CN111234017B - Anti-human PD-L1 antibody and antigen binding fragment thereof, preparation method and application - Google Patents

Abstract

The invention provides an anti-human PD-L1 antibody or an antigen binding fragment thereof, a preparation method and application. The invention also provides vectors comprising the isolated polynucleotides of the invention. The invention also provides the use of an antibody or antigen-binding fragment thereof of the invention in the manufacture of a medicament for the treatment and/or prevention of a condition that would benefit from an enhanced immune response and/or from exposure to interferon, wherein the condition is cancer or a viral infection, for example a hepatitis b virus infection.

Description

Anti-human PD-L1 antibody and antigen binding fragment thereof, preparation method and application

Technical Field

The invention belongs to the technical field of biological immunity, and particularly relates to an anti-human PD-L1 antibody and an antigen binding fragment thereof, which can specifically bind to human PD-L1 and block the binding with programmed death molecule L (PD-L).

Background

Programmed cell death factor ligand 1 (PD-L1), also known as CD274 or B7-H1, is a 40kDa type 1 transmembrane protein consisting of 290 amino acids. PD-L1 belongs to B7 family, has IgV and IgC like region, transmembrane region and cytoplasmic tail, and PD-L1 interacts with Programmed cell death factor 1 (PD-1) on T cells and plays a major role in suppressing the immune system. The combination of PD-L1 and PD-1 can generate an inhibition signal, inhibit the proliferation of antigen-specific T cells and reduce the apoptosis of regulatory T cells (Treg cells). Therefore, tumor cells that highly express PD-L1 are able to evade immune surveillance by this immunosuppressive effect.

In the normal immune system, PD-L1 plays an extremely important role in immune tolerance. Studies have shown that PD-L1 is expressed on the surface of periodontal ligament cells, promotes apoptosis of T cells and inhibits the damaging effect of inflammatory cells on periodontal ligament cells (Zhang Jiehua et al, proceedings of the ninth national institute of immunology, 2014).

In the tumor, expression of PD-L1 was detected in most tumor cell lines, such as lymphoma, choriocarcinoma, melanoma, esophageal cancer, etc., resulting in apoptosis of T cells and elimination of activated T cells (Dong H, et al, Nat Med,2002,8(8): 793-. PD-L1 is up-regulated in various tumors such as Breast Cancer (Muenst S, Breast Cancer Res Treat,2014,146(1):15-24.), gastric Cancer (Kim J W. gastric Cancer,2014oma [ J ]. Ann oncol,2014,25(11):2178-2184), and low or no expression in normal tissues.

Tumor cell immune escape is one of the important reasons for tumor evasion and immune system clearance. Binding of PD-L1 expressed on the surface of tumor cells to PD-1 inhibits activation of T cells, rendering them unable to find tumors. The anti-PD-L1 monoclonal antibody can effectively bind to PD-L1 on the surface of cancer cells, block a signal path, activate T cells and kill tumor cells. Blocking the PD-L1/PD-1 pathway can increase T cell responses. The research finds that the expression level of PD-L1 in non-muscle invasive bladder cancer cells is related to the pathological stage of tumors, and the treatment effect can be improved by selecting the medicament according to the expression of PD-L1. The expression of mRNA of lung adenocarcinoma A549 cell PD-L1 is down-regulated, the killing effect of killer cells on A549 cells can be enhanced (Jiang, modern tumor medicine 2015.23(12):1641-1643), and PD-L1 protein is an important index (Liuming Dong, modern tumor medicine 2017.885-889) for predicting the postoperative progression of tumors in non-small cell lung cancer. The prognostic role of PD-L1 has also been reported in studies of other tumors.

In the case of chronic viral infectious diseases, the results of the study showed that in PD-L1 knockout mice, a large number of CD8+ T cells accumulated in the liver, which decreased the clearance of activated T cells, resulting in an increased incidence of autoimmune hepatitis (Dong H., Immunity,2004,20(3): 327-) -336.). It was found that interferon up-regulates the expression of PD-L1 on hepatocytes in activated T cells or during the viral infection phase and that co-action with PD-1 expressed on T cells induces T cell apoptosis. The research on the mouse infected by the chronic lymphocytic choriomeningitis virus shows that the gene expression analysis comparison of the CD8+ T cells specific to the function-impaired virus shows that the PD-L1 is also highly expressed on the infected cells, and the blocking of the PD-1/PD-L1 signal channel can enhance the reactivity of the T cells and restore partial functions of the CD8+ T cells. This mechanism helps to develop therapeutic strategies for chronic infectious diseases.

The antibody medicine is prepared by using an antibody engineering technology which takes a cell engineering technology and a genetic engineering technology as main bodies, has the advantages of high specificity, clear mechanism, obvious curative effect, less toxic or side effect and the like, and has very wide prospect in the treatment of various diseases, particularly the treatment of tumors. Many PD-L1 inhibitors are being developed as immuno-oncology therapies, and the currently approved anti-PD-L1 monoclonal antibody drug by the U.S. FDA is Atezolizumab (Tecnriq) by Roche/Genentech, clinically used for the treatment of bladder cancer and non-small cell lung cancer. The marketed PD-L1 antibodies also include Atezolizumab and Avelumab.

Based on this, there is currently a need for more potent, more specific anti-PD-L1 antibodies.

Disclosure of Invention

Based on the defects of the prior art, the main purpose of the invention is to provide a high-efficiency and safe anti-human PD-L1 antibody. The invention also provides a preparation method and application of the antibody, and the anti-human PD-L1 antibody can be used for treating various cancers or virus infection by regulating human immune function.

In one aspect, the invention provides an anti-human PD-L1 antibody or antigen-binding fragment thereof comprising one or more heavy chain variable regions selected from the group consisting of:

a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO. 1;

a VH CDR2 comprising an amino acid sequence set forth as SEQ ID NO. 2; and

VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO. 3.

Preferably, the anti-human PD-L1 antibody or antigen-binding fragment thereof further comprises one or more light chain variable regions selected from the group consisting of:

VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO. 4;

VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO. 5; and

VL CDR3 comprising the amino acid sequence shown in SEQ ID NO. 6.

In one embodiment according to the present invention, the anti-human PD-L1 antibody or antigen-binding fragment thereof further comprises a human heavy chain constant region and/or a human light chain constant region linkage; preferably, the human heavy chain constant region is selected from a human IgG1, IgG2, IgG3 or IgG4 heavy chain constant region and the human light chain constant region is selected from a human IgG1, IgG2, IgG3 or IgG4 light chain constant region; more preferably, the human heavy chain constant region is a human IgG1 heavy chain constant region and the human light chain constant region is a kappa chain.

In one embodiment according to the invention, the anti-human PD-L1 antibody or antigen binding fragment thereof is a camelized single domain antibody, scFv dimer, BsFv, dsFv2, dsFv-dsFv ', Fv fragment, Fab ', F (ab ')2, ds diabody, nanobody, domain antibody, or diabody.

In one embodiment according to the present invention, the heavy chain variable region of the anti-human PD-L1 antibody or antigen-binding fragment thereof comprises the amino acid sequence shown in SEQ ID NO. 7.

In one embodiment according to the present invention, the variable region of the light chain of the anti-human PD-L1 antibody or antigen-binding fragment thereof comprises the amino acid sequence shown in SEQ ID NO. 8.

In one embodiment according to the present invention, the heavy chain of the anti-human PD-L1 antibody or antigen-binding fragment thereof comprises the amino acid sequence shown in SEQ ID NO. 9.

In one embodiment according to the present invention, the light chain of the anti-human PD-L1 antibody or antigen-binding fragment thereof comprises the amino acid sequence shown in SEQ ID NO. 10.

In one embodiment according to the present invention, the heavy chain variable region of anti-human PD-L1 antibody or antigen binding fragment thereof comprises the amino acid sequence shown in SEQ ID NO. 13.

In one embodiment according to the present invention, the variable region of the light chain of the anti-human PD-L1 antibody or antigen-binding fragment thereof comprises the amino acid sequence shown in SEQ ID NO. 14.

In one embodiment according to the invention, the heavy chain of an anti-human PD-L1 antibody or antigen-binding fragment thereof comprises the amino acid sequence shown in SEQ ID NO. 15.

In one embodiment according to the present invention, the light chain of the anti-human PD-L1 antibody or antigen-binding fragment thereof comprises the amino acid sequence shown in SEQ ID NO 16.

In another embodiment according to the present invention, the heavy chain and the light chain of said anti-human PD-L1 antibody or antigen-binding fragment thereof antibody are linked by a self-cleaving sequence selected from the group consisting of 2A peptide fragments. The self-cutting sequence 2A peptide fragment is derived from foot-and-mouth disease viruses (FMDV). The 2A peptide segment or the similar peptide segment thereof consists of 18-22 amino acids, and the C-terminal sequence is highly conserved: -D _X E _X NPGP-. When the ribosome translates into this sequence, an automatic cleavage (self-cleavage) without protease is achieved because "skipping" occurs. Self-cleavage occurs between the C-terminal G-P residues. (see "development of a multigene expression vector based on 2A peptide strategy", China Biotechnology,2013,33(1):104- "108). Preferably, the 2A peptide fragment is selected from P2A (SEQ ID NO:25ATNFSLLKQAGDVEENPGP), T2A (SEQ ID NO:26 EGRGSLLTCGDVEENPGP) or E2A (SEQ ID NO:27 QCTNYALLKLAGDVESNPGP); more preferably, the self-splicing sequence is P2A, because the efficiency of self-splicing is highest.

In another aspect, the present invention also provides an isolated polynucleotide encoding the above-described anti-human PD-L1 antibody or antigen-binding fragment thereof;

preferably, the polynucleotide comprises a nucleotide sequence shown as SEQ ID NO. 11;

preferably, the polynucleotide comprises a nucleotide sequence as set forth in SEQ ID NO. 12;

preferably, the polynucleotide comprises a nucleotide sequence as set forth in SEQ ID NO. 17;

preferably, the polynucleotide comprises the nucleotide sequence shown as SEQ ID NO. 18.

In yet another aspect, the invention also provides a vector comprising the isolated polynucleotide of the invention.

In yet another aspect, the present invention also provides a host cell comprising the vector of the present invention, preferably, the host cell is a mammalian cell, more preferably a human, murine, ovine, equine, canine or feline cell, and even more preferably a chinese hamster ovary cell.

Further, the present invention also provides a method for producing the antibody or antigen-binding fragment thereof of the present invention, which comprises culturing the host cell under conditions capable of expressing the polynucleotide of the present invention.

Further, the present invention also provides a kit comprising the antibody or antigen-binding fragment thereof of the present invention.

Further, the present invention also provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of the present invention and a pharmaceutically acceptable carrier.

Further, the invention also provides the use of an antibody or antigen-binding fragment thereof comprising the invention in the manufacture of a medicament for the prevention and/or treatment of a condition which benefits from enhancing an immune response and/or from exposure to interferon.

In one embodiment according to the present invention, wherein the condition is cancer or a viral infection, such as hepatitis B virus infection.

Specifically, the preparation method of the anti-human PD-L1 antibody or antigen combination thereof provided by the invention comprises the following steps:

1) taking a Balb/c mouse immunized by using a CHO cell strain over-expressing PD-L1 as an antigen as a material, extracting splenocytes, and fusing the splenocytes with a mouse myeloma cell line SP2/0-AG14 to obtain a hybridoma cell strain capable of expressing an anti-human PD-L1 antibody or antigen combination thereof;

2) cloning and expressing a gene combined by the anti-human PD-L1 antibody or the antigen thereof in the hybridoma cell strain obtained in the step 1);

3) providing an expression vector, wherein the expression vector comprises the gene cloned in the step 2) and an expression regulation and control sequence operatively connected with the gene;

4) transforming a host cell with the expression vector of step 3);

5) culturing the host cell obtained in the step 4);

6) separating and purifying to obtain the monoclonal antibody.

In another aspect, the present invention provides a hybridoma cell line for use in the above preparation method.

The invention prepares human-mouse hybridoma secreting specific anti-human PD-L1 antibody, clones the heavy chain and light chain sequences (one hundred percent of human genes) of the antibody by utilizing molecular biology technology, constructs the anti-human PD-L1 human monoclonal antibody, and expresses and produces the antibody by CHO cells. The antibodies have stronger binding capacity and specificity compared with the existing antibodies as medicaments.

Drawings

FIG. 1 shows the results of ELISA binding experiments of the antibody of the present invention to PD-L1 protein. (A) Binding of the antibody to human PD-L1 protein; (B) binding of the antibody to cynomolgus monkey PD-L1 protein;

FIG. 2 shows the binding of the antibody of the present invention to cells expressing human PD-L1.

FIG. 3 shows the ability of the antibodies of the invention to inhibit the binding of Raji-PD-L1 cells to PD-1 protein.

FIG. 4 shows that the antibodies of the invention antagonize the inhibition of the NFAT signaling pathway in Jurkat-PD-1 cells by PD-L1.

FIG. 5 shows the potency of the antibodies of the invention in a mouse model of CT26-PD-L1 subcutaneous tumor suppressor.

Detailed Description

The following description of the present application is intended to be illustrative of various embodiments of the present application. Therefore, the specific modifications discussed herein should not be construed as limitations on the scope of the application. Numerous equivalents, changes, and modifications will readily occur to those skilled in the art without departing from the scope of the present application, and it is intended that such equivalents be included within the scope of the present invention. All documents, including publications, patents, and patent applications, cited in this application are incorporated by reference in their entirety.

Definition of

The term "antibody" as used herein includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multispecific antibody or bispecific (bivalent) antibody that binds a particular antigen. A natural intact antibody comprises two heavy chains and two light chains. Each heavy chain consists of a variable region and first, second and third constant regions; each light chain consists of a variable region and a constant region. Mammalian heavy chains can be classified as α, δ, ε, γ, and μ, and mammalian light chains as λ or κ. The antibody is "Y" shaped, with the neck of the Y structure consisting of the second and third constant regions of the two heavy chains, which are bound by disulfide bonds. Each arm of the "Y" configuration includes the variable region and the first constant region of one of the heavy chains, which is associated with the variable and constant regions of one of the light chains. The variable regions of the light and heavy chains determine the binding of the antigen. The variable region of each chain contains three hypervariable regions, called Complementarity Determining Regions (CDRs). The CDRs of the light chain (L) comprise LCDR1, LCDR2, LCDR3, and the CDRs of the heavy chain (H) comprise HCDR1, HCDR2, HCDR 3. The CDR boundaries of the antibodies and antigen-binding fragments disclosed herein can be named or identified by the Kabat, Chothia or Al-Lazikani nomenclature. (AI-Lazikani, B., Chothia, C., Lesk, A.M., J. mol. biol.,273 (4): 927 (1997); Chothia, C., et al, J.mol. biol.,186 (3): 651-63 (1985); Chothia, C.and Lesk, A.M., J.mol. biol., 196: 901 (1987); Chothia, C. et al, Nature,342 (6252): 877-83 (1989); Kabat, E.A. et al, National instruments of Health, Bethesda, Md. (1991)). Where three CDRs are separated by flanking continuous portions called Framework Regions (FRs) that are more highly conserved than the CDRs and form a scaffold-supported hypervariable loop. The constant regions of the heavy and light chains are not involved in antigen binding, but have multiple effector functions. Antibodies can be divided into several classes depending on the amino acid sequence of the heavy chain constant region. Depending on whether it contains alpha, delta, epsilon, gamma and mu heavy chains, antibodies can be classified into five main classes or isoforms, respectively: IgA, IgD, IgE, IgG and IgM. Several major antibody classes can also be divided into subclasses, such as IgG1(γ 1 heavy chain), IgG2(γ 2 heavy chain), IgG3(γ 3 heavy chain), IgG4(γ 4 heavy chain), IgA1(α 1 heavy chain), or IgA2(α 2 heavy chain), among others.

The term "antigen-binding fragment" as used herein refers to an antibody fragment formed from a portion of an antibody that contains one or more CDRs or any other antibody fragment that binds an antigen but does not have an intact antibody structure. Examples of antigen binding fragments include, but are not limited to, such as diabodies (diabodies), fabs ', F (ab ')2, Fv fragments, disulfide stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv '), disulfide stabilized diabodies (ds diabodies), single chain antibody molecules (scFv), scFv dimers (diabodies), bivalent single chain antibodies (BsFv), multispecific antibodies, camelized single domain antibodies (camelized single domain antibodies), nanobodies, domain antibodies, and bivalent domain antibodies. The antigen-binding fragment may bind to the same antigen as the maternal antibody. In certain embodiments, an antigen-binding fragment can comprise one or more CDRs from a particular human antibody grafted to a framework region from one or more different human antibodies.

An "Fab" fragment of an antibody refers to the portion of the antibody molecule that is disulfide bonded to one light chain (which includes both the variable and constant regions) and to the variable and partial constant regions of one heavy chain.

By "Fab'" fragment is meant a Fab fragment comprising part of the hinge region.

"F (ab')2" refers to a dimer of Fab.

The Fc portion of an antibody is responsible for a variety of different effector functions such as ADCC and CDC, but is not involved in antigen binding.

The "Fv" segment of an antibody refers to the smallest antibody fragment that contains the entire antigen-binding site. The Fv fragment consists of the variable region of one light chain and the variable region of one heavy chain.

"Single chain Fv antibody" or "scFv" refers to an engineered antibody comprising a light chain variable region joined directly to a heavy chain variable region or via a peptide chain (Huston JS et al, Proc Natl Acad Sci USA, 85: 5879 (1988)).

"Single chain antibody Fv-Fc" or "scFv-Fc" refers to an engineered antibody consisting of an scFv linked to an Fc portion of an antibody.

"Camelidized single domain antibody", "Heavy chain antibody" or "HCAb (Heavy-chain-only antibodies, HCAb)" all refer to antibodies that contain two VH domains but no light chain (Riechmann L. and Muydermans S., J Immunol methods.231 (1-2): 25-38 (1999); Muydermans S., J Biotechnol.74 (4): 277-302 (2001); W094/04678; W094/25591; U.S. patent No.6,005,079). Heavy chain antibodies were originally derived from camelidae (camels, dromedary and llamas). Despite the absence of the light chain, camelized antibodies (camelized antibodies) have a positive full function of antigen binding (polymers Casterman C. et al, Nature 363 (6428): 446-8 (1993); Nguyen VK. et al, "Heavy-chain antibodies in camelids: a case of evolution innovation, immunogenetics.54 (1): 39-47 (2002); Nguyen VK. et al, immunology.109 (1): 93-101 (2003)). The variable region (VH domain) of heavy chain antibodies is the smallest known antigen-binding unit produced by acquired immunity (Koch-Nolte F. et al, FASEB J.21 (13): 3490-8.Epub (2007)).

"Nanobody" refers to an antibody fragment consisting of one VH domain from a heavy chain antibody and two constant regions, CH2 and CH 3.

"bifunctional antibodies" (diabodies) include small antibody fragments with two antigen-binding sites, wherein the fragment contains a VH domain and a VL domain linked on the same polypeptide chain (see Holliger P. et al, Proc Natl Acad Sci U S A.90 (14): 6444-8 (1993); EP 404097; W093/11161). The linker between the two domains is so short that the two domains on the same chain do not pair with each other, thereby forcing the two domains to pair with the complementary domains of the other chain, forming two antibody binding sites. The two antibody binding sites may be targeted to bind to the same or different antigens (or epitopes).

"Domain antibody" refers to an antibody fragment containing only one heavy chain variable region or one light chain variable region. In some cases, two or more VH domains are covalently bound by one polypeptide linker and form a bivalent domain antibody. The two VH domains of a bivalent domain antibody may be targeted to the same or different antigens.

In certain embodiments, the "(dsFv)2" comprises three peptide chains: two VH genes are linked by a polypeptide linker and are linked to two VL groups by disulfide bonds.

In certain embodiments, a "bispecific ds bifunctional antibody" comprises VL1-VH2 (linked by a polypeptide linker) and VH1-VL2 (also linked by a polypeptide linker) which are bound by a disulfide bond between VH1 and VLl.

A "bispecific dsFv" or "dsFv-dsFv" comprises three polypeptide chains: VH1-VH2 groups in which the heavy chains of both are linked by polypeptide linkers (e.g.long flexible linkers) and are bound by disulfide bonds to VL1 and VL2 groups respectively, each pair of heavy chain and light chain paired by a disulfide bond having a different antigen specificity.

In certain embodiments, an "scFv dimer" is a bivalent diabody or bivalent single chain antibody (BsFv) comprising two VH-VL (joined by a polypeptide linker) groups that dimerize, wherein the VH of two groups cooperates with the VL of another group to form two binding sites that can be targeted to bind to the same antigen (or epitope) or to different antigens (or epitopes). In other embodiments, an "scFv dimer" is a bispecific diabody comprising interconnected V _L1 -V _H2 (ligated by polypeptide linker) and V _H1 -V _L2 (ligated by polypeptide linkers) wherein V _H1 And V _L1 Cooperation, V _H2 And V _L2 In cooperation, and each cooperative pair has a different antigen specificity.

The term "fully human" as used herein, when applied to an antibody or antigen-binding fragment, means that the antibody or antigen-binding fragment has or consists of an amino acid sequence that corresponds to that of an antibody produced by a human or human immune cell or derived from a non-human source, e.g., a transgenic non-human animal that utilizes a human antibody repertoire, or other sequence encoding a human antibody. In certain embodiments, the fully human antibody does not comprise amino acid residues (particularly antigen binding residues) derived from a non-human antibody.

The term "humanized" as used herein, when applied to an antibody or antigen-binding fragment, refers to an antibody or antigen-binding fragment that includes CDRs derived from a non-human animal, FR regions derived from a human, and constant regions derived from a human, where applicable. Because the humanized antibody or antigen binding fragment has reduced immunogenicity, it may be used in certain embodiments as a therapeutic agent in humans. In some embodiments, the non-human animal is a mammal such as a mouse, rat, rabbit, goat, sheep, guinea pig, or hamster. In some embodiments, the humanized antibody or antigen-binding fragment consists essentially entirely of sequences of human origin, except for the CDR sequences which are of non-human origin. In some embodiments, the human-derived FR region may include the same amino acid sequence as the human-derived antibody from which it is derived, or it may include some amino acid changes, e.g., no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid change. In some embodiments, the amino acid change may be present in only the heavy chain FR region, only the light chain FR region, or both chains. In some preferred embodiments, the humanized antibody comprises human FRl-3 and human JH and JK.

The term "chimeric" as used herein refers to an antibody or antigen-binding fragment having a portion of a heavy and/or light chain derived from one species, and the remainder of the heavy and/or light chain derived from a different species. In an illustrative example, a chimeric antibody can include constant regions derived from a human and variable regions derived from a non-human animal, such as a mouse.

The term "PD-L1" refers to a ligand that binds to PD-1. Others are designated as CD274 or B7-H1, etc. It has a molecular weight of 40kDa and is stored in SwissProt under the accession number Q9NZQ 7.

"specific binding" or "specific binding" in this application refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen. In certain embodiments, the antibodies or antigen binding fragments thereof of the present application specifically bind to human and/or monkey PD-L1, and their binding affinity (K) _D )≤10 ^-6 M。K in the present application _D Refers to the ratio of dissociation rate to association rate (k) _off /k _on ) It can be determined by means of surface plasmon resonance, for example using an instrument such as Biacore.

As used herein, "MT02-L4" refers to a human-murine chimeric antibody having a heavy chain as shown in SEQ ID NO. 9, a light chain as shown in SEQ ID NO. 10, and the constant regions of the heavy and light chains are human IgG1 and kappa chains, respectively.

As used herein, "MT02-C4" refers to a humanized antibody having a heavy chain as shown in SEQ ID NO. 15, a light chain as shown in SEQ ID NO. 16, and the heavy and light chain constant regions are human IgG1 and kappa chains, respectively.

In the present application when "conservative substitution" is used for an amino acid sequence, it is meant that one amino acid residue is substituted with another amino acid residue having a side chain with similar physicochemical properties. For example, conservative substitutions may be made between hydrophobic side chain amino acid residues (e.g., Met, Ala, VaL, Leu, and Ile), between neutral hydrophilic side chain residues (e.g., Cys, Ser, Thr, Asn, and Gln), between acidic side chain residues (e.g., Asp, Glu), between basic side chain amino acids (e.g., His, Lys, and Arg), or between aromatic side chain residues (e.g., Trp, Tyr, and Phe). It is known in the art that conservative substitutions do not generally result in significant changes in the conformational structure of a protein, and therefore the biological activity of the protein can be retained.

"percent sequence identity," when used with respect to an amino acid sequence (or nucleic acid sequence), refers to the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to a reference sequence to the amino acid (or nucleic acid) residues in the candidate sequence after alignment and, if necessary, introduction of a spacer to maximize the number of identical amino acids (or nucleic acids). Conservative substitutions of such amino acid residues may or may not be considered identical residues. Sequences can be aligned by means disclosed in the art to determine the percent sequence identity of amino acid (or nucleic acid) sequences. One skilled in the art can use default parameters for the tool or adjust the parameters appropriately according to the needs of the alignment, for example by choosing an appropriate algorithm.

"T cells" as used herein include CD4+ T cells, CD8+ T cells, T helper type 1T cells, T helper type 2T cells, T helper type 17T cells, and suppressor T cells.

"effector function" as used herein refers to the biological activity of the Fc region of an antibody to bind its effectors such as the C1 complex and Fc receptor. Exemplary effector functions include Complement Dependent Cytotoxicity (CDC) induced by interaction of the antibody with C1q on the C1 complex, antibody dependent cell mediated cytotoxicity (ADCC) induced by binding of the Fc region of the antibody to Fc receptors on effector cells, and phagocytosis.

By "cancer" or "cancerous condition" in the present application is meant any medical condition that is mediated by the growth, proliferation or metastasis of neoplastic or malignant cells and that causes solid and non-solid tumors, such as leukemia. The term "tumor" as used herein refers to a solid substance of a tumor and/or malignant cells.

"treating" or "treatment" of a condition includes preventing or alleviating the condition, reducing the rate at which a condition develops or develops, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with a condition, reducing or terminating symptoms associated with a condition, producing a complete or partial reversal of a condition, curing a condition, or a combination thereof. "treating" or "therapy" with respect to cancer may refer to inhibiting or slowing the growth, reproduction, or metastasis of a tumor or malignant cell, or some combination thereof. "treating" or "therapy" with respect to a tumor includes eliminating all or part of the tumor, inhibiting or slowing tumor growth and metastasis, preventing or delaying the development of the tumor, or some combination thereof.

The "isolated" material has been artificially altered from its natural state. If a "separated" substance or component occurs in nature, it has been altered or removed from its original state, or both. For example, a polynucleotide or polypeptide naturally present in a living animal is not isolated, but is considered to be "isolated" if the polynucleotide or polypeptide is sufficiently isolated from substances with which it coexists in its natural state and is present in a sufficiently pure state. In certain embodiments, the antibodies and antigen-binding fragments are at least 90%, 93%, 95%, 96%, 97%, 98%, 99% pure, as determined by electrophoretic methods (e.g., SDS-PAGE, isoelectric focusing, capillary electrophoresis), or chromatographic methods (e.g., ion exchange chromatography or reverse phase HPLC).

A "vector" in the present invention refers to a vehicle into which a polynucleotide encoding a protein is operably inserted and the protein is expressed. The vector may be used to transform, transduce or transfect a host cell so that the genetic material elements it carries are expressed in the host cell. By way of example, the carrier includes: plasmids, phagemids, cosmids, artificial chromosomes such as Yeast Artificial Chromosomes (YACs), Bacterial Artificial Chromosomes (BACs) or P1-derived artificial chromosomes (PACs), bacteriophages such as lambda phage or M13 bacteriophages, animal viruses, and the like. Examples of animal virus species for use as vectors are retroviruses (including lentiviruses, adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papilloma viruses, papilloma virus vacuolium (e.g., SV 40)). The vector may contain a variety of elements which control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may contain a replication origin. The carrier may also include components that facilitate its entry into the cell, including, but not limited to, viral particles, liposomes, or protein coats.

The term "host cell" as used herein refers to a cell into which an exogenous polynucleotide and/or vector has been introduced.

As used herein, a "therapeutically effective amount" or "effective dose" refers to a dose or concentration of a drug effective to treat a disease. For example, for use of the antibodies or antigen-binding fragments thereof disclosed in the present invention, a therapeutically effective amount is at that dose or concentration, and the antibody or antigen-binding fragment thereof can eliminate all or a portion of the tumor, inhibit or slow tumor growth, inhibit tumor cell metastasis, reduce any symptoms or markers associated with the tumor or cancer condition, prevent or delay the development of the tumor or cancer condition, inhibit or eliminate virus or virus-infected cells, or some combination thereof.

By "pharmaceutically acceptable" is meant a carrier, vehicle, diluent, adjuvant and/or salt that is generally chemically and/or physically compatible with the other ingredients of the formulation and physiologically compatible with the recipient.

Anti-human PD-L1 antibody of the present invention

In certain embodiments, the present application provides an exemplary anti-human PD-L1 antibody MT 02-C4.

It will be appreciated by those skilled in the art that the foregoing CDR sequences may be modified to include substitutions of one or more amino acids to result in improved biological activity, such as improved binding affinity to human PD-L1. For example, libraries of antibody variants (e.g., Fab or FcFv variants) can be produced and expressed using phage display technology, and subsequently screened for antibodies having affinity for human PD-L1. In another example, computer software can be used to mimic the binding of the antibody to human PD-L1 and to identify the amino acid residues on the antibody that form the binding interface. Substitutions of these residues can be avoided to prevent a decrease in binding affinity, or substitutions can be targeted to these residues to form stronger binding. In certain embodiments, at least one (or all) substitution in a CDR sequence is a conservative substitution.

In certain embodiments, the antibody or antigen-binding fragment comprises one or more CDR sequences having sequence identity to at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) of the sequence of SEQ ID NOs 1-6 while retaining similar or even greater binding affinity to human PD-L1 as its parent antibody. The parent antibodies have substantially identical sequences, but the corresponding CDR sequences have 100% sequence identity to the sequences set forth in SEQ ID Nos. 1-6.

In some embodiments, the antibodies or antigen binding fragments described herein can have a size of ≦ 10 ^-7 Binding affinity of M (K) _D ) Specifically binds to human PD-L1, as measured by surface plasmon resonance. The binding affinity value may be represented by K _D The value is expressed as the ratio of the off-rate to the on-rate (k) at which the binding of antigen and antigen binding molecule reaches equilibrium _off /k _on ) MeterAnd (5) calculating to obtain. Binding affinity of the antigen (e.g., K) _D ) May be suitably determined by suitable methods known in the art, including, for example, plasmon resonance binding using an instrument such as Biacore.

In certain embodiments, the antibodies or antigen-binding fragments described herein bind to human PD-L1 at an EC50 (i.e., half the binding concentration) of 10ng/mL to 10 μ g/mL. Binding of the antibody or antigen binding fragment to human PD-L1 can be determined by methods known in the art such as sandwich assays such as ELISA, Western blot, FACS or other binding assays. In an illustrative example, a test antibody (i.e., a primary antibody) is bound to immobilized human PD-L1 or cells expressing human PD-L1, followed by washing away unbound antibody, and introducing a labeled secondary antibody that is capable of binding to the primary antibody and thus detecting the bound secondary antibody. The detection can be performed on a microplate reader plate when immobilized PD-L1 is used, or can be performed using FACS analysis when cells expressing human PD-L1 are used.

In certain embodiments, an antibody or antigen-binding fragment described herein binds to human PD-L1 with an EC50 (i.e., an effective concentration of 50%) of 0.1 μ g/mL to 10 μ g/mL (as determined using FACS analysis).

The antibody is specific for human PD-L1. In certain embodiments, the antibody does not bind to murine PD-L1, but binds to monkey PD-L1 with similar binding affinity as human PD-L1.

In some embodiments, the antibody has a constant region of the IgG2 isotype with reduced or eliminated effector function. Equivalent functions such as ADCC and CDC can result in cytotoxicity to cells expressing PD-L1. Some normal cells are capable of expressing PD-L1. To avoid potential undesirable toxicity to these normal cells, certain embodiments of the antibodies of the invention have reduced or even eliminated effector function. Numerous assays are known for assessing ADCC or CDC activity, such as Fc receptor binding assays, complement Clq binding assays and cell lysis methods, which can be readily selected by one of skill in the art. Without wishing to be bound by theory, it is believed that antibodies with reduced or eliminated effector functions such as ADCC and CDC do not cause or minimize cytotoxicity to cells expressing PD-L1 (e.g., those normal cells), thus avoiding undesirable side effects.

In some embodiments, the antibodies or antigen-binding fragments described herein have reduced side effects. For example, the anti-PD-L1 antibodies and antigen-binding fragments thereof may have fully human IgG sequences and are therefore less immunogenic than humanized antibodies. As another example, the antibodies and antigen binding fragments thereof may have an IgG2 or IgG4 format to eliminate ADCC and CDC.

In some embodiments, the antibodies described herein are advantageous in that they can be used in combination with immunogenic agents, such as tumor cells, purified tumor antigens and cells transfected with an encoding immunostimulatory factor, tumor vaccines. In addition, the anti-PD-L1 antibodies and antigen-binding fragments thereof can be included in combination therapies, including standard chemotherapy and radiation therapy, target-based small molecule therapy, other emerging immune checkpoint modulator therapies. In some embodiments, the antibodies and antigen-binding fragments thereof can be used as the base molecule for antibody-drug conjugates, bispecific or multivalent antibodies.

In some embodiments, the antibodies and antigen-binding fragments thereof described herein are camelized single domain antibodies (camelized single domain antibodies), diabodies (diabodies), scfvs, scFv dimers, BsFv, dsFv, (dsFv)2, dsFv-dsFv ', Fv fragments, Fab ', F (ab ')2, ds diabodies (ds diabodies), nanobodies, domain antibodies, or diabodies.

In some embodiments, the antibodies described herein comprise an immunoglobulin constant region. In some embodiments, the immunoglobulin constant region comprises a heavy chain and/or light chain constant region. The heavy chain constant region comprises the CH1, CH1-CH2, or CH1-CH3 regions. In some embodiments, the immunoglobulin constant region may further comprise one or more modifications to achieve a desired property. For example, the constant region may be modified to reduce or eliminate one or more effector functions to enhance FcRn receptor binding or to introduce one or more cysteine residues.

In certain embodiments, the antibodies and antigen-binding fragments thereof further comprise a conjugate. It is contemplated that the antibodies or antigen-binding fragments thereof of the present invention can be linked to a variety of conjugates (see, e.g., "Conjugate Vaccines", constraints to Microbiology and Immunology, j.m. house and r.e. lewis, Jr. (eds.), Carger Press, New York (1989)). These conjugates may be attached to the antibody or antigen conjugate by covalent binding, affinity binding, intercalation, coordinate binding, complexation, binding, mixing or addition, among other means. In certain embodiments, the antibodies and antigen-binding fragments disclosed herein can be engineered to contain specific sites other than the epitope-binding portion that can be used to bind to one or more conjugates. For example, such sites may comprise one or more reactive amino acid residues, such as cysteine and histidine residues, for facilitating covalent attachment to the conjugate. In certain embodiments, the antibody may be linked indirectly to the conjugate, or via another conjugate. For example, the antibody or antigen-binding fragment thereof can bind to biotin and then indirectly bind to a second conjugate, which is linked to avidin. The conjugate may be a detectable label, a pharmacokinetic modification moiety, a purification moiety, or a cytotoxic moiety. Examples of detectable labels may include fluorescent labels (e.g., fluorescein, rhodamine, dansyl, phycoerythrin or texas red), enzyme substrate labels (e.g., horseradish peroxidase, alkaline phosphatase, luciferase, glucoamylase, lysozyme, carbohydrate oxidase or β -D-galactoxiiase), stable isotopes or radioisotopes, chromophore moieties, digoxin, biotin/avidin, DNA molecules or gold for detection. In certain embodiments, the conjugate can be a pharmacokinetic modifying moiety such as PEG, which helps to extend the half-life of the antibody. Other suitable polymers include, for example, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, ethylene glycol/propylene glycol copolymers, and the like. In certain embodiments, the conjugate can be a purification moiety such as a magnetic bead. A "cytotoxic moiety" may be any agent that is harmful to or may damage or kill a cell. Examples of cytotoxic moieties include, but are not limited to, paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthrax dione, mitoxantrone, mithramycin, actinomycin D, l-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin and analogs thereof, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine glance sideways at, cytarabine, 5 fluorouracil dacarbazide alkylating agents) (e.g., mechlorethamine, thiotepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, mitoxantrone, doxycycline, meclomethamine, vincristine, vinblastine, vincristine, levofloxacin, doxycycline, and doxycycline, and one, and one, and one, and one, and one, and one, and one, streptozotocin, mitomycin C and cis-dichlorodiammineplatinum (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly known as actinomycin), bleomycin, mithramycin and Amikacin (AMC)), and antimitotic agents (e.g., vincristine and vinblastine).

Polynucleotides and recombinant methods

The amino acid sequences of the antibodies and antigen-binding fragments thereof described herein can be converted to the corresponding DNA coding sequences using genetic engineering techniques well known in the art. Due to the degeneracy of the genetic code, the resulting DNA sequences can be completely identical while the encoded protein sequence remains unchanged.

Vectors comprising polynucleotides encoding the antibodies and antigen-binding fragments thereof may be introduced into host cells for cloning (amplification of DNA) or gene expression using recombinant techniques well known in the art. In another embodiment, the antibodies and antigen binding fragments thereof can be made by methods of homologous recombination as are well known in the art. Various carriers are available for selection. Carrier components generally include, but are not limited to, two or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer sequence, a promoter (e.g., SV40, CMV, EF-1a) and a transcription termination sequence.

In some embodiments, the vector system comprises a mammalian, bacterial, yeast system, etc., and will include plasmids such as, but not limited to, pALTER, pBAD, pcDNA, pCal, pL, pELpGEMEX, pGEX, pCLpCMV, pEGFP, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS420, pLexA, pACT2, etc., other vectors available from the laboratory or commercially available. Suitable vectors may include plasmids or viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses).

Vectors comprising polynucleotides encoding the antibodies and antigen-binding fragments thereof can be introduced into host cells for cloning or gene expression. Host cells suitable for cloning or expressing the DNA in the vector of the present invention are prokaryotic cells, yeast or the above-mentioned higher eukaryotic cells. Prokaryotic cells suitable for use in the present invention include eubacteria, such as gram-negative or gram-positive bacteria, for example Enterobacteriaceae (e.g., E.coli), Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescens, and Shigella, and bacilli, e.e., Bacillus subtilis and Bacillus licheniformis, Pseudomonas, e.g., Peucedanum and Streptomyces.

In addition to prokaryotic cells, eukaryotic microorganisms such as filamentous fungi or yeast may also be used as host cells for cloning or expressing vectors encoding the antibodies. Saccharomyces cerevisiae, or Saccharomyces cerevisiae, is the most commonly used lower eukaryotic host microorganism. However, many other genera, species and strains are commonly used and are suitable for use in the present invention, such as Schizosaccharomyces pombe; kluyveromyces hosts such as Kluyveromyces lactis, Kluyveromyces fragilis (ATCC12424), Kluyveromyces bulgaricus (ATCC16045), Kluyveromyces williamsii (ATCC24178), Kluyveromyces lactis (ATCC56500), Kluyveromyces drosophilus (ATCC36906), Kluyveromyces thermotolerans, and Kluyveromyces marxianus: yarrowia lipolytica (EP 402226); pichia pastoris (EP 183070); candida spp: trichoderma reesei (EP 244234); performing Neurospora; western flourishing yeasts such as: schwann yeast western; and filamentous fungi, such as: neurospora, Penicillium, Tolypocladium and Aspergillus, such as: aspergillus nidulans and Aspergillus niger.

The host cells provided in the present invention that are suitable for expressing glycosylated antibodies or antigen binding fragments thereof are derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. Various baculovirus strains (bacterial strains) and variants thereof, as well as corresponding permissive insect host cells (permissive insect host cells), have been found to be derived from hosts such as: spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori. A variety of viral strains for transfection are publicly available, such as Autographa californica nuclear polyhedrosis virus and Bm-5 variants of Bombyx mori nuclear polyhedrosis virus, all of which can be used in the present invention, particularly for transfecting Spodoptera frugiperda cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco may also be used as hosts.

However, the most interesting are the vertebral cells, and the culture of the vertebral cells (tissue culture) has become a routine procedure. As examples of mammalian host cells which may be used, there are SV40 transformed monkey kidney cell CV1 line (COS-7, ATCC CRL 1651); human embryonic kidney cell lines (293 or 293 cell subclones in suspension culture, Graham et al, Gen Virol.36: 59 (1977)); baby hamster kidney cells (B blood, ATCC CCL 10); chinese hamster ovary cells/-DHFR (CHO, Urlaub et al, Proc.Natl. Acad.Sci.USA 77: 4216 (1980)); mouse testicular support cells (TM4, Mather, biol. reprod.23: 243-251 (1980)); monkey kidney cells (CV1ATCC CCL 70); african green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical cancer cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumors (MMT 060562, ATCC CCL 51); TRI cells (Mather et al, Annals N. Y.Acad.Sci.383: 44-68 (1982)); MRC 5 cells; FS4 cells; and human hepatoma cell line HepG 2). In certain preferred embodiments, the host cell is a 293F cell.

Host cells are transformed with the above-described expression or cloning vectors that produce the antibodies and antigen-binding fragments thereof and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformed cells, or amplifying genes encoding the sequences of interest.

The host cells used in the invention for producing the antibodies and antigen-binding fragments thereof can be cultured in a variety of media well known in the art. The medium may also contain any other necessary additives at appropriate concentrations known in the art. The conditions of the medium, such as temperature, pH, and the like, which have been previously used to select host cells for expression, are well known to those of ordinary skill.

When using recombinant techniques, the antibodies can be produced intracellularly, in the periplasmic space, or secreted directly into the culture medium. If the antibody is produced intracellularly, the particulate debris of the host cells or lysed fragments is first removed, for example, by centrifugation or sonication. Carter et al, Bio/Technology 10: 163-167(1992) describes a method for isolating antibodies secreted into the E.coli wall membrane space. Briefly, the cell paste (cell paste) was lysed in the presence of uranium acetate (pH 3.5), EDTA, and phenylmethanesulfonic fluoride (PMSF) for about 30 minutes or more. Cell debris was removed by centrifugation. If the antibody is secreted into the culture medium, the supernatant of the expression system is usually first concentrated using commercially available protein concentration filters, such as the lAmicon or Millipore Pellicon ultrafiltration units. Protease inhibitors such as PMSF may be added in any of the foregoing steps to inhibit proteolytic degradation, as well as antibiotics to prevent the growth of adventitious contaminants.

The antibody produced from the cells can be purified by purification methods such as hydroxyapatite chromatography, gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography, ammonium sulfate precipitation, salting out, and affinity chromatography, with affinity chromatography being a preferred purification technique. The class of the antibody and the presence of the Fc domain of any immunoglobulin in the antibody determines whether protein a is suitable as an affinity ligand. Protein a can be used to purify antibodies based on human gamma 1, gamma 2 or gamma 4 heavy chains (Lindmark et al, j. lmmunol. meth.62: 13 (1983)). Protein G is applicable to all murine isoforms and human gamma 3(Guss et al, EMBO J.5: 15671575 (1986)). Agarose is the most commonly used affinity ligand attachment matrix, but other matrices may be used. Mechanically stable matrices such as controlled pore glass or poly (styrene) benzene can achieve faster flow rates and shorter processing times than can be achieved with agarose. If the antibody contains a CH3 domain, it can be purified using Bakerbond ABX. TM. resin (J.T.Baker, Phillipsburg, N.J.). Other techniques for protein purification may also be determined depending on the antibody to be obtained, such as fractionation in ion exchange columns, ethanol precipitation, reverse phase HPLC, silica gel chromatography, heparin sepharose chromatography based on anion or cation exchange resins (e.g.polyaspartic acid columns), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation.

After any preliminary purification step, the mixture containing the antibody of interest and impurities can be treated by low pH hydrophobic interaction chromatography, using an elution buffer at a pH of about 2.5-4.5, preferably at low salt concentrations (e.g., from about 0 to 0.25M salt concentration).

Reagent kit

Kits comprising the antibodies or antigen binding fragments thereof are provided. In some embodiments, the kit is used to detect the presence or level of PD-L1 in a biological sample. The biological sample may comprise a cell or tissue.

In some embodiments, the kit comprises an antibody or antigen-binding fragment thereof conjugated to a detectable label. In some embodiments, the kit comprises an unlabeled antibody and further comprises a secondary antibody capable of binding the label to the unlabeled antibody. The kit may further include instructions for use and packaging separating each component in the kit.

In some embodiments, the antibody is linked to a substrate or instrument for use in a sandwich assay such as an ELISA or immunochromatographic assay. Suitable substrates or instruments may be, for example, microplates and test strips.

Pharmaceutical compositions and methods of treatment

The application further provides pharmaceutical compositions comprising the antibodies and one or more pharmaceutically acceptable carriers.

Pharmaceutically acceptable carriers for use in the pharmaceutical compositions disclosed herein can include, for example, pharmaceutically acceptable liquids, gels or solid carriers, aqueous media, non-aqueous media, antimicrobial substances, isotonic substances, buffers, antioxidants, anesthetics, suspending/dispersing agents, integrating agents, diluents, adjuvants or nontoxic auxiliary substances, other components well known in the art, or various combinations thereof.

Suitable ingredients may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavoring agents, thickening agents, coloring agents, emulsifying agents, or stabilizing agents such as sugars and cyclodextrins. Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, thioglycerol, thioglycolic acid, mercaptosorbitol, butyl methyl anisole, butylated hydroxytoluene, and/or propyl gallate. The inclusion of one or more antioxidants, such as methionine, in a composition comprising an antibody of the present disclosure will reduce oxidation of the antibody. The reduction in oxidation prevents or reduces the reduction in binding affinity, thereby improving antibody stability and extending shelf life.

Further, pharmaceutically acceptable carriers may include, for example, aqueous media such as sodium chloride injection, ringer's solution injection, isotonic glucose injection, sterile water injection, or dextrose and lactate ringer's solution injection, non-aqueous media such as: non-volatile oils of vegetable origin, cottonseed oil, corn oil, sesame oil or peanut oil, antibacterial substances at bacteriostatic or fungistatic concentrations, isotonizing agents such as sodium chloride or glucose, buffers such as phosphate or citrate buffers, antioxidants such as sodium bisulfate, local anesthetics such as procaine hydrochloride, suspending and dispersing agents such as sodium carboxymethylcellulose, hydroxypropylmethylcellulose or polyvinylpyrrolidone, emulsifying agents such as polysorbate 80 (tween-80), integrating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethyleneglycol bis (2-aminoethylether) tetraacetic acid), ethanol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid or lactic acid. The antibacterial agent as a carrier may be added to the pharmaceutical composition in a multi-dose container, which includes phenols or cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoate esters, thimerosal, benzalkonium chloride and benzethonium chloride. Suitable excipients may include, for example, water, salt, glucose, glycerol or ethanol. Suitable non-toxic auxiliary substances may include, for example, emulsifiers, pH buffers, stabilizers, solubilizers, or substances such as sodium acetate, sorbitan laurate, triethanolamine oleate or cyclodextrins.

The pharmaceutical composition may be a liquid solution, suspension, emulsion, pill, capsule, tablet, sustained release formulation or powder. Oral formulations may include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinyl pyrrolidone, sodium saccharin, cellulose, magnesium carbonate, and the like.

In certain embodiments, the pharmaceutical composition is formulated as an injectable composition. Injectable pharmaceutical compositions may be prepared in any conventional form, for example, as liquid solvents, suspending agents, emulsifying agents, or solid forms suitable for the production of liquid solvents, suspending agents or emulsifying agents. Injectable preparations can include ready-to-use sterile and/or pyrogen-free solutions, sterile dried solubles, such as lyophilized powders, including subcutaneous tablets, sterile suspensions ready for injection, sterile dried insoluble products, which are combined with a vehicle prior to use, and sterile and/or pyrogen-free emulsions. The solvent may be aqueous or non-aqueous.

In certain embodiments, a unit dose of an injectable formulation is packaged in an ampoule, a manifold, or a syringe with a needle. It is well known in the art that all formulations for injection administration should be sterile and pyrogen free.

In certain embodiments, sterile lyophilized powders can be prepared by dissolving an antibody or antigen-binding fragment thereof disclosed herein in an appropriate solvent. The solvent may contain a compound that enhances the stability of the powder or reconstituted solution prepared from the powder, or improves other pharmacological components of the powder or reconstituted solution. Suitable excipients include, but are not limited to, water, glucose, sorbitol, fructose, corn syrup, xylitol, glycerol, glucose, brown sugar, or other suitable materials. The solvent may contain a buffer, such as citric acid buffer, sodium or potassium phosphate buffer or other buffers known to those skilled in the art, and in one embodiment, the pH of the buffer is neutral. Subsequent sterile filtration of the solution is carried out under standard conditions well known in the art and then lyophilized to produce the desired formulation. In one embodiment, the resulting solvent is dispensed into vials for lyophilization. Each vial can contain a single dose or multiple doses of the anti-PD-L1 antibody or antigen-binding fragment thereof or composition thereof. The loading per vial may be slightly higher than required for each dose or for multiple doses (e.g., 10% excess), thereby ensuring accurate sampling and accurate administration. The lyophilized powder may be stored under appropriate conditions, such as in the range of about 4 ℃ to room temperature.

And re-dissolving the freeze-dried powder with water for injection to obtain the preparation for injection administration. In one embodiment, the lyophilized powder can be reconstituted by addition to sterile pyrogen-free water or other suitable liquid carrier. The precise amount will be determined by the therapy chosen and may be determined empirically.

Also provided are methods of treatment comprising administering a therapeutically effective amount of an antibody described herein to a subject in need thereof.

The therapeutically effective dose of the antibody provided herein depends on a variety of factors well known in the art, such as weight, age, past medical history, current treatment, the health status and potential for cross-infection of the subject, allergies, hypersensitivity and side effects, as well as the route of administration and the extent of tumor development. One skilled in the art (e.g., a physician or veterinarian) can proportionately lower or raise the dosage based on these or other conditions or requirements.

In certain embodiments, the antibodies provided herein can be administered at a therapeutically effective dose of between about 0.0lmg/kg to about 100 mg/kg. In certain embodiments, the antibody is administered at a dose of about 50mg/kg or less, and in certain embodiments, 10mg/kg or less, 5mg/kg or less, 1mg/kg or less, 0.5mg/kg or less, or 0.1mg/kg or less. A particular dose can be administered at multiple intervals, such as once daily, twice or more monthly, once weekly, once biweekly, once every three weeks, once monthly, or once every two or more months. In certain embodiments, the dosage administered may vary with the course of treatment. For example, in certain embodiments, the initial dose may be higher than the subsequent dose. In certain embodiments, the dosage administered is adjusted during the course of treatment according to the response of the subject to whom it is administered.

The dosage regimen may be adjusted to achieve an optimal response (e.g., therapeutic response). For example, a single dose may be administered or multiple divided doses may be administered over a period of time.

The antibodies disclosed in the present invention can be administered by administration means well known in the art, such as injection (e.g., subcutaneous injection, intraperitoneal injection, intravenous injection, including intravenous drip, intramuscular injection, or intradermal injection) or non-injection (e.g., oral, nasal, sublingual, rectal, or topical administration).

In certain embodiments, the antibodies may be used to treat disorders associated with their molecular mechanisms, including tumors and cancers, such as non-small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymus cancer, leukemia, lymphoma, myeloma, granulomatosis (mycoses fungoids), merkel cell carcinoma, and other hematological malignancies, such as Classical Hodgkin Lymphoma (CHL), primary mediastinal large B-cell lymphoma, T-cell/histiocytic B-rich lymphoma, EBV positive and negative PTLD, and EBV-associated diffuse large B-cell lymphoma (DLBCL), plasmablast lymphoma, extranodal/T-cell lymphoma, and the like, Nasopharyngeal carcinoma and HHV 8-associated primary effusion lymphoma, hodgkin's lymphoma, Central Nervous System (CNS) tumors, e.g., primary CNS lymphoma, spinal axis tumors, brain stem glioma. In certain embodiments, the antibody-treated condition comprises a chronic viral infection, such as hepatitis b, hepatitis c, herpes virus, Epstein-Barr virus, aids virus, cytomegalovirus, herpes simplex virus type I, herpes simplex virus type 2, human papilloma virus, viral infection of adenovirus, kaposi's sarcoma-associated herpes virus epidemic, thin-ring virus (Torquetenovirus), JC virus, or BK virus, among others.

Application method

The application further provides methods of using the antibodies.

In some embodiments, the present application provides a method of treating a condition or disorder associated with the antibody mechanism in an individual comprising administering a therapeutically effective amount of an antibody described herein.

The antibodies disclosed herein can be administered alone or in combination with one or more other therapeutic means or substances. For example, the antibodies disclosed herein can be used in combination with chemotherapy, radiation therapy, surgery for cancer treatment (e.g., tumor resection), antiviral drugs, one or more anti-emetic drugs or other therapies for complications resulting from chemotherapy, or any other therapeutic substance for cancer or viruses. In certain such embodiments, the antibodies disclosed herein, when used in combination with one or more therapeutic agents, may be administered concurrently with the one or more therapeutic agents, and in certain such embodiments, the antibodies may be administered concurrently as part of the same pharmaceutical composition. However, an antibody that is "in combination" with another therapeutic substance need not be administered simultaneously or in the same composition as the therapeutic substance. The meaning of "in combination" in the present invention also includes that an antibody administered before or after another therapeutic agent is also considered to be "in combination" with the therapeutic agent, even if the antibody and the second agent are administered by different administration means. Where possible, other therapeutic substances used in combination with the antibodies disclosed herein may be administered by Reference to the methods of the product specifications for the other therapeutic substance, or by Reference to, surgeon's docket No. 2003(Physicians' Desk Reference,57th Ed; Medical Economics Company; ISBN: 1563634457; 57th edition (11 months 2002)), or by other methods known in the art.

In certain embodiments, the therapeutic substance is capable of inducing or enhancing an immune response against the cancer. For example, tumor vaccines can be used to induce an immune response to certain tumors or cancers. Cytokine therapy can be used to enhance the presentation of tumor antigens to the immune system. Examples of cytokine therapy include, but are not limited to, interferons such as alpha, beta and gamma interferons, colony stimulating factors such as macrophage CSF, granulocyte macrophage CSF and granulocyte CSF, interleukins such as 1L-L, 1L-1a, 1L-2, 1L 3, 1L-4, 1L-5, 1L-6, 1L-7, 1L-8, 1L-9, 1L-10, 1L-ll and 1L-12, and tumor necrosis factors such as TNF-alpha and TNF-beta. Agents that inactivate immunosuppressive targets, such as PD-1 antibodies, TGF- β inhibitors, IL-10 inhibitors, and Fas ligand inhibitors, may also be used. Another group of agents include those that activate an immune response against a tumor or cancer cell, e.g., those that enhance T cell activation (e.g., T cell costimulatory signaling pathways such as CTLA-4, ICOS, OX40, 4-1BB, etc.) as well as those that enhance dendritic cell function and antigen presentation.

The following examples are intended to better illustrate the invention and should not be construed as limiting the scope of the invention. All of the specific compositions, materials and methods described below, in whole or in part, are within the scope of the invention. These specific compositions, materials and methods are not intended to limit the invention but merely to illustrate specific embodiments within the scope of the invention. Those skilled in the art may develop equivalent compositions, materials, and methods without adding inventive step and without departing from the scope of the invention. It will be appreciated that various modifications to the method of the invention may still be included within the scope of the invention. The inventors intend such variations to be included within the scope of the present invention.

Example 1: obtaining of mouse anti-human PD-L1 monoclonal antibody

The present inventors constructed a CHO cell line overexpressing PD-L1 and immunized mice therewith. Spleen cells of immunized mice are taken and fused with SP2/0-AG14 cells to form hybridoma cells, and a proper amount of fused cells are paved on a 96-well plate. Supernatants from each well were collected 10 days after the fusion, and the binding activity of mouse antibody secreted from hybridoma cells to human PD-L1 and the inhibitory activity thereof on the binding of PD-1/PD-L1 were examined by ELISA, as shown in Table 1, to obtain a series of hybridoma cells having high activity. Selecting hybridoma cell 13B8 with best activity, and sequencing to obtain heavy chain variable region cDNA sequence and light chain variable region cDNA sequence corresponding to the secretion antibody, wherein the encoded heavy chain amino acid sequence is shown in SEQ ID NO. 7; the coded light chain amino acid sequence is shown as SEQ ID NO. 8. The variable region of the heavy chain and the variable region of the light chain of the mouse antibody are respectively connected with the constant region of the heavy chain and the constant region of the kappa chain of human IgG1 to obtain the human-mouse chimeric antibody MT02-L4, the heavy chain sequence of which is shown as SEQ ID NO. 9, and the light chain sequence of which is shown as SEQ ID NO. 10.

TABLE 1 screening of Positive hybridomas

The heavy chain amino acid sequence of hybridoma cell 13B8 is shown below;

SEQ ID NO:7:

EVQLQESGPSLVKPSQTLSLTCSVTGDSITSGYWSWIRKFPGNNLDY MGYISYTGSTYYSPSLIRRMSINRDTSKNQFYLQLNSVTTEDTATYYCAK YENWKEGYFDIWGAGTTVTVSS

the light chain amino acid sequence of hybridoma cell 13B8 is shown below;

SEQ ID NO:8：

DIVMTRSQKFMSTSVGDRVSVTCKASQNVGINVAWYQQKAGQSPK ALIHSATQRYSGVPDRFTGGGSGTDFTLTIDDVQSADLAEYFCQQYYGYP LTFGAGTRLELK

the amino acid sequence of the heavy chain of the human-mouse chimeric antibody MT02-L4 is shown as follows;

SEQ ID NO:9：

EVQLQESGPSLVKPSQTLSLTCSVTGDSITSGYWSWIRKFPGNNLDY MGYISYTGSTYYSPSLIRRMSINRDTSKNQFYLQLNSVTTEDTATYYCAK YENWKEGYFDIWGAGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK

the light chain amino acid sequence of the human-mouse chimeric antibody MT02-L4 is shown as follows;

SEQ ID NO:10：

DIVMTRSQKFMSTSVGDRVSVTCKASQNVGINVAWYQQKAGQSPK ALIHSATQRYSGVPDRFTGGGSGTDFTLTIDDVQSADLAEYFCQQYYGYP LTFGAGTRLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC

example 2: humanization of antibodies

The sequences of the mouse antibody heavy chain variable region and the light chain variable region obtained in example 1 were analyzed to obtain the heavy chain complementarity determining regions CDR having the following sequences:

SEQ ID NO:1GDSITSGYWS

2YISYTGSTYYSPSLIR and SEQ ID NO

SEQ ID NO:3AKYENWKEGYFDI；

The light chain complementarity determining region has the following sequence:

SEQ ID NO:4KASQNVGINVA

SEQ ID NO 5SATQRYS and

SEQ ID NO:6QQYYGYPLT。

the human germline antibody sequence database (IGMT) is searched to respectively obtain human germline antibody sequences with higher homology with the heavy chain/light chain variable regions of the mouse antibody, the framework regions of the human germline antibody sequences are combined with the CDRs of the mouse antibody, namely CDR grafting, and partial amino acids of the framework regions are subjected to back mutation, so that the humanized antibody MT02-C4 is finally obtained, wherein the heavy chain sequence is shown as SEQ ID NO:15, and the light chain sequence is shown as SEQ ID NO: 16.

The heavy chain variable region sequence of the humanized antibody MT02-C4 is as follows:

SEQ ID NO:13

QVQLQESGPGLVKPSETLSLTCTVSGDSITSGYWSWIRQPPGKGLEYMGY ISYTGSTYYSPSLIRRMTISRDTSKNQFSLKLSSVTAADTAVYYCAKYEN WKEGYFDIWGQGTLVTVSS

the light chain variable region sequence of humanized antibody MT02-C4 is as follows:

SEQ ID NO:14

DIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGKAPKA LIYSATQRYSGVPSKFSGSGSGTDFTLTISSLQPEDFATYFCQQYYGYPLTF GQGTRLEIK

the heavy chain sequence of the humanized antibody MT02-C4 is as follows:

SEQ ID NO:15

QVQLQESGPGLVKPSETLSLTCTVSGDSITSGYWSWIRQPPGKGLEY MGYISYTGSTYYSPSLIRRMTISRDTSKNQFSLKLSSVTAADTAVYYCAK YENWKEGYFDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK

the light chain sequence of the humanized antibody MT02-C4 is as follows:

SEQ ID NO:16

DIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGKAPKA LIYSATQRYSGVPSKFSGSGSGTDFTLTISSLQPEDFATYFCQQYYGYPLTF GQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT HQGLSSPVTKSFNRGEC

example 3: preparation of antibodies

The cDNA sequences encoding the heavy chain and the light chain of MT02-L4 are shown as SEQ ID NO. 11 and SEQ ID NO. 12, respectively;

the cDNA sequence encoding the heavy chain of MT02-L4 is shown below:

SEQ ID NO:11

GAGGTGCAGCTGCAGGAGAGCGGCCCTAGCCTGGTGAAGCCTTC CCAGACACTGAGCCTGACCTGTTCCGTGACCGGCGATAGCATCACATC CGGCTACTGGAGCTGGATCAGAAAGTTCCCCGGCAATAACCTGGACT ACATGGGCTACATCTCCTACACAGGCTCCACCTACTACAGCCCTAGCC TGATCAGAAGAATGAGCATCAACAGAGACACCTCCAAGAACCAGTTC TACCTGCAGCTGAACTCCGTGACAACAGAGGACACCGCCACCTACTA CTGCGCCAAGTACGAGAATTGGAAGGAGGGCTACTTTGATATCTGGG GCGCCGGCACCACAGTGACCGTGTCCTCC

GCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCC AAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGA CTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGAC CAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTA CTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCA GACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGG ACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCA CCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTC CCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTC ACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTT CAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGC CGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTC ACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAA GGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCA AAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCA TCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGT CAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATG GGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCC GACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGG TGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTG CACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA

the cDNA sequences encoding the light chain of MT02-L4 are shown below:

SEQ ID NO:12

GACATCGTGATGACCAGAAGCCAGAAGTTCATGAGCACCTCCGT GGGCGACAGAGTGTCCGTGACATGTAAGGCCAGCCAGAACGTGGGCA TCAACGTGGCCTGGTATCAACAGAAGGCCGGCCAGAGCCCCAAGGCC CTGATCCACAGCGCCACCCAGAGATACTCCGGCGTGCCCGACAGGTTT ACAGGCGGCGGCAGCGGCACCGATTTTACACTGACAATCGACGATGT GCAGAGCGCCGACCTGGCCGAGTACTTCTGTCAGCAGTACTACGGCT ACCCTCTGACCTTCGGCGCCGGCACAAGACTGGAGCTGAAGCGAACG GTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTG AAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCC AGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGG TAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCT ACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAA CACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCC CGTCACAAAGAGCTTCAACAGGGGAGAGTGT

the cDNA sequence encoding the MT02-C4 heavy chain is shown below:

SEQ ID NO:17

CAGGTGCAGCTGCAGGAGTCCGGCCCTGGCCTGGTGAAGCCTAG CGAGACACTGTCCCTGACATGCACCGTGAGCGGCGATTCCATCACCTC CGGCTACTGGTCCTGGATCAGGCAGCCCCCCGGCAAGGGCCTGGAGT ACATGGGCTACATCTCCTACACAGGCTCCACATACTACAGCCCCTCCC TGATCAGAAGAATGACCATCTCCAGAGACACATCCAAGAACCAGTTT AGCCTGAAGCTGTCCTCCGTGACAGCCGCCGACACCGCCGTGTACTAC TGCGCCAAGTACGAGAACTGGAAGGAGGGCTACTTCGACATCTGGGG CCAGGGCACACTGGTGACAGTGTCCTCCGCTAGCACCAAGGGCCCAT CGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAG CGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACG GTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCG GCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACC GTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAAT CACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATC TTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCT GGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCT CATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGA GCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTG GAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACA GCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGC TGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCA GCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGA ACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGA ACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACA TCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAA GACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAG CAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCT CATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAG AGCCTCTCCCTGTCTCCGGGTAAA

the cDNA sequences encoding the light chain of MT02-c4 are shown below:

SEQ ID NO:18

GACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCTCCGT GGGAGACAGGGTGACCATCACATGTAAGGCCAGCCAGAACGTGGGCA TCAACGTGGCCTGGTATCAACAGAAGCCCGGCAAGGCCCCCAAGGCC CTGATCTACAGCGCCACCCAGAGGTACAGCGGCGTGCCTAGCAAGTT TAGCGGCAGCGGCTCCGGCACCGACTTCACACTGACCATCAGCAGCC TGCAGCCTGAGGATTTTGCCACATACTTTTGTCAGCAGTACTACGGCT ACCCTCTGACCTTCGGCCAGGGCACCAGGCTGGAGATCAAGCGAACG GTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTG AAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCC AGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGG TAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCT ACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAA CACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCC CGTCACAAAGAGCTTCAACAGGGGAGAGTGT

the cDNA sequences were cloned separately into the mammalian cell expression vector pcDNA3.4. The heavy chain expression plasmid and the light chain expression plasmid were expressed as 2: 1 molar ratio was transfected into HEK293 cells using Lipofectamine 2000 transfection reagent (Invitrogen) and cultured at 37 ℃ under 5% carbon dioxide for 7 days. The culture supernatant was collected, and the antibody in the supernatant was purified by Protein A affinity chromatography. The purified antibody was dialyzed against PBS solution, freeze-dried, concentrated, and stored at-20 ℃.

Example 4: binding of antibodies to PD-L1 protein

A solution of human PD-L1 protein at a concentration of 1. mu.g/mL was coated onto a 96-well high affinity plate at 100. mu.L/well, shaken overnight at 4 ℃. The next day, the cells were washed 3 times with 300. mu.L of PBST (Tween 20: 0.5 ‰), then blocked with 100. mu.L/well of 5% BSA/PBS for 2 hours, and shaken at room temperature. 300 u L PBST washing 3 times. A gradient dilution of the antibody sample was made with PBS. Add to 96 well plate at 100. mu.L/well and shake for 1 hour at room temperature. 300 u L PBST washing 3 times. A solution of a secondary antibody, goat anti-mouse (goat anti-mouse) IgG HRP or goat anti-human (goat anti-human) IgG HRP, was prepared, added to a 96-well plate at 100. mu.L/well, and shaken at room temperature for 1 hour. 300 u L PBST washing 4 times. TMB was added at 100. mu.L/well and the color was developed for 20 min. Adding 100 mu L/well of 0.6N H ₂ SO ₄ Stopping the color development and detecting the OD ₄₅₀ nm。

The results are shown in FIG. 1A, and the EC50 of the chimeric antibody MT02-L4 combined with human PD-L1 is 215.6ng/mL, and the EC50 of the humanized antibody MT02-C4 combined with human PD-L1 is 65.09 ng/mL.

Similarly, the binding ability of atezolizumab (available from Baiying Biotechnology Limited, Tay., Inc.) to human PD-L1 was tested by ELISA and showed that atezolizumab bound to human PD-L1 with an EC50 of 128.8ng/mL (not shown).

Similarly, the binding ability of the antibody to cynomolgus monkey PD-L1 protein was tested by ELISA. The results showed that chimeric antibody MT02-L4 bound to cynomolgus monkey PD-L1 with an EC50 of 42.18ng/mL (FIG. 1B).

Example 5: binding of antibodies to cells expressing PD-L1

CT26 mouse tumor cells were transfected with an expression plasmid encoding human PD-L1 protein and cultured for 48 hours. The antibody concentration gradient solution was prepared with PBS to make 10 × working solution of final concentration. CT26 cells over-expressing PD-L1 or wild-type HCC827 human tumor cells were collected, washed once with PBS, counted and diluted to 2x10 ⁶ A cell suspension; respectively adding 10 μ l of antibody working solution into 100 μ l of cell suspension, and incubating at 4 deg.C in dark for 30 min; after washing with PBS for 2 times, adding corresponding fluorescence labeled secondary antibody, incubating for 30min at 4 ℃ in the dark, after washing with PBS once, suspending with 400. mu.l FACS buffer, and detecting the binding condition of the antibody and the cells by a flow cytometer.

As shown in FIG. 2, the results showed that MT02-L4, MT02-C4 bound to CT26 cells overexpressing PD-L1, and the EC50 thereof was 0.2095 and 0.3780. mu.g/mL (FIG. 2A), respectively; MT02-L4 and MT02-C4 bind to HCC827 cells naturally over-expressing PD-L1, and the EC50 of the HCC827 cells is 44.50 and 86.42ng/mL respectively (FIG. 2B).

Example 6: antibodies inhibit the binding of Raji-PD-L1 cells to PD-1 protein

Firstly, a Raji stable transfectant cell strain which over-expresses PD-L1 is constructed. In the experiment, the antibody was diluted with PBS in a gradient manner to prepare a working solution of 4 Xfinal concentration. Collecting Raji-PD-L1 cells, centrifuging, suspending in culture medium, adjusting density to 8x10 ⁶ PermL, add appropriate amount of FcR blocker and incubate on ice for 15 min. Subsequently, 25. mu.L of cell suspension was added to a 96-well round bottom plate, and 25. mu.L of the antibody solution was added in a gradient and incubated on ice for 30 min. At the end, 50. mu.L of 2. mu.g/mL human PD-1-Fc protein (Sino Biological) was added to each well and incubation on ice was continued for 30 min. The cells were then washed three times with PBS and 100. mu.L of anti-PD-1-APC antibody was added and incubated on ice for 30 min. After the incubation was completed, the cells were washed with PBSThe cells were suspended three times in 400. mu.l FACS buffer and the proportion of cells labeled with APC and the signal intensity were measured by flow cytometry.

As shown in fig. 3, PD-1 protein can bind to Raji-PD-L1 cells, which can be inhibited by either chimeric antibody MT02-L4 or humanized antibody MT02-C4, with IC50 of 480.7ng/mL and 298.2ng/mL, respectively. Similarly, applicants used atezolizumab (available from Baiying Biotech, Inc., Thai) to inhibit the binding of Raji-PD-L1 cells to PD-1 protein with an IC50 of 677.0 ng/mL.

Example 7: the antibody antagonizes the inhibition of the NFAT signaling pathway in Jurkat-PD-1 cells by PD-L1

Binding of PD-L1 to PD-1 inhibited T cell activation, and in order to evaluate whether the antibody of the present invention could antagonize this inhibitory activity of PD-L1, the inventors constructed CHO-PDL1-OKT3 cell line and Jurkat-PD-1/NFAT-luciferase reporter cell line, respectively. CHO-PDL1-OKT3 cells at 1.2X10 ⁴ cells/15. mu.L/well were seeded in 384-well plates and cultured overnight. The following day, 15 μ L of the gradient diluted MT02-C4 antibody solution was added to the wells, incubated for 30 minutes, and then reporter cells were added to each well on a 6000 cells/15 μ L/well basis. After 4 hours of culture, 45. mu.L of One-glo (Promega) reagent was added to the wells and the luciferase bioluminescence intensity was measured by Pheragar.

The results show that co-incubation with Jurkat-PD-1/NFAT-luciferase cells did not effectively activate the reporter gene activity of Jurkat cells due to overexpression of PD-L1 on CHO-PDL1-OKT3 cells. However, when the inhibitory effect of PD-L1 antibody on Jurkat cells was released by the action of PD-L1, the reporter gene activity was activated effectively, and a clear dose effect was exhibited (FIG. 4). EC50 for MT02-C4 was calculated to be 83.81 ng/mL. Similarly, applicants used the effect of atezolizumab (synthesized by Baiying Biotechnology, Inc., Tanzozu), calculated to have an EC50 of 121ng/mL, with PD-L1 being released from its inhibitory effect on Jurkat cells, the reporter gene activity being effectively activated, and a clear dose effect.

Example 8: antigen antibody affinity assaySide survey

Affinity detection was performed according to the general method by Nanjing Kinsry Biotechnology Ltd. Briefly, MT02-C4 at a concentration of 4. mu.g/mL was incubated with protein A sensor chip for 60s for antibody capture. In the antigen binding phase, the PD-L1 protein diluted in gradient was used as mobile phase for 180s binding with MT02-C4 antibody captured on a sensor chip. During the dissociation phase, the elution was continued with HBS-EP buffer for 1200 s. The binding of PD-L1 to the antibody on the sensor chip was quantitatively determined using Biacore 8k (GE healthcare). The results are shown in Table 2, and the affinity of MT02-C4 is 0.4 nM.

TABLE 2 affinity and kinetic data for MT02-C4 with PD-L1

Example 9: mouse tumor drug effect model

CT26 cell is a cell strain derived from mouse colon cancer, and the endogenous PD-L1 of a mouse is low-expression. The invention discloses a CT26 cell line CT26-hPD-L1 which over-expresses human PD-L1. Press 10 ⁶ Cell/cell amount CT26-hPD-L1 cells were inoculated subcutaneously in female BALB/c mice. When the tumor grows to about 160mm ³ Thereafter, MT02-C4 antibody or IgG1 control (5mg/kg) was administered intravenously to 8 mice per group. The administration was once every 3 days for a total of 4 times.

As shown in fig. 5, 11 days after the start of administration, MT02-C4 had a very significant drug effect, and the inhibition rate reached 55%, compared to the control group.

While specific embodiments of the present invention have been illustrated and described in detail, it should be appreciated that the invention is not limited by the specific embodiments. Various modifications, adaptations, and variations of the present invention can be made without departing from the spirit and scope of the invention, and these are within the scope of the invention.

Sequence listing

<110> Nanjing Youmei Biotechnology Ltd

<120> anti-human PD-L1 antibody and antigen binding fragment derivative thereof, preparation method and application

<130> DIC18110069

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Ser Ala Thr Gln Arg Tyr Ser

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Gln Gln Tyr Tyr Gly Tyr Pro Leu Thr

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<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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Glu Val Gln Leu Gln Glu Ser Gly Pro Ser Leu Val Lys Pro Ser Gln

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Thr Leu Ser Leu Thr Cys Ser Val Thr Gly Asp Ser Ile Thr Ser Gly

20 25 30

Tyr Trp Ser Trp Ile Arg Lys Phe Pro Gly Asn Asn Leu Asp Tyr Met

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Gly Tyr Ile Ser Tyr Thr Gly Ser Thr Tyr Tyr Ser Pro Ser Leu Ile

50 55 60

Arg Arg Met Ser Ile Asn Arg Asp Thr Ser Lys Asn Gln Phe Tyr Leu

65 70 75 80

Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys Ala

85 90 95

Lys Tyr Glu Asn Trp Lys Glu Gly Tyr Phe Asp Ile Trp Gly Ala Gly

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Thr Thr Val Thr Val Ser Ser

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Asp Ile Val Met Thr Arg Ser Gln Lys Phe Met Ser Thr Ser Val Gly

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Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Asn Val Gly Ile Asn

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Val Ala Trp Tyr Gln Gln Lys Ala Gly Gln Ser Pro Lys Ala Leu Ile

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His Ser Ala Thr Gln Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr Gly

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Gly Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asp Asp Val Gln Ser

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Thr Phe Gly Ala Gly Thr Arg Leu Glu Leu Lys

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Glu Val Gln Leu Gln Glu Ser Gly Pro Ser Leu Val Lys Pro Ser Gln

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Thr Leu Ser Leu Thr Cys Ser Val Thr Gly Asp Ser Ile Thr Ser Gly

20 25 30

Tyr Trp Ser Trp Ile Arg Lys Phe Pro Gly Asn Asn Leu Asp Tyr Met

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Gly Tyr Ile Ser Tyr Thr Gly Ser Thr Tyr Tyr Ser Pro Ser Leu Ile

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Arg Arg Met Ser Ile Asn Arg Asp Thr Ser Lys Asn Gln Phe Tyr Leu

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Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys Ala

85 90 95

Lys Tyr Glu Asn Trp Lys Glu Gly Tyr Phe Asp Ile Trp Gly Ala Gly

100 105 110

Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

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Asp Ile Val Met Thr Arg Ser Gln Lys Phe Met Ser Thr Ser Val Gly

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Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Asn Val Gly Ile Asn

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Val Ala Trp Tyr Gln Gln Lys Ala Gly Gln Ser Pro Lys Ala Leu Ile

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His Ser Ala Thr Gln Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Gly Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asp Asp Val Gln Ser

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Ala Asp Leu Ala Glu Tyr Phe Cys Gln Gln Tyr Tyr Gly Tyr Pro Leu

85 90 95

Thr Phe Gly Ala Gly Thr Arg Leu Glu Leu Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

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Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

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Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 11

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<213> Artificial Sequence (Artificial Sequence)

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gaggtgcagc tgcaggagag cggccctagc ctggtgaagc cttcccagac actgagcctg 60

acctgttccg tgaccggcga tagcatcaca tccggctact ggagctggat cagaaagttc 120

cccggcaata acctggacta catgggctac atctcctaca caggctccac ctactacagc 180

cctagcctga tcagaagaat gagcatcaac agagacacct ccaagaacca gttctacctg 240

cagctgaact ccgtgacaac agaggacacc gccacctact actgcgccaa gtacgagaat 300

tggaaggagg gctactttga tatctggggc gccggcacca cagtgaccgt gtcctccgct 360

agcaccaagg gcccatcggt cttccccctg gcaccctcct ccaagagcac ctctgggggc 420

acagcggccc tgggctgcct ggtcaaggac tacttccccg aaccggtgac ggtgtcgtgg 480

aactcaggcg ccctgaccag cggcgtgcac accttcccgg ctgtcctaca gtcctcagga 540

ctctactccc tcagcagcgt ggtgaccgtg ccctccagca gcttgggcac ccagacctac 600

atctgcaacg tgaatcacaa gcccagcaac accaaggtgg acaagaaagt tgagcccaaa 660

tcttgtgaca aaactcacac atgcccaccg tgcccagcac ctgaactcct ggggggaccg 720

tcagtcttcc tcttcccccc aaaacccaag gacaccctca tgatctcccg gacccctgag 780

gtcacatgcg tggtggtgga cgtgagccac gaagaccctg aggtcaagtt caactggtac 840

gtggacggcg tggaggtgca taatgccaag acaaagccgc gggaggagca gtacaacagc 900

acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa tggcaaggag 960

tacaagtgca aggtctccaa caaagccctc ccagccccca tcgagaaaac catctccaaa 1020

gccaaagggc agccccgaga accacaggtg tacaccctgc ccccatcccg ggatgagctg 1080

accaagaacc aggtcagcct gacctgcctg gtcaaaggct tctatcccag cgacatcgcc 1140

gtggagtggg agagcaatgg gcagccggag aacaactaca agaccacgcc tcccgtgctg 1200

gactccgacg gctccttctt cctctacagc aagctcaccg tggacaagag caggtggcag 1260

caggggaacg tcttctcatg ctccgtgatg catgaggctc tgcacaacca ctacacgcag 1320

aagagcctct ccctgtctcc gggtaaa 1347

<210> 12

<211> 642

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

gacatcgtga tgaccagaag ccagaagttc atgagcacct ccgtgggcga cagagtgtcc 60

gtgacatgta aggccagcca gaacgtgggc atcaacgtgg cctggtatca acagaaggcc 120

ggccagagcc ccaaggccct gatccacagc gccacccaga gatactccgg cgtgcccgac 180

aggtttacag gcggcggcag cggcaccgat tttacactga caatcgacga tgtgcagagc 240

gccgacctgg ccgagtactt ctgtcagcag tactacggct accctctgac cttcggcgcc 300

ggcacaagac tggagctgaa gcgaacggtg gctgcaccat ctgtcttcat cttcccgcca 360

tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat 420

cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag 480

gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg 540

ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 600

ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gt 642

<210> 13

<211> 119

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 13

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Asp Ser Ile Thr Ser Gly

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Tyr Met

35 40 45

Gly Tyr Ile Ser Tyr Thr Gly Ser Thr Tyr Tyr Ser Pro Ser Leu Ile

50 55 60

Arg Arg Met Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Lys Tyr Glu Asn Trp Lys Glu Gly Tyr Phe Asp Ile Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 14

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 14

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val Gly Ile Asn

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Ala Leu Ile

35 40 45

Tyr Ser Ala Thr Gln Arg Tyr Ser Gly Val Pro Ser Lys Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Tyr Tyr Gly Tyr Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100 105

<210> 15

<211> 449

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 15

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Asp Ser Ile Thr Ser Gly

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Tyr Met

35 40 45

Gly Tyr Ile Ser Tyr Thr Gly Ser Thr Tyr Tyr Ser Pro Ser Leu Ile

50 55 60

Arg Arg Met Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Lys Tyr Glu Asn Trp Lys Glu Gly Tyr Phe Asp Ile Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210> 16

<211> 214

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 16

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val Gly Ile Asn

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Ala Leu Ile

35 40 45

Tyr Ser Ala Thr Gln Arg Tyr Ser Gly Val Pro Ser Lys Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Tyr Tyr Gly Tyr Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 17

<211> 1347

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

caggtgcagc tgcaggagtc cggccctggc ctggtgaagc ctagcgagac actgtccctg 60

acatgcaccg tgagcggcga ttccatcacc tccggctact ggtcctggat caggcagccc 120

cccggcaagg gcctggagta catgggctac atctcctaca caggctccac atactacagc 180

ccctccctga tcagaagaat gaccatctcc agagacacat ccaagaacca gtttagcctg 240

aagctgtcct ccgtgacagc cgccgacacc gccgtgtact actgcgccaa gtacgagaac 300

tggaaggagg gctacttcga catctggggc cagggcacac tggtgacagt gtcctccgct 360

agcaccaagg gcccatcggt cttccccctg gcaccctcct ccaagagcac ctctgggggc 420

acagcggccc tgggctgcct ggtcaaggac tacttccccg aaccggtgac ggtgtcgtgg 480

aactcaggcg ccctgaccag cggcgtgcac accttcccgg ctgtcctaca gtcctcagga 540

ctctactccc tcagcagcgt ggtgaccgtg ccctccagca gcttgggcac ccagacctac 600

atctgcaacg tgaatcacaa gcccagcaac accaaggtgg acaagaaagt tgagcccaaa 660

tcttgtgaca aaactcacac atgcccaccg tgcccagcac ctgaactcct ggggggaccg 720

tcagtcttcc tcttcccccc aaaacccaag gacaccctca tgatctcccg gacccctgag 780

gtcacatgcg tggtggtgga cgtgagccac gaagaccctg aggtcaagtt caactggtac 840

gtggacggcg tggaggtgca taatgccaag acaaagccgc gggaggagca gtacaacagc 900

acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa tggcaaggag 960

tacaagtgca aggtctccaa caaagccctc ccagccccca tcgagaaaac catctccaaa 1020

gccaaagggc agccccgaga accacaggtg tacaccctgc ccccatcccg ggatgagctg 1080

accaagaacc aggtcagcct gacctgcctg gtcaaaggct tctatcccag cgacatcgcc 1140

gtggagtggg agagcaatgg gcagccggag aacaactaca agaccacgcc tcccgtgctg 1200

gactccgacg gctccttctt cctctacagc aagctcaccg tggacaagag caggtggcag 1260

caggggaacg tcttctcatg ctccgtgatg catgaggctc tgcacaacca ctacacgcag 1320

aagagcctct ccctgtctcc gggtaaa 1347

<210> 18

<211> 642

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

gacatccaga tgacccagag ccccagcagc ctgagcgcct ccgtgggaga cagggtgacc 60

atcacatgta aggccagcca gaacgtgggc atcaacgtgg cctggtatca acagaagccc 120

ggcaaggccc ccaaggccct gatctacagc gccacccaga ggtacagcgg cgtgcctagc 180

aagtttagcg gcagcggctc cggcaccgac ttcacactga ccatcagcag cctgcagcct 240

gaggattttg ccacatactt ttgtcagcag tactacggct accctctgac cttcggccag 300

ggcaccaggc tggagatcaa gcgaacggtg gctgcaccat ctgtcttcat cttcccgcca 360

tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat 420

cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag 480

gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg 540

ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 600

ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gt 642

Claims (28)

1. An anti-human PD-L1 antibody or antigen-binding fragment thereof, having heavy and light chain complementarity determining regions as follows:

VH CDR1, the amino acid sequence of which is shown in SEQ ID NO: 1;

VH CDR2, the amino acid sequence of which is shown in SEQ ID NO. 2;

VH CDR3, the amino acid sequence of which is shown in SEQ ID NO. 3;

VL CDR1, the amino acid sequence of which is shown in SEQ ID NO. 4;

VL CDR2, the amino acid sequence of which is shown in SEQ ID NO. 5; and

VL CDR3, the amino acid sequence of which is shown in SEQ ID NO. 6.

2. The anti-human PD-L1 antibody or antigen-binding fragment thereof according to claim 1, wherein the anti-human PD-L1 antibody or antigen-binding fragment thereof further comprises a human heavy chain constant region and/or a human light chain constant region.

3. The anti-human PD-L1 antibody or antigen-binding fragment thereof of claim 2, wherein the human heavy chain constant region is selected from the heavy chain constant regions of human IgG1, IgG2, IgG3, or IgG 4.

4. The anti-human PD-L1 antibody or antigen-binding fragment thereof according to claim 2, wherein the human heavy chain constant region is the heavy chain constant region of human IgG1 and the human light chain constant region is a kappa chain.

5. The anti-human PD-L1 antibody or antigen-binding fragment thereof according to claim 1, wherein said anti-human PD-L1 antibody or antigen-binding fragment thereof is an Fv fragment, Fab ', F (ab') 2.

6. The anti-human PD-L1 antibody or antigen-binding fragment thereof of claim 5, wherein the anti-human PD-L1 antibody or antigen-binding fragment thereof is an scFv, scFv dimer, BsFv, dsFv2, dsFv-dsFv'.

7. The anti-human PD-L1 antibody or antigen-binding fragment thereof according to claim 1, wherein the amino acid sequence of the heavy chain variable region of the anti-human PD-L1 antibody or antigen-binding fragment thereof is as set forth in SEQ ID NO. 7.

8. The anti-human PD-L1 antibody or antigen-binding fragment thereof according to claim 1, wherein the amino acid sequence of the light chain variable region of the anti-human PD-L1 antibody or antigen-binding fragment thereof is represented by SEQ ID NO. 8.

9. The anti-human PD-L1 antibody or antigen-binding fragment thereof according to claim 1, wherein the amino acid sequence of the heavy chain of the anti-human PD-L1 antibody or antigen-binding fragment thereof is represented by SEQ ID NO 9.

10. The anti-human PD-L1 antibody or antigen-binding fragment thereof according to claim 1, wherein the amino acid sequence of the light chain of the anti-human PD-L1 antibody or antigen-binding fragment thereof is shown as SEQ ID NO. 10.

11. The anti-human PD-L1 antibody or antigen-binding fragment thereof according to claim 1, wherein the amino acid sequence of the heavy chain variable region of the anti-human PD-L1 antibody or antigen-binding fragment thereof is represented by SEQ ID NO 13.

12. The anti-human PD-L1 antibody or antigen-binding fragment thereof according to claim 1, wherein the amino acid sequence of the light chain variable region of the anti-human PD-L1 antibody or antigen-binding fragment thereof is represented by SEQ ID NO. 14.

13. The anti-human PD-L1 antibody or antigen-binding fragment thereof according to claim 1, wherein the amino acid sequence of the heavy chain of the anti-human PD-L1 antibody or antigen-binding fragment thereof is represented by SEQ ID NO. 15.

14. The anti-human PD-L1 antibody or antigen-binding fragment thereof according to claim 1, wherein the amino acid sequence of the light chain of the anti-human PD-L1 antibody or antigen-binding fragment thereof is represented by SEQ ID NO 16.

15. An isolated polynucleotide encoding an anti-human PD-L1 antibody or antigen-binding fragment thereof according to any one of claims 1-14.

16. The isolated polynucleotide of claim 15, wherein the polynucleotide comprises the nucleotide sequence set forth in SEQ ID No. 11.

17. The isolated polynucleotide of claim 15, wherein the polynucleotide comprises a nucleotide sequence set forth in SEQ ID No. 12.

18. The isolated polynucleotide of claim 15, wherein the polynucleotide comprises a nucleotide sequence set forth in SEQ ID No. 17.

19. The isolated polynucleotide of claim 15, wherein the polynucleotide comprises a nucleotide sequence set forth in SEQ ID No. 18.

20. A vector comprising the isolated polynucleotide of any one of claims 15-19.

21. A host cell comprising the vector of claim 20.

22. The host cell of claim 21, wherein the host cell is a mammalian cell.

23. The host cell of claim 22, wherein the host cell is a human, murine, ovine, equine, canine, or feline cell.

24. The host cell of claim 22, wherein the host cell is a chinese hamster ovary cell.

25. A method of producing an anti-human PD-L1 antibody or antigen-binding fragment thereof according to any one of claims 1-14, comprising culturing the host cell of any one of claims 21-24 under conditions capable of expressing the anti-human PD-L1 antibody or antigen-binding fragment thereof according to any one of claims 1-14.

26. A kit comprising an anti-human PD-L1 antibody or antigen-binding fragment thereof according to any one of claims 1-14.

27. A pharmaceutical composition comprising an anti-human PD-L1 antibody or antigen-binding fragment thereof according to any one of claims 1-14 and a pharmaceutically acceptable carrier.

28. Use of an anti-human PD-L1 antibody or antigen-binding fragment thereof according to any one of claims 1-14 in the manufacture of a medicament for the prevention and/or treatment of a cancer selected from the group consisting of small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic adenocarcinoma, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, prostate cancer, cervical cancer, thymus cancer, mercke's cell carcinoma, and brain stem glioma.

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