antigen-binding molecule of claim 8, the chimeric antigen receptor of claim 9, the immune effector cell of claim 10, the isolated nucleic acid molecule of claim 11, the expression vector of claim 12, the cell of claim 13, the product made by the process of claim 14 or 15, or the pharmaceutical composition of claim 16; optionally, instructions for use are also included.