Antibodies specifically binding to CD147 and uses thereof

Abstract

The present disclosure relates to antibodies, or antigen-binding fragments thereof, that specifically bind CD147, and methods of treating a disease or condition in a subject in need thereof, the method comprises administering to the subject an anti-CD147 antibody or antigen binding fragment, or a polynucleotide encoding the anti-CD147 antibody or antigen binding fragment, or a pharmaceutical composition comprising the anti-CD147 antibody or antigen binding fragment or the polynucleotide; the use of the anti-CD147 antibody or antigen-binding fragment thereof, or the polynucleotide in the manufacture of a medicament or kit for the treatment of a disease or condition; and the use of the anti-CD147 antibody or antigen-binding fragment thereof, or a combination of the polynucleotide with an MCT inhibitor, in the manufacture of a medicament or kit for the treatment of a disease or condition.

Description

Technical Field

[0001] The present disclosure relates to the biomedical field. In particular, the present disclosure relates to anti-CD147 antibodies and uses thereof. Background Art

[0002] Monocarboxylates play important roles in a wide range of activities including energy metabolism and cell signaling. The movement of monocarboxylates across membranes in mammals is catalyzed by two types of solute carriers (SLCs): proton-coupled monocarboxylate transporters (MCTs) belonging to the solute carrier 16 (SLC16) family and sodium-coupled monocarboxylate transporters (SMCTs) belonging to the solute carrier 5 (SLC5) family. Among the 14 identified MCTs, MCTs 1-4 have been extensively studied because they selectively transport products of the glycolytic cycle, including lactate, pyruvate, and ketone bodies. The net direction of substrate transport is determined by the transmembrane concentration gradients of protons and monocarboxylates.

[0003] Lactic acid, or lactate, produced by aerobic glycolysis plays an important role in tumor growth and microenvironment maintenance. MCTs1-4 are the major lactate shuttles in the human body and are therefore physiologically critical for lactate homeostasis at both the tissue and organismal levels. At the tissue level, circulating lactate concentrations are tightly controlled to avoid lactic acidosis, a serious medical condition. Furthermore, lactate exchange between the circulation and tissues regulates not only circulating lactate concentrations but also lactate localization in tissues, allowing independent operation of glycolysis and the TCA cycle in tissues. At the organismal level, lactate shuttling between glycolytic and oxidative cells maintains a tight balance between glycolytic production and TCA cycle consumption.

[0004] MCT1 and MCT4 are overexpressed in many cancers and promote tumor growth and invasiveness in multiple ways. Tumor cells often establish a metabolic symbiosis with lactate, whereby oxidative cancer cells preferentially use lactate as an oxidative fuel, thereby freeing up glucose for glycolytic cancer cells or cancer-associated fibroblasts (CAFs), which in turn convert glucose to lactate for oxidative cancer cells. MCT1 and MCT4 are key lactate shuttles that maintain this symbiosis. In addition, MCT1 and MCT4 import lactate into vascular endothelial cells to promote tumor angiogenesis through the NF-κB / IL-8 signaling pathway and export lactate to the extracellular niche of tumors to suppress antitumor immune responses. Inhibiting the transport activity of MCT1 and MCT4 in cancer cells to prevent the release of lactate into the tumor microenvironment (TME) enhances the activity of effector T cells and improves checkpoint therapy.

[0005] Due to the important roles of MCT1 and MCT4 in lactate homeostasis, inhibition of these MCTs is a promising therapeutic target for cancer treatment. Previous studies have shown that the anchoring and stability of MCT1 and MCT4 on the plasma membrane requires their physical interaction with basigin (BSG). BSG, also known as cluster of differentiation 147 (CD147), is a single-pass transmembrane protein of the immunoglobulin (Ig) superfamily that plays a key role in reproduction, neurological function, pathogen infection, and T cell activation and proliferation. BSG-2, which encodes two extracellular Ig domains, is the most predominant splice variant of BSG and binds tightly to MCT1 or MCT4. BSG-2 is highly expressed in many cancer cells and stimulates the production of vascular endothelial growth factor (VEGF) and the expression of matrix metalloproteinases to promote tumor angiogenesis and proliferation. BSG-2 has become a target for the treatment of hepatocellular carcinoma (HCC) in clinical trials.

[0006] There is an unmet need to provide therapeutic agents that inhibit MCT1, MCT4 and / or CD147 for treating diseases or disorders. Summary of the invention

[0007] The present disclosure relates to anti-CD147 antibodies or antigen-binding fragments thereof, and uses of the anti-CD147 antibodies or antigen-binding fragments thereof for treating diseases or disorders.

[0008] Certain aspects of the present disclosure relate to an anti-CD147 antibody or an antigen-binding fragment thereof, comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 2, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 3, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 6.

[0009] Certain aspects of the present disclosure relate to polynucleotides encoding the antibodies or antigen-binding fragments thereof according to the present disclosure.

[0010] Certain aspects of the present disclosure relate to a pharmaceutical composition comprising an anti-CD147 antibody or antigen-binding fragment thereof according to the present disclosure or a polynucleotide according to the present disclosure, and a pharmaceutically acceptable carrier.

[0011] Certain aspects of the present disclosure relate to a kit comprising an anti-CD147 antibody or antigen-binding fragment according to the present disclosure or a polynucleotide according to the present disclosure or a pharmaceutical composition according to the present disclosure. In some embodiments, the kit provided herein comprises an MCT inhibitor and / or an immune checkpoint blocking therapeutic agent.

[0012] Certain aspects of the present disclosure relate to a method for preventing, diagnosing or treating a disease or condition in a subject in need thereof, the method comprising administering to the subject an anti-CD147 antibody or antigen-binding fragment, or a polynucleotide encoding the anti-CD147 antibody or antigen-binding fragment, or a pharmaceutical composition comprising the anti-CD147 antibody or antigen-binding fragment or the polynucleotide.

[0013] Certain aspects of the present disclosure relate to the use of an anti-CD147 antibody or antigen-binding fragment thereof, or a polynucleotide encoding the anti-CD147 antibody or antigen-binding fragment, or a pharmaceutical composition provided herein in the manufacture of a medicament / therapeutic agent or a kit for diagnosing, preventing or treating a disease or condition. Certain aspects of the present disclosure relate to the use of an anti-CD147 antibody or antigen-binding fragment thereof, or a polynucleotide encoding the anti-CD147 antibody or antigen-binding fragment, or a combination of a pharmaceutical composition and an MCT inhibitor provided herein in the manufacture of a medicament or a kit for treating a disease or condition.

[0014] Certain aspects of the present disclosure relate to the use of an anti-CD147 antibody or antigen-binding fragment thereof, or a polynucleotide encoding the anti-CD147 antibody or antigen-binding fragment, or a pharmaceutical composition provided herein in combination with an immune checkpoint blockade therapeutic agent in the manufacture of a medicament or kit for treating a disease or condition. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Amino acid sequence alignment of human BSG-2 with other vertebrate orthologs (cow, chicken, hamster, rat, rabbit, mouse) is shown.

[0016] Figure 2 H+ influx mediated by MCT1 and MCT1 / BSG-2 in the presence of WZHAb10 or AZD3965 or both is shown, demonstrating the inhibitory efficacy of WZHAb10 and AZD3965 on MCT1.

[0017] Figure 3 H+ influx mediated by MCT4 and MCT4 / BSG-2 in the presence of WZHAb10 is shown, demonstrating the inhibitory efficacy of WZHAb10 on MCT4.

[0018] Figure 4 The transport activity of MCT1 / BSG-2 variants in the absence or presence of WZHAb10 compared to IgG control is shown. The transport activity of MCT1 / BSG-2 variants was normalized to that of WT MCT1 / BSG-2. All data are mean ± SD (n = 3). *, p < 0.05. **, p < 0.01. ***, p < 0.001. ns, not statistically significant.

[0019] Figure 5 Shown are the viability of A20 cells in the presence of AZD3965 and / or WZHAb10 and the IC50 measurements of WZHAb10 on A20 cells.

[0020] Figure 6 Shown are the fold changes in lactate concentration in A20 cells treated with WZHAb10 and / or AZD3965.

[0021] Figure 7 Viability of A549 cells in the presence of WZHAb10 and / or AZD3965, and fold changes in lactate concentration in A549 cell cultures treated with WZHAb10 and / or AZD3965 are shown. All data are mean ± SD (n = 3). *, p < 0.05.

[0022] Figure 8 Shown are the viability of B16F10 cells in the presence of AZD3965 and / or WZHAb10, and the fold change in lactate concentration in B16F10 cell cultures treated with WZHAb10 and / or AZD3965. *, p<0.05.

[0023] Fig. 9 Shown are the BSG-2, MCT1, and MCT4 expressions of A549 cells cultured in normoxia (O2, 21%) and hypoxia (O2, 1%), and in the presence of different concentrations of WZHAb10.

[0024] Fig.10 Shown are the expressions of BSG-2, MCT1, and MCT4 of A20 cells in the presence of different concentrations of WZHAb10.

[0025] Fig.11 Superposition of the Ig2 domains of mBSG-2 and hBSG-2 is shown to demonstrate the structural similarity between the two BSG-2s.hBSG-2 does not have equivalent amino acid residues at G147, A158 and N159 in mBSG-2.

[0026] Fig.12 The changes in mouse body weight during WZHAb10 treatment of A20 tumor mice are shown, and WZHAb10 inhibits the growth of A20 tumors in mice. The mouse tumor model was treated with 5 mg / kg WZHAb10 or IgG (ip) every other day when tumors appeared.

[0027] Fig.13Shown are the changes in mouse body weight during WZHAb10 treatment of A549 xenograft mice, and WZHAb10 inhibits the growth of A549 xenograft tumors in mice. Mouse tumor models were treated with 5 mg / kg WZHAb10 or IgG (ip) every other day when tumors appeared.

[0028] Fig.14 Shown is the appearance of xenograft A549 tumors at the end of the course of treatment with IgG or WZHAb10 (5 mg / kg).

[0029] Fig.15A Dose-response curves generated from NSCLC organoids derived from patient 1 treated with AZD3965, WZHAb10 or GSK2837808A are shown. Viability was measured by CellTiter-Glo 3D cell viability assay. Each assay was performed in triplicate for each individual organoid line.

[0030] Fig. 15B Shown is a dose-response curve for PBMCs from patient 1 in the presence of AZD3965.

[0031] Fig. 15C Shown is the dose-response curve of PBMCs from patient 1 in the presence of WZHAb10.

[0032] Fig.15D Shown is the dose-response curve of PBMCs from patient 1 in the presence of GSK2837808A.

[0033] Fig.16A Dose-response curves generated from NSCLC organoids derived from patient 2 treated with AZD3965, WZHAb10 or GSK2837808A are shown. Viability was measured by CellTiter-Glo 3D cell viability assay. Each assay was performed in triplicate for each individual organoid line.

[0034] Fig. 16B Shown is a dose-response curve for PBMCs from patient 2 in the presence of AZD3965.

[0035] Fig. 16C Shown is the dose-response curve of PBMCs from patient 2 in the presence of WZHAb10.

[0036] Fig.16D Shown is the dose-response curve of PBMCs from patient 2 in the presence of GSK2837808A.

[0037] Fig.17ADose-response curves generated from NSCLC organoids derived from patient 3 treated with AZD3965, WZHAb10 or GSK2837808A are shown. Viability was measured by CellTiter-Glo 3D cell viability assay. Each assay was performed in triplicate for each individual organoid line.

[0038] Fig. 17B Shown is a dose-response curve for PBMCs from patient 3 in the presence of AZD3965.

[0039] Fig. 17C Shown is the dose-response curve of PBMCs from patient 3 in the presence of WZHAb10.

[0040] Fig.17D Shown is the dose-response curve of PBMCs from patient 3 in the presence of GSK2837808A.

[0041] Fig.18 Shown are cell viability assays of NSCLC organoids derived from patient 1, co-cultured with PBMCs from patient 1, and treated with pembrolizumab, WZHAb10, or a combination thereof. *, p<0.05. **, p<0.01. ns, not statistically significant.

[0042] Fig.19 Shown is a cell viability assay of NSCLC organoids derived from patient 2, co-cultured with PBMCs from patient 2 and treated with pembrolizumab, GSK2837808A, WZHAb10, or a combination thereof.

[0043] Fig. 20 Shown is a cell viability assay of NSCLC organoids derived from patient 3, co-cultured with PBMCs from patient 3 and treated with pembrolizumab, AZD3965, WZHAb10, or a combination thereof. DETAILED DESCRIPTION

[0044] In order to more easily understand the present disclosure, some terms are first defined. As used in this application, unless otherwise expressly provided herein, each of the following terms should have the meaning set forth below. Additional definitions are set forth throughout the application.

[0045] It should be noted that the absence of quantifiers ("a" / "an") covers the situation of one or more / one or more of the entities; for example, "nucleotide sequence" is understood to mean one or more / one or more nucleotide sequences. Similarly, the absence of quantifiers, "one or more / one or more" and "at least one / at least one" are used interchangeably herein.

[0046] In addition, when used in this article, "and / or" should be understood as a specific disclosure of each of the two specified features or components with or without the other. Therefore, the term "and / or" as used in phrases such as "A and / or B" in this article is intended to include "A and B", "A or B", "A" (alone) and "B" (alone). Similarly, the term "and / or" as used in phrases such as "A, B and / or C" is intended to cover each of the following aspects: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0047] The term "about" is used herein to mean approximately, roughly, approximately, or around. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values ​​set forth above.

[0048] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure relates.

[0049] The term "antibody" refers broadly to an immunoglobulin (Ig) molecule typically composed of four polypeptide chains (two heavy (H) chains and two light (L) chains), or any functional fragment, mutant, variant or derivative thereof that retains the essential target binding characteristics of an Ig molecule. Such mutant, variant or derivative antibody forms are known in the art.

[0050] As used herein, the term "CDR" refers to the complementarity determining region within the variable sequence of an antibody. There are three CDRs in each variable region of the heavy chain (HC) and light chain (LC), designated as CDR1, CDR2 and CDR3 (or specifically, HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2 and LC CDR3) for each variable region.

[0051] The term "amino acid" is intended to encompass all molecules, whether natural or synthetic, that include both an amino functional group and an acid functional group and that can be included in a polymer of naturally occurring amino acids. Exemplary amino acids include naturally occurring amino acids; analogs, derivatives, and homologs thereof; amino acid analogs with variant side chains; and all stereoisomers of any of the foregoing. As used herein, the term "amino acid" includes D- or L-optical isomers and peptide mimetics. A "conservative amino acid substitution" is a substitution in which an amino acid residue is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0052] The terms "polypeptide," "peptide," and "protein" (if single chain) are used interchangeably herein to refer to amino acid polymers of any length. The polymer may be linear or branched, it may contain modified amino acids, and it may be interrupted by non-amino acids. These terms also encompass amino acid polymers that have been modified; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component. A polypeptide may be isolated from a natural source, may be produced by recombinant techniques from a eukaryotic or prokaryotic host, or may be the product of a synthetic procedure.

[0053] The terms "nucleic acid", "nucleic acid sequence", "nucleotide sequence" or "polynucleotide sequence" and "polynucleotide" are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. A polynucleotide may be single-stranded or double-stranded, and if single-stranded, may be a coding strand or a non-coding (antisense) strand. A polynucleotide may contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. The nucleotide sequence may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, for example by conjugation with a labeling component. A nucleic acid may be a recombinant polynucleotide, or a polynucleotide of genomic, cDNA, semisynthetic or synthetic origin that does not occur in nature or is linked to another polynucleotide in a non-natural arrangement.

[0054] The term "activity" includes the binding specificity / affinity etc. activities of the antibody.

[0055] The terms “proton-coupled monocarboxylate transporter” and “MCTs” belong to the solute carrier 5 (SLC5) family. Among the 14 identified MCTs, MCTs 1-4 have been extensively studied because they selectively transport products of the glycolytic cycle, including lactate, pyruvate, and ketone bodies. MCTs 1-4 are the major lactate shuttles in humans and are therefore physiologically critical for lactate homeostasis at both the tissue and organismal levels.

[0056] MCT1 and MCT4 are overexpressed in several cancers and promote tumor growth and invasiveness in multiple ways. Tumor cells often establish a metabolic symbiosis with lactate, whereby oxidative cancer cells preferentially use lactate as an oxidative fuel, thereby freeing up glucose for glycolytic cancer cells or cancer-associated fibroblasts (CAFs), which in turn convert glucose to lactate for oxidative cancer cells. MCT1 and MCT4 are key lactate shuttles that maintain this symbiosis. Due to the important roles of MCT1 and MCT4 in lactate homeostasis, inhibition of these MCTs is a promising therapeutic target for cancer treatment.

[0057] The anchoring and stability of MCT1 and MCT4 at the plasma membrane requires their physical interaction with basic immunoglobulin (BSG).

[0058] The terms "BSG," "basic immunoglobulin," and "CD147" are used to describe single-pass transmembrane proteins of the immunoglobulin (Ig) superfamily that play key roles in reproduction, neurological function, pathogen infection, and T-cell activation and proliferation. BSG-2, encoding two extracellular Ig domains, is the most predominant splice variant of BSG and binds tightly to MCT1 or MCT4. BSG-2 is highly expressed in many cancer cells, stimulating the production of vascular endothelial growth factor (VEGF) and the expression of matrix metalloproteinases to promote tumor angiogenesis and proliferation. BSG-2 has become a target for the treatment of hepatocellular carcinoma (HCC) in clinical trials. The CD147 molecule may be closely associated with tumor progression and recurrence.

[0059] The terms "cancer" and "tumor" can be used interchangeably. For example, both terms encompass solid tumors and liquid tumors. As used herein, the terms "cancer" or "tumor" include precancerous as well as malignant cancers and tumors.

[0060] The terms "DLBCL" and "diffuse large B-cell lymphoma" are used to describe aggressive (fast-growing) non-Hodgkin lymphoma (NHL) that affects B lymphocytes. Lymphocytes are a type of white blood cell. B cells are lymphocytes that produce antibodies to fight infection and are an important part of the lymphatic system.

[0061] Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma (NHL) in the United States and worldwide, accounting for approximately 22% of newly diagnosed B-cell NHL cases in the U.S. More than 18,000 people are diagnosed with DLBCL each year.

[0062] The term "NSCLC" refers to any type of epithelial lung cancer other than small cell lung cancer (SCLC). The most common types of NSCLC are squamous cell carcinoma, large cell carcinoma, and adenocarcinoma, but there are several other types that occur less frequently, and all can occur in unusual histologic variants.

[0063] The term "melanoma" refers to a form of skin cancer characterized by abnormal expression of melanocytes, which are responsible for producing pigmentation in the skin. Melanoma is the most deadly form of skin cancer, with a five-year survival rate of less than 20% for cases diagnosed in the late stages. Approximately half of all melanoma diagnoses are considered invasive, and incidence levels have increased significantly over the past few decades. Many therapeutic agents are being developed to reduce incidence levels and improve standards of care, but highly effective treatments for advanced melanoma remain to be achieved.

[0064] The term "transfection" refers to the process of introducing an agent into a cell. The list of agents that can be transfected is long and includes, but is not limited to, siRNA, sense and / or antisense sequences, DNA encoding one or more genes and organized into expression plasmids, proteins, protein fragments, etc. There are many methods for transfecting agents into cells, including but not limited to electroporation, calcium phosphate-based transfection, DEAE-dextran-based transfection, lipid-based transfection, molecular conjugate-based transfection (e.g., polylysine-DNA conjugate), microinjection, etc.

[0065] As used herein, the term "combination therapy" refers to the administration of two or more therapeutic substances, such as an anti-CD147 antibody or antigen-binding fragment and an additional therapeutic agent. The additional therapeutic agent can be administered simultaneously with, before, or after the administration of the anti-CD147 antibody or antigen-binding fragment. In one embodiment, the anti-CD147 antibody or antigen-binding fragment of the present invention is administered in combination with one or more inhibitors for the treatment of tumors.

[0066] As used herein, the term "subject" refers to a human or non-human animal subject. Exemplary subjects include humans and mammals. Exemplary subjects include humans, monkeys, dogs, cats, mice, rats, cattle, chickens, hamsters, horses, camels, goats, rabbits, pigs, and sheep. In certain embodiments, the subject is a human, cattle, chickens, hamsters, rats, rabbits, and mice. In certain embodiments, the subject is a human. In some embodiments, the subject suffers from a disease or condition that can be treated with the antibodies provided herein.

[0067] Certain aspects of the present disclosure relate to an anti-CD147 antibody or an antigen-binding fragment thereof, comprising a heavy chain CDR1 having an amino acid sequence as shown in SEQ ID NO: 1, a heavy chain CDR2 having an amino acid sequence as shown in SEQ ID NO: 2, a heavy chain CDR3 having an amino acid sequence as shown in SEQ ID NO: 3, a light chain CDR1 having an amino acid sequence as shown in SEQ ID NO: 4, a light chain CDR2 having an amino acid sequence of X1X2VSX3X4IX5 (SEQ ID NO: 5), and a light chain CDR3 having an amino acid sequence as shown in SEQ ID NO: 6. In some embodiments, X1, X2, X3, X4, and X5 are independently any amino acids.

[0068] In some embodiments, the anti-CD147 antibody or its antigen-binding fragment comprises a heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO:7, a heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO:8, a heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO:9, a light chain CDR1 having the amino acid sequence shown in SEQ ID NO:10, a light chain CDR2 having the amino acid sequence shown in SEQ ID NO:11, and a light chain CDR3 having the amino acid sequence shown in SEQ ID NO:12.

[0069] In some embodiments, the anti-CD147 antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:13 and a light chain variable region having the amino acid sequence shown in SEQ ID NO:14.

[0070] In some embodiments, the anti-CD147 antibody or antigen-binding fragment thereof comprises a heavy chain constant region having the amino acid sequence shown in SEQ ID NO:15 and a light chain constant region having the amino acid sequence shown in SEQ ID NO:16.

[0071] In some embodiments, the anti-CD147 antibody or antigen-binding fragment thereof comprises a heavy chain having the amino acid sequence shown in SEQ ID NO:17 and a light chain having the amino acid sequence shown in SEQ ID NO:18.

[0072] Certain aspects of the present disclosure relate to polynucleotides encoding the antibodies or antigen-binding fragments thereof according to the present disclosure.

[0073] Certain aspects of the present disclosure relate to a pharmaceutical composition comprising the above-mentioned anti-CD147 antibody or antigen-binding fragment thereof or the above-mentioned polynucleotide, and a pharmaceutically acceptable carrier.

[0074] Typically, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 5-8, preferably about 6-8, although the pH can be varied depending on the properties of the formulated substance and the disease condition to be treated. The formulated pharmaceutical composition can be administered by conventional routes, including but not limited to intratumoral administration, intraperitoneal administration, intravenous administration or topical administration.

[0075] The pharmaceutical composition of the present disclosure contains a safe and effective amount (such as 0.001-99wt%, preferably 0.01-95wt%, more preferably 0.1-90wt%) of the provided single domain antibody or fusion protein, and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to) saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should match the mode of administration. The pharmaceutical composition of the present application can be prepared in an injectable form, for example, by preparing the pharmaceutical composition by conventional methods with physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions (such as injections and solutions) should be manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, such as about 10 μg / kg body weight to about 100 mg / kg body weight per day.

[0076] In some embodiments, the pharmaceutical composition comprises an additional therapeutic agent.

[0077] In some embodiments, the additional therapeutic agent comprises an MCT inhibitor.

[0078] In some embodiments, the MCT inhibitor is an MCT1 inhibitor and / or an MCT4 inhibitor.

[0079] In some embodiments, the pharmaceutical composition includes an immune checkpoint blockade therapeutic.

[0080] The term "immune checkpoint" refers to a component of the immune system that provides inhibitory signals to other components to regulate immune responses. Known immune checkpoint proteins include CTLA-4, PD1 and its ligands PD-L1 and PD-L2, and also include LAG-3, BTLA, B7H3, B7H4, TIM3, KIR. Pathways involving LAGS, BTLA, B7H3, B7H4, TIM3 and KIR are considered in the art to constitute immune checkpoint pathways similar to CTLA-4 and PD-1 dependent pathways (see, for example, Pardoll, 2012. Nature Rev Cancer [Natural Cancer Review] 12: 252-264; Mellman et al., 2011. Nature [Natural] 480: 480-489).

[0081] An immune checkpoint blockade therapeutic agent refers to any compound that inhibits the function of an immune checkpoint protein. Inhibition includes both reduced function and complete blockade. In particular, the immune checkpoint protein is a human immune checkpoint protein.

[0082] In some embodiments, the pharmaceutical composition comprises anti-PD1 antibody, anti-PDL1 antibody, anti-CTLA4 antibody, anti-PDL2 antibody, anti-LAG3 antibody, anti-BTLA antibody, anti-B7H3 antibody, anti-B7H4 antibody, anti-TIM3 antibody and / or anti-KIR antibody. In some embodiments, the pharmaceutical composition comprises anti-PD1 antibody or anti-PDL1 antibody.

[0083] Certain aspects of the present disclosure relate to a kit comprising an anti-CD147 antibody or antigen-binding fragment according to the present disclosure or a polynucleotide according to the present disclosure or a pharmaceutical composition according to the present disclosure.

[0084] In some embodiments, the kit includes an MCT inhibitor, such as an MCT1 inhibitor or an MCT4 inhibitor.

[0085] In some embodiments, the kit includes an immune checkpoint blockade therapeutic agent, which includes but is not limited to an anti-PD1 antibody or an anti-PDL1 antibody.

[0086] Certain aspects of the present disclosure relate to a method of preventing, diagnosing or treating a disease or condition in a subject in need thereof, the method comprising administering to the subject an anti-CD147 antibody or antigen-binding fragment according to the present disclosure, or a polynucleotide according to the present disclosure, or a pharmaceutical composition according to the present disclosure.

[0087] In some embodiments, the method comprises administering to the subject a therapeutically effective amount of the above-described anti-CD147 antibody or antigen-binding fragment, polynucleotide, or pharmaceutical composition.

[0088] A therapeutically effective amount reduces the severity of disease symptoms and increases the frequency and duration of symptom-free periods of a disease, disorder or condition, or prevents injury or disability due to the disease or affliction. For example, for the treatment of tumors (including, for example, melanoma, lymphoma, bladder cancer, non-small cell lung cancer, head and neck cancer, and colon cancer), a "therapeutically effective amount" preferably inhibits cell growth or tumor growth by at least about 10%, preferably at least about 20%, more preferably at least about 30%, more preferably at least about 40%, more preferably at least about 50%, more preferably at least about 60%, more preferably at least about 70%, and more preferably at least about 80%, relative to an untreated subject.

[0089] In some embodiments, the disease or condition comprises a disease with CD147, MCT1, MCT3 and / or MCT4 expression. In some embodiments, the disease or condition comprises a disease with high expression of CD147, MCT1, MCT3 and / or MCT4.

[0090] In some embodiments, the disease or disorder comprises a disease with immune checkpoint (optionally, PD1 and / or PDL1) expression.

[0091] In some embodiments, the diseases or conditions include tumors and infectious diseases.

[0092] In some embodiments, the tumor comprises prostate cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, gastric cancer, lymphoma, esophageal cancer, intestinal cancer, bone cancer, wherein the infectious disease comprises inflammatory bowel disease, new virus pneumonia, enteritis caused by Escherichia coli and Salmonella, measles, listeriosis and falciparum malaria.

[0093] In some embodiments, the method comprises administering to the subject an MCT inhibitor prior to, subsequent to, or concurrently with administering an anti-CD147 antibody or antigen-binding fragment, or polynucleotide, or pharmaceutical composition provided herein.

[0094] In some embodiments, the method comprises administering to the subject an immune checkpoint blockade therapeutic agent before, after, or simultaneously with the administration of an anti-CD147 antibody or antigen-binding fragment, or polynucleotide, or pharmaceutical composition provided herein.

[0095] Certain aspects of the present disclosure relate to the use of the anti-CD147 antibodies or antigen-binding fragments, or the polynucleotides provided by the present disclosure in the manufacture of a medicament / therapeutic agent or kit for diagnosing, preventing or treating a disease or disorder.

[0096] Provided herein is the use of the above-mentioned anti-CD147 antibody or antigen-binding fragment thereof, or the above-mentioned polynucleotide, or the above-mentioned pharmaceutical composition in combination with an MCT inhibitor in the manufacture of a medicament / therapeutic agent or kit for diagnosing, preventing or treating a disease or condition.

[0097] Provided herein is the use of the above-mentioned anti-CD147 antibody or antigen-binding fragment thereof, or the above-mentioned polynucleotide, or the above-mentioned pharmaceutical composition in combination with an immune checkpoint blockade therapeutic agent in the manufacture of a therapeutic agent or kit for diagnosing, preventing or treating a disease or condition.

[0098] In some embodiments, the disease or condition comprises a disease with expression of CD147, MCT1, MCT3 and / or MCT4.

[0099] In some embodiments, the disease or disorder comprises a disease with immune checkpoint (eg, PD1) expression.

[0100] In some embodiments, the diseases or conditions include tumors and infectious diseases.

[0101] In some embodiments, the tumor comprises prostate cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, gastric cancer, lymphoma, esophageal cancer, intestinal cancer, bone cancer, wherein the infectious disease comprises inflammatory bowel disease, new virus pneumonia, enteritis caused by Escherichia coli and Salmonella, measles, listeriosis and falciparum malaria.

[0102] In some embodiments, the subject comprises a human or non-human animal, such as a cow, chicken, hamster, rat, rabbit, mouse.

[0103] The present disclosure will be further illustrated in the following non-limiting working examples, it should be understood that these examples are for illustration only, and the present disclosure is not intended to be limited to the materials, conditions, process parameters, etc. described herein. Unless otherwise stated, all proportions are by weight.

[0104] Example

[0105] Example 1: Amino Acid Sequence of BSG Ig2 Domain

[0106] The amino acid sequence of the BSG Ig2 domain is as follows Figure 1 Most residues of BSG-2 at the interface are highly or moderately conserved among vertebrate orthologs ( Figure 1 ), which indicates that anti-CD147 antibodies can bind to these orthologs. Therefore, the antibodies can be used in humans and other animals.

[0107] Selected functionally important amino acid residues in BSG-2 that interact with WZHAb10 are annotated. Residues highlighted in grey are involved in extensive polar and non-polar interactions between BSG-2 and WZHAb10. Residues labeled by circles, triangles, and diamonds each contact multiple residues of WZHAb10 in polar interactions, respectively, and if mutated to alanine residues, may impair the binding of BSG-2 and WZHAb10. WZHAb10 comprises a heavy chain having the amino acid sequence shown in SEQ ID NO:17 and a light chain having the amino acid sequence shown in SEQ ID NO:18.

[0108] Example 2: Preparation of anti-CD147 antibodies

[0109] A monoclonal antibody capable of binding to BSG-2 was developed using hybridoma technology. MCT1 / BSG-2 was selected as the antigen.

[0110] (1) Expression and purification of WT MCTs, complexes of WT MCTs and WT BSG-2, or MCT1 / BSG-2 mutant variants

[0111] The coding sequences of mouse wild-type MCT1 (MCT1), MCT4 (MCT4), and BSG-2 (BSG-2), as well as their mutant or truncated variants, were each cloned into the pEG BacMam vector, a gift from Eric Gouaux's laboratory. Unless otherwise indicated, the 8xHis-Flag tag was located at the C-terminus of the protein and was connected by a rhinovirus 3C protease-cleavable linker. In this study, WT MCTs, complexes of WT MCTs with WT BSG-2, and MCT1 / BSG-2 variants with point mutations in one or both proteins were expressed in HEK293S GnTI- (N-acetylglucosaminyltransferase I negative) cells (American Type Culture Collection, catalog number CRL-3022). The cells were collected by centrifugation (5,000 × g, 10 min, 4°C) and thawed by KIMBLE Dounce tissue grinder (Merck Millipore) in 4% paraformaldehyde supplemented with 5.2 μg / mL aprotinin, 2 μg mL -1The cells were disrupted in a buffer (50 mM HEPES pH 7.5, 150 mM NaCl) containing leupeptin and 1.4 μg / mL pepstatin A (both from Merck Millipore). Cell debris was removed by centrifugation (10,000 × g, 25 min, 4° C.). The membrane fraction was collected by ultracentrifugation (100,000 × g, 1 hr, 4° C.) and dissolved in a buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 1% (w / v) n-dodecyl-D-maltoside (DDM, Anatrace), 0.1% (w / v) cholesterol hemisuccinate (CHS, Anatrace)) for 2.5 h at 4° C. Insoluble material was removed by ultracentrifugation (100,000 × g, 1 hr, 4° C.). The detergent-soluble fraction was incubated with Ni-NTA resin (Qiagen) and incubated at 4°C for 2 hr. The beads were eluted with elution buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.03% DDM, 0.003% CHS and 250 mM imidazole) and further incubated for 2 hours with anti-DYKDDDDK G1 affinity resin (GenScript Biotech Co., Ltd.) to improve protein purity. The protein sample was then eluted with DYKDDDDK peptide (GenScript Biotech Co., Ltd.) and purified by size exclusion chromatography (SEC) on a Superdex 200Increase 10 / 300GL column (GE Healthcare), which was balanced with SEC buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.03% DDM, 0.003% CHS). Peak fractions of protein samples were collected and concentrated to 2.0-5.0 mg / mL using 100 kDa MWCO Amicon centrifugal filters (Merck Millipore). Each protein or protein complex was snap frozen and stored in liquid nitrogen for further use.

[0112] (2) Expression and purification of Saposin A

[0113] Saposin A in the pNIC28-Bsa4 vector is expressed in Escherichia coli strain Rosetta-gami BDE3 with an N-terminal 6xHis-tag and a thrombin cleavage site. Cell cultures were first grown in TB medium at 37°C until OD600 was 1.0, and then induced overnight with 0.4mM IPTG at 18°C ​​for protein expression. After harvesting the cells, the precipitate was resuspended in a buffer (50mM HEPES pH 7.5, 300mM NaCl, 1% (w / v) Triton X-100 (Merck Millipore)) and then lysed by a homogenizer (ATS Engineering Limited, China). The cell suspension was heated to 85°C for 10min to precipitate all heat-labile components. Insoluble matter was removed by centrifugation (16000×g, 20min, 4°C). The supernatant containing Saposin A was incubated with Ni-NTA resin at 4°C for 3 hours. The beads were washed and the protein was eluted with a buffer (20mM HEPES pH 7.5, 300mM NaCl, 50mM sodium cholate, 30 / 400mM imidazole) with increasing concentrations of imidazole. Thrombin (Merck Millipore) was added to the eluted protein and dialyzed overnight at 4°C with a dialysis buffer (20mM HEPES pH 7.5, 300mM NaCl, 5% glycerol). The cleaved protein was concentrated and purified by SEC on a Superdex200Increase 10 / 300GL column (General Medical Group) balanced with SEC buffer (20mM HEPES pH7.5, 150mM NaCl). Pure Saposin A was concentrated to 1.0-2.0mg / mL and stored at -80°C for further use.

[0114] (3) Reconstitution of WT MCT1 / BSG-2 into Salipro nanoparticles and preparation of WT MCT1 / BSG-2 and WZHAb10Fab complexes

[0115] WT MCT1 / BSG-2 was reconstituted into Salipro nanoparticles according to the established protocol. Briefly, a lipid mixture of 1-palmitoyl-2-oleoyl-sn-glycero-3-PC (POPC): 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE): L-α-phosphatidylglycerol (POPG) (3:1:1, all from Avanti) was first resuspended from a dried lipid film in 15 mM cholate to obtain a 10 mM lipid stock solution. The MCT1 / BSG-2: Saposin A: POPC / POPE / POPG lipid mixture at a molar ratio of 1:8:10 was then mixed in a buffer containing 20 mM HEPES pH 7.5, 150 mM NaCl and incubated at 4°C for 2 hours. Salipro formation was initiated by adding 160 mg / mL equilibrated SM2 bio-beads (Bio-Rad) to remove detergent and stirring continuously overnight at 4°C. Subsequently, the sample was incubated twice with 160 mg / mL equilibrated SM2 bio-beads (Bio-Rad) at 4°C for 30 minutes each to remove residual detergent. The sample was then filtered through a 0.22 μm pore centrifugal filter and SEC was performed on a Superose 6 Increase 10 / 300 column (GE Healthcare) equilibrated with SEC buffer (20 mM HEPES pH 7.5, 150 mM NaCl). The SEC peak fractions were combined and concentrated to 2.0 mg / mL and stored at -80°C for further use.

[0116] To prepare the WT MCT1 / BSG-2 complex with WZHAb10Fab, fresh WZHAb10Fab (see below) and WT MCT1 / BSG-2 embedded in Salipro nanoparticles were incubated with 1.3 molar excess of WZHAb10Fab for 2 hours on an ice-water bath. The mixture was concentrated to 0.5-1.0 mL using a 100,000 MWCO Amico centrifugal filter and then SEC was performed on a Superose6Increase 10 / 300 column (GE Healthcare) equilibrated with SEC buffer (20 mM HEPES pH 7.5, 150 mM NaCl). The fraction from the center of the gel filtration peak was concentrated to 0.7 mg / mL. The final sample was centrifuged at 20,000 × g for 5 min at 4°C to precipitate any debris, and then the sample was vitrified as quickly as described below.

[0117] (4) Antibody selection and production

[0118] In order to produce mouse anti-MCT1 / BSG-2 monoclonal antibody WZHAb10, C57BL / 6 mice were immunized with 50 μg MCT1 / BSG-2 complexes reconstructed into Salipro nanoparticles as described above. After the first immunization, 4 booster immunizations were performed, each with 50 μg of protein. About 2000 hybridoma cells were produced by fusing SP2 / 0-Ag14 mouse myeloma cells with spleen B lymphocytes from immunized animals. Culture supernatants were screened for reactivity with ELISA. Using MCT1 / BSG-2 embedded in Salipro nanoparticles, ELISA screening was performed under detergent-free conditions and captured on MaxiSorp surface plates (Thermofisher). 20 ELISA-positive, immunoblot-negative hybridomas were subcloned by serial dilution three times, and their antibodies were purified by affinity chromatography using protein A Sepharose 4Fast Flow columns. The eluted antibodies were dialyzed overnight against mAb dialysis buffer (20 mM Na3PO4 pH 7.5 and 150 mM NaCl) and assayed for binding to the MCT1 / BSG-2 complex by gel filtration. Five antibodies showed strong binding to MCT1 / BSG-2, but only one (called WZHAb10, subclass IgG2) was shown to inhibit MCT1 transport activity.

[0119] Fab sequencing of IgG-WZHAb10 is as follows: RNA was extracted from hybridoma cells using HiPure RNA mini columns (Magen) according to the manufacturer's instructions. RNA concentration and integrity were measured using NanoDrop and gel electrophoresis. According to the manufacturer's instructions, cDNA was synthesized from RNA using SMARTScribe reverse transcriptase (Takara) with oligonucleotide-dT and template switching oligonucleotide (TSO). The resulting cDNA was diluted for amplification. The forward primer was anchored to TSO, and the reverse primer was bound to the constant region of the heavy or light chain. The 5' ends of the forward and reverse primers were labeled with partial P5 and P7 adapters (Illumina), respectively. In the first stage PCR, heavy and light chain fragments were amplified in separate reactions. The PCR products were purified with magnetic beads. During the second stage PCR, index primers were attached to both ends of the first stage PCR products to form a TruSeq dual index library. The library was purified with magnetic beads and quantified with a Qubit 4 fluorometer (Thermo Fisher), and finally sequenced on an Illumina MiSeq (double-end 2×300bp) according to the manufacturer's user manual (Illumina). The original fastq file was first quality assessed. Adaptors and bases with low quality scores (Q values ​​less than 20) were deleted using Trimmomatic (v0.36) to generate clean data (trimmed data). Pandaseq (2.10) was used to merge double-end reads. The merged sequences were processed by IgBLAST software to identify V(D)J sequences. The reference sequence was obtained in the IMGT database (4). Mouse WZHAb10 was humanized using the "humanized yeast" platform.

[0120] To generate WZHAb10Fab, the full-length antibody was digested with 1:100 w / w papain in mAb dialysis buffer (see above) supplemented with 1 mM EDTA, 10 mM L-cysteine ​​hydrochloride and 50 mM Na3PO4 pH 7.5 at 37°C for 5 hours. After digestion, papain was inactivated with 30 mM iodoacetamide on ice for 15 min, and the digested Fab was subjected to SEC on a Superdex200Increase 10 / 300 column (GE Healthcare) equilibrated with buffer (20 mM HEPES pH 7.5, 150 mM NaCl). The purified Fab was immediately used for MCT1 / BSG-2 binding experiments, including cryoEM grid preparation, or was otherwise rapidly frozen by liquid nitrogen and stored at -80°C for further use.

[0121] Example 3: Monoclonal antibody WZHAb10 binds to BSG-2 and inhibits the transport activity of MCT 1 and 4

[0122] (1) Preparation of protein-free liposomes or proteoliposomes encapsulating HPTS

[0123] Proteoliposomes were prepared using the following purified protein samples: WT MCT1, WT MCT1 / BSG-2, WT MCT4, WT MCT4 / BSG-2, or MCT1 / BSG-2 mutant and truncated variants. A lipid mixture of 3:1:1:1:1 ratio (w / w) dissolved in chloroform, POPC, POPE, POPG, and cholesterol (all lipids from Avanti) was first dried into a lipid film under an argon stream and then vacuumed for 16 hours to completely remove the chloroform. The dried lipids were resuspended in a buffer containing 20mMTris pH 8.5 and 150mM NaCl by ultrasonic treatment. 12mM N-octyl-β-D-maltopyranoside (OM, Anatras) was added and mixed with a 10mg / mL lipid suspension at room temperature for 2 hours. Protein samples purified in DDM were added to the lipid / OM mixture at a protein to lipid molar ratio of 1:1000. After incubation at room temperature for 4 hours, SM2 bio-beads (Bio-Rad) were added and the detergent was removed by incubating the mixture overnight at 4°C. Subsequently, the mixture was incubated three more times with SM2 bio-beads at 4°C for 30 min each time to remove residual detergent. The mixture was then loaded onto a manually filled Sephadex G-50 column (Sephadex G-50 Superfine, Cytiva), which was pre-balanced with reconstitution buffer. Fractions that could be stained with Bradford dye (Bio-Rad) were collected and concentrated to a final concentration of 2.5 mg / mL using an Amicon Ultra centrifugal filter (0.5 mL, 100 kDa cutoff). The protein concentration after reconstitution was measured in the same manner as described above and further confirmed by Coomassie blue staining and western blotting.

[0124] In order to load pH-sensitive fluorescent dye 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS, Merck Millipore) into proteoliposomes, proteoliposomes were mixed with 0.1mM HPTS and sonicated in an ice-water bath, wherein multiple 1min on and 1min off cycles were performed to prevent overheating. Then, using a lipid extruder (Avanti), the mixture was extruded (25 times) through a polycarbonate filter with a pore size of 0.2 μm. After extrusion, uncaptured dyes were removed by a Sephadex G-50 column and eluted with a buffer solution containing 20mM Tris pH 8.5 and 150mM NaCl. The eluted fractions of the proteoliposomes containing loaded HPTS were collected and concentrated to 2.0mg / mL or 2.5mg / mL. Protein-free liposomes (referred to as empty liposomes) were prepared in parallel in the same manner in the absence of protein. Within 48 hours, flux determination was performed using freshly prepared proteoliposomes and empty liposomes encapsulating HPTS.

[0125] (2) Fluorescence-based flux measurement

[0126] All fluorescence-based flux assays were performed at 25° C. Empty liposomes encapsulating HPTS were run in parallel as negative controls in the same manner as proteoliposomes unless otherwise stated.

[0127] (3) Substrate selectivity determination

[0128] Liposomes were reconstituted with WT MCT1, WT MCT1 / BSG-2, WT MCT4, or WT MCT4 / BSG-2 (2.0 mg / mL each). The assay was performed using a SpectraMax i3 (Molecular Devices) plate reader based on previously published methods (5) and modified. Each well of a Nunc MicroWell 96-well plate (Thermofisher Scientific) was first filled with 85 μL of extralipid buffer containing 20 mM HEPES pH 7.0 and 150 mM NaCl, and then 10 μL of HPTS-loaded proteoliposomes were slowly mixed. After incubation for 10 min, baseline fluorescence F was recorded every 15 s. 开始 , for 8 cycles, with excitation and emission wavelengths of 450nm and 510nm, respectively. Then 5μL of the test substance at a final concentration of 25mM was added to initiate H+ influx, and the fluorescence signal F was monitored every 15s for 60 cycles. After monitoring the flux signal for 15min, the proteoliposomes were sonicated to allow H+ and the test substance to pass through the membrane and reach equilibrium. The final fluorescence value F was then recorded. 结束The test substances used were: sodium chloride, sodium citrate, sodium D-lactate, sodium L-lactate or sodium pyruvate. Each test substance was adjusted to pH 7.0 using HCl or NaOH.

[0129] (4) H+ influx under different H+ gradients

[0130] Proteoliposomes were reconstituted with WT MCT1 or WT MCT1 / BSG-2 (2.0 mg / mL each). H+ influx assays were performed following the same procedure described above for the substrate selectivity assay, with two changes. First, the extraliposome buffer was replaced with one of the following test buffers containing 150 mM NaCl: 20 mM Tris pH 8.5, 20 mM HEPES pH 7.5, 20 mM HEPES pH 7.0, or 20 mM MES pH 6.5. Second, the F 开始 Afterwards, H+ influx was initiated by 25 mM pyruvate adjusted at the corresponding pH value.

[0131] (5) H+ influx under different pyruvate or L-lactate gradients

[0132] To test the effect of a pyruvate gradient on H+ influx, proteoliposomes were reconstituted with WT MCT1 or WT MCT1 / BSG-2 (2.0 mg / mL each). The assay was performed following the same procedure as described above for the substrate selectivity assay, with one change: while recording F 开始 Afterwards, H+ influx was initiated by pyruvate pH 7.0, each at a final concentration of 0 mM, 5 mM, 25 mM or 100 mM. To test the effect of an L-lactate gradient on H+ influx, proteoliposomes were reconstituted with WT MCT4 or WT MCT4 / BSG-2. The assay was performed following the same procedure described above for the pyruvate gradient, but pyruvate was replaced with L-lactate pH 7.0 at a final concentration of 0 mM, 5 mM, 25 mM or 100 mM, respectively.

[0133] (6) H+-pyruvate symport mediated by MCT1 / BSG-2 mutant variants

[0134] To compare H+ influx mediated by WT MCT1 or WT MCT1 / BSG-2 with that mediated by MCT1 / BSG-2 mutant variants, proteoliposomes were reconstituted with WT MCT1, or WT MCT1 / BSG-2 or MCT1 / BSG-2 variants (2.0 mg / mL each). The assay was performed following the same procedure as described above for the substrate selectivity assay, with one modification: when recording F 开始 Afterwards, H+ influx was initiated by 25 mM pyruvate pH 7.0.

[0135] (7) H+-pyruvate symport mediated by WT MCT1 or WT MCT1 / BSG-2 in the presence of WZHAb10, AZD3965, or both

[0136] The following stock solutions were prepared for the assay: 200 μM WZHAb10 or IgG negative control, each prepared in a buffer containing 20 mM Tris pH 8.5, 150 mM NaCl; 20 mM AZD3965 in pure dimethyl sulfoxide (DMSO, Merck Millipore); HPTS-encapsulated proteoliposomes reconstituted with WT MCT1 or WT MCT1 / BSG-2 (each at 2.5 mg / mL in a buffer containing 20 mM Tris pH 8.5 and 150 mM NaCl).

[0137] In order to measure H+-pyruvate symport in the presence of WZHAb10, AZD3965 or both, the proteoliposomes encapsulating HPTS at a final concentration of 2.0 mg / mL were mixed with WZHAb10 or AZD3965 at a final concentration of 3 μM, or 6 μM or a combination thereof (3 μM+3 μM or 6 μM+6 μM) in a buffer containing 20 mM Tris pH 8.5 and 150 mM NaCl. The mixture was incubated at 4°C for 2 hours under gentle rotation. After incubation, the mixture was immediately used for determination, which was carried out according to the same procedure described above for the determination of H+-pyruvate symport mediated by MCT1 / BSG-2 mutant variants. The proteoliposomes encapsulating HPTS at 2.0 mg / mL in the presence of 6 μM IgG and 0.03% DMSO were used as controls and were carried out in parallel in the same manner.

[0138] (8) H+-pyruvate symport mediated by MCT1 / BSG-2 mutant and truncated variants in the presence of WZHAb10

[0139] The following stock solutions were prepared for the assay: 200 uM WZHAb10 or IgG control, each prepared in a buffer containing 20 mM Tris pH 8.5, 150 mM NaCl; 2.5 mg / mL HPTS-encapsulated proteoliposomes reconstituted with WT MCT1 or MCT1 / BSG-2 mutant and truncated variants (in a buffer containing 20 mM Tris pH 8.5 and 150 mM NaCl, respectively).

[0140] To measure the inhibitory effect of WZHAb10 on proteoliposome-mediated H+-pyruvate symport, WZHAb10 at a final concentration of 6 μM was mixed with 2.0 mg / mL HPTS-encapsulated proteoliposomes in a buffer containing 20 mM Tris pH 8.5 and 150 mM NaCl. The mixture was incubated at 4 ° C for 2 hours under gentle rotation. After incubation, the mixture was immediately used for determinations, which were performed according to the same procedures described above for H+-pyruvate symport determinations mediated by MCT1 / BSG-2 mutant variants. 2.0 mg / mL HPTS-encapsulated proteoliposomes in the presence of 6 μM IgG were used as controls and were performed in parallel in the same manner.

[0141] (9) Immunofluorescence staining

[0142] Treated cells were cultured on small round coverslips, fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, blocked with 5% bovine serum, and treated with mouse anti-EMMPRIN / CD147 antibody (Santa Cruz) and rabbit anti-MCT4 or MCT1 antibody (Thermo Fisher Scientific) overnight at 4°C. After incubation with corresponding fluorescently labeled secondary antibodies (Alexa Fluor 555-labeled goat anti-mouse IgG (H+L) and Alexa Fluor 488-labeled goat anti-rabbit IgG (H+L; both from Thermo Fisher Scientific), samples were visualized under an OLYMPUS IX83 confocal fluorescence microscope to evaluate the colocalization of BSG-2 and MCT1 or MCT4.

[0143] (10) H+-L-lactate symport mediated by WT MCT4 and WT MCT4 / BSG-2 in the presence of WZHAb10

[0144] The following stock solutions were prepared for the assay: 200 uM WZHAb10 or IgG control, each prepared in a buffer containing 20 mM Tris pH 8.5, 150 mM NaCl; 2.5 mg / mL HPTS-encapsulated proteoliposomes reconstituted with WT MCT4 or WT MCT4 / BSG-2 (in a buffer containing 20 mM Tris pH 8.5 and 150 mM NaCl, respectively).

[0145] To measure the inhibitory effect of WZHAb10 on proteoliposome-mediated H+-L-lactate symport, WZHAb10 at a final concentration of 6 μM was mixed with 2.0 mg / mL HPTS-encapsulated proteoliposomes in a buffer containing 20 mM Tris pH 8.5 and 150 mM NaCl. The mixture was incubated at 4°C for 2 hours with gentle rotation. After incubation, the mixture was immediately used for assays, which were performed according to the same procedures described above for the H+-pyruvate symport assay mediated by MCT1 / BSG-2 mutant variants (BSG-2D136A, BSG-2E146A, BSG-2S145A, BSG-2Q195A, BSG-2D194A), but with one modification: the F was recorded at 4 °C for 2 h. 开始 Afterwards, H+ influx was initiated by 25 mM L-lactate pH 7.0. 2.0 mg / mL HPTS-encapsulated proteoliposomes in the presence of 6 μM IgG were used as a control and were run in parallel in the same manner.

[0146] (11) Data analysis

[0147] For flux assays, fluorescence data were first normalized to eliminate baseline fluorescence fluctuations using the following equation:

[0148]

[0149] where F 归一化 is the normalized fluorescence plotted in the flux assay graph, F is the fluorescence measured in arbitrary units, and F 开始 is the average of the fluorescence measured before the addition of the monocarboxylate substrate to initiate H + influx, and F 结束 is the endpoint fluorescence measured after disruption of proteoliposomes by sonication. Normalization was performed using Excel (Microsoft Corporation).

[0150] Prism software (GraphPad) was used to analyze flux assays, cell viability, lactate concentration, and tumor growth. Statistical significance (p<0.05) was assessed using the Student's T test. Error bars represent SD.

[0151] WZHAb10 reduced the transport activities of MCT1 / BSG-2 and MCT4 / BSG-2 by approximately 75% and 50%, respectively ( Figure 2-3 ). Notably, WZHAb10 did not affect the transport activity of MCT1 or MCT4 that were not coupled to BSG-2, suggesting that WZHAb10 allosterically inhibited MCT1 or MCT4 by binding to BSG-2.

[0152] To determine whether WZHAb10 has an inhibitory effect on the transport activity of MCTs similar to that of small molecule inhibitors, the inhibitory efficacy of WZHAb10 and AZD3965 (a selective and potent small molecule MCT1 inhibitor developed by AstraZeneca and currently in clinical trials (https: / / clinicaltrials.gov / ct2 / show / NCT01791595)) on MCT1 was compared. Figure 2 ), as selective and potent small molecule inhibitors of MCT4 are not yet commercially available. Interestingly, WZHAb10 produced a similar, or even stronger, inhibition of MCT1 / BSG-2-mediated H+ influx. We further investigated whether WZHAb10 and AZD3965 had a combined effect on MCT1 inhibition. Indeed, the simultaneous presence of half the concentration of WZHAb10 and AZD3965 was as effective in inhibiting MCT1 / BSG-2-mediated H+ influx as either AZD3965 or WZHAb10 alone ( Figure 2 ), suggesting the possibility of utilizing both orthosteric and allosteric inhibitors of the MCT1 / BSG-2 complex.

[0153] WZHAb10 was as effective as other known MCT inhibitors on the transport activity of MCTs. For comparison, AZD3965, a potent orthosteric inhibitor of MCT1 in clinical trials, was chosen for testing. Indeed, WZHAb10 alone or in combination with AZD3965 inhibited the transport activity of MCT1 / BSG-2 to a similar extent as that conferred by AZD3965 ( Figure 2 ), suggesting that allosteric modulation could be explored alone or in combination with orthosteric modulation to intervene in the activity of the transporters MCT1, MCT3, and MCT4 that use BSG-2 as their chaperone.

[0154] Figure 4 The transport activity of MCT1 / BSG-2 variants in the absence or presence of WZHAb10 compared to IgG control is shown. The results suggest that the key residues for the interaction of BSG-2 with WZHAb10 may affect its inhibitory activity on the transport activity of MCTs.

[0155] The transport activity of MCT1 / BSG-2 variants was normalized to that of WT MCT1 / BSG-2. All data are mean ± SD (n = 3). *, p < 0.05. **, p < 0.01. ***, p < 0.001. ns, not statistically significant. Intraliposome buffer (buffer inside liposomes): 150 mM NaCl, 0.1 mM HPTS, 20 mM Tris pH 8.5; extraliposome buffer: 150 mM NaCl, 25 mM pyruvate, 20 mM HEPES pH 7.0. Empty liposomes were negative controls.

[0156] Example 4: WZHAb10 inhibits the growth of cancer cells in vitro

[0157] (1) Cell culture and transfection

[0158] A549 (ATCC No. CCL-185), A20 (ATCC No. TIB-208) and B16F10 (ATCC No. CCL-6475) cells were cultured in RPMI1640 (Merck Millipore) with 10% fetal bovine serum (FBS, Thermo Fisher Scientific), penicillin (100 U / ml, Merck Millipore) and streptomycin (100 μg / ml, Merck Millipore). A20 cells were cultured in normoxia (21% O2, 5% CO2 and 37°C) because cell viability was observed to drop sharply in hypoxia. A549 and B16F10 cells were cultured in normoxia and hypoxia (1% O2, 5% CO2 and 37°C). For siRNA transfection, Lipofectamine RNAiMax (Thermo Fisher Scientific) was used for A549 cells, and NEPA21 electroporator (NEPA GENE) was used for A20 cells.

[0159] (2) Cell viability assay

[0160] Cells were seeded in 96-well culture dishes in culture medium (3,000 cells per well for A20 cells; 1,500 cells per well for A549 cells and B16F10 cells). A20 cells were seeded and treated with the indicated concentrations of AZD3965 and / or WZHAb10 in complete culture medium for 72 hours. IgG and DMSO groups served as controls. Cell viability was then determined using the CCK8 kit in SpectraMax i3 (Molecular Instruments). For A549 cells and B16F10 cells, cells were incubated overnight to adhere and treated under normoxia or hypoxia for 72 hours as indicated, and then cell viability was measured.

[0161] (3) Measurement of lactate concentration

[0162] A20 cells were seeded and treated with the indicated concentrations of AZD3965 and / or WZHAb10 in complete medium for 72 hours. IgG and DMSO groups were used as controls. Lactate concentration was measured using a lactate colorimetric / fluorimetric assay kit (BioVision catalog number K607-100) in a SpectraMax i3 (Molecular Instruments). For A549 cells and B16F10 cells, cells were incubated overnight to adhere and treated for 72 hours under normoxia or hypoxia as indicated, and then lactate concentration was determined.

[0163] WZHAb10 inhibits cell proliferation of A20 lymphoma cells, A549 non-small cell lung (NSCL) cancer cells, and B16F10 melanoma cells ( Figure 5-8 ). WZHAb10 exhibited an additive effect with AZD3965 in the low micromolar range, suggesting that cancer cells can be treated with a combination of MCT small molecule inhibitors and MCT / BSG antibodies. Different cancer cells exhibited different expression levels of MCT1 and MCT4 under normoxic and hypoxic conditions. Therefore, the reduction in lactate concentration was different in the culture medium, which may confirm that the transport activity of MCT1 was correspondingly inhibited in the presence of AZD3965, or the transport activity of the complex of MCTs and BSG was correspondingly inhibited in the presence of WZHAb10, or a combination thereof. Consistently, half-concentrations of WZHAb10 and AZD3965 showed different inhibitory effects on cancer cell growth when combined together, suggesting that small molecule inhibitors and biological inhibitors in different molar ratios can be used for effective cancer treatment under normoxic and hypoxic conditions.

[0164] After observing allosteric regulation of mMCT1 and mMCT4 by the antibody, WZHAb10 was investigated for its ability to inhibit tumor survival in vitro and in vivo. Murine A20 lymphoma cells have high glycolytic activity under normoxia (45), as well as human A549 non-small cell lung (NSCL) cancer cells and murine B16F10 melanoma cells under hypoxia. As expected, WZHAb10 inhibited the proliferation of A20 cells under normoxia ( Figure 5-6 ), and inhibiting A549 cells under hypoxia ( Figure 7 ) and B16F10 cells ( Figure 8 ) cell survival and lactate transport.

[0165] The inhibition of survival of these human (A549) and murine (A20 and B16F10) cancer cells by WZHAb10 is consistent with the high structural similarity between the extracellular Ig2 domains of human and murine BSG-2s, which bind tightly to WZHAb10 ( Fig.11), which suggests a common regulatory mechanism of WZHAb10 on human and mouse MCT / BSG-2 transporter complexes (e.g., MCT1 / BSG-2, MCT4 / BSG-2, and MCT3 / BSG-2).

[0166] Although WZHAb10 showed similar efficacy to AZD3965 in inhibiting mMCT1 activity at the molecular level ( Figure 2 ), but it has a stronger inhibitory effect on cancer cell survival and lactate transport than AZD3965 ( Figure 5-8 ), indicating that WZHAb10 inhibits the activity of multiple MCTs in cells. In fact, MCT4 is expressed in A20 and A549 cells ( Figure 9-10 ). Furthermore, the combination of allosteric WZHAb10 and orthosteric AZD3965 resulted in additive inhibition of cancer cell growth and lactate flux ( Figure 5-8 ), thus providing a potential alternative approach for treating glycolytic cancer cells.

[0167] Example 5: WZHAb10 inhibits the growth of cancer cells in vivo

[0168] Xenograft Models

[0169] Three to four-week-old male or female BALB / c nude mice were purchased from the Animal Center of Guangxi Medical University. A20 cells and A549 cells were suspended in complete culture medium. Then 100 μL of cells (1×10 6 The mice were injected subcutaneously with WZHAb10 (5 mg / kg) suspended in PBS every other day (6-8 mice per group). IgG was used as a control (vehicle). Tumor volume was measured with a digital caliper using the following formula: Volume (mm 3 )=(length×width 2 ) / 2.

[0170] The present disclosure further examined the antitumor efficacy of WZHAb10 on A20 (mouse tumor) and A549 (human tumor) xenografts in nude mice. Administration of WZHAb10 (5 mg / kg, ip) significantly inhibited tumor growth, but had little effect on body weight during treatment ( Fig.12 , 13).

[0171] Fig.14 Shown is the appearance of xenograft A549 tumors at the end of the course of treatment with IgG or WZHAb10 (5 mg / kg).WZHAb10 treatment was not associated with any overt toxicity.

[0172] Example 6: The combination of WZHAb10 and anti-PD1 antibody showed higher anti-tumor efficacy than these antibodies alone in an ex vivo NSCLC model

[0173] Non-small cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancers and is often diagnosed at an advanced stage. Several anti-PD-L1 and anti-PD1 antibodies have been approved for the treatment of patients with metastatic NSCLC with high PD-L1 expression. Despite this, most NSCLC patients are resistant or acquire resistance to immunotherapy. To investigate whether preventing the release of lactate into the TME by blocking both MCT 1 and MCT4 of cancer cells can enhance the activity of effector T cells and improve checkpoint therapy, three patient-derived organoids (PDOs) were used to investigate the combined antitumor effects of WZHAb10 and pembrolizumab (anti-PD1 antibody).

[0174] (1) PDO culture

[0175] All procedures involving human participants were approved by the Ethics Committee of Shanghai Jiao Tong University School of Medicine and conformed to the ethical standards of the Declaration of Helsinki (revised in 2013). All donors signed informed consent. After harvesting, freshly resected tumor samples from patients were kept in MACS tissue storage solution (Miltenyi) at 4°C. Tumor tissue was cut into small pieces on ice and then incubated for three hours at 37°C in DMEM / F12 medium (Thermo Fisher) with 0.001% DNase (Sigma-Aldrich, Missouri, USA), 1 mg / mL collagenase / dispase (Roche, Indiana, USA), 200 U / mL penicillin, 200 mg / mL streptomycin and 0.5 mg / mL amphotericin B (2% antibiotics, Sigma), while gently stirring and resuspending intermittently. After this, the tissue suspension of digestion is repeatedly ground via pipetting, and passed through a 70 μm filter. The filtered suspension was centrifuged for three minutes at 112 × g, and lysis buffer (00443357, Thermo Fisher Scientific) was used to lyse red blood cells for five minutes. Precipitation is resuspended in 100 μL serum-free culture media (SFM, Thermo Fisher Scientific) supplemented with 20 ng / mL bFGF (Thermo Fisher Scientific), 50 ng / mL human EGF (Thermo Fisher Scientific), N2 (Thermo Fisher Scientific), B27 (Thermo Fisher Scientific), 10 μM ROCK inhibitor (Enzo Life Sciences (Enzo Life Sciences)) and 1% penicillin-streptomycin (Thermo Fisher Scientific). Thereafter, 200 μL of Matrigel (Corning) was added to 100 μL of cell suspension to establish organoids, and the resulting suspension was solidified at 37 ° C on a preheated 6-well culture plate (Corning) for 30 minutes. After gelation, 3 mL of SFM was added to the wells. The culture medium was replaced every four days. Organoids were passaged every two weeks at a ratio of 1:2 or 1:3. For organoid passage, organoids were first harvested by incubating with cold PBS for one hour at 4 ° C and dissociated using 1 × TrypLe (Thermo Fisher). The dissociated organoids were then mixed in SFM+Matrigel (1:3 ratio) and re-seeded in a culture dish, and then the culture medium was added after gelation. Early passage organoids (<3 generations) were frozen in liquid nitrogen for further study.

[0176] (2) Organoid drug response assay

[0177] Organoids were harvested after two weeks of culture and dissociated using 1×TrypLe (Thermo Fisher Scientific). The dissociated organoids were mixed in SFM+Matrigel (1:3 ratio) and seeded onto 384-well white plates. After gelation, 30 μL of SFM was added to each well. The organoids were cultured for 48 hours. The average diameter of the organoids was set to 50 μm as the minimum requirement for drug screening. Thereafter, a dilution series of each compound (MCT1 inhibitor AZD3965, 0.001, 0.01, 0.1, 1, 10, 100 μM; LDHA inhibitor GSK2837808A (CAS No. 1445879-21-9), 0.0001, 0.001, 0.01, 0.1, 1, 10, 100 μM; or WZHAb10 (0.000006, 0.00006, 0.0006, 0.006, 0.06 μM)) was dispensed, and after three days of drug incubation, cell viability was determined using CellTiter-Glo (Promega). Fig.15A , 16A and 17A). The plates were stirred at room temperature (10°C-30°C) for 30 min before measuring luminescence. As expected, single-agent treatment exerted a moderate antiproliferative effect in all models, although the extent of response varied among patients. These results are consistent with those observed in the xenograft models ( Figure 12-14 ), suggesting that blocking lactate metabolism is robust in cancer therapy.

[0178] To investigate whether preventing lactate release into the TME of cancer cells could improve immune checkpoint blockade therapy, the toxicity of AZD3965, GSK2837808A, and WZHAb10 on peripheral blood mononuclear cells (PBMCs) obtained from the same patients was determined ( Fig. 15B -D, 16B-D, 17B-D), and found that 0.1 μM AZD3965, 1 μM GSK2837808A, and 0.0006 μM WZHAb10 had little effect on T cell growth in these patients. Organoids were then treated with any of these agents (0.1 μM AZD3965, 1 μM GSK2837808A, and 0.0006 μM WZHAb10) in combination with PBMCs in the presence of 5 μg / mL pembrolizumab ( Fig.18 , 19 and 20). Fig.18 IgG was used as a control. Figure 19-20 IgG and / or vehicle were used as controls.

[0179] In all cases, the WZHAb10 and pembrolizumab combination showed a much stronger antiproliferative effect than pembrolizumab alone in all models, suggesting that WZHAb10 improves immune checkpoint blockade therapy by preventing lactate release into the TME of cancer cells.

Claims

1. An anti-CD147 antibody or an antigen-binding fragment thereof, comprising a heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO: 1, a heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO: 2, a heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO: 3, a light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 4, a light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 5, and a light chain CDR3 having the amino acid sequence shown in SEQ ID NO:

6.

2. The anti-CD147 antibody or antigen-binding fragment thereof according to claim 1, wherein: (1) the heavy chain CDR1 has the amino acid sequence shown in SEQ ID NO:7; (2) the heavy chain CDR2 has the amino acid sequence shown in SEQ ID NO: 8; (3) the heavy chain CDR3 has the amino acid sequence shown in SEQ ID NO:9; (4) the light chain CDR1 has the amino acid sequence shown in SEQ ID NO: 10; (5) the light chain CDR2 has the amino acid sequence shown in SEQ ID NO: 11; and (6) The light chain CDR3 has the amino acid sequence shown in SEQ ID NO:

12.

3. The anti-CD147 antibody or antigen-binding fragment thereof according to claim 1 or 2, comprising: (1) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 13; and (2) A light chain variable region having the amino acid sequence shown in SEQ ID NO:

14.

4. The anti-CD147 antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, comprising: (1) a heavy chain constant region having the amino acid sequence shown in SEQ ID NO: 15; and (2) A light chain constant region having the amino acid sequence shown in SEQ ID NO:

16.

5. The anti-CD147 antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, comprising: (1) a heavy chain having the amino acid sequence shown in SEQ ID NO: 17; and (2) A light chain having the amino acid sequence shown in SEQ ID NO:

18.

6. A polynucleotide encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5.

7. A pharmaceutical composition comprising the anti-CD147 antibody or antigen-binding fragment thereof according to any one of claims 1 to 5 or the polynucleotide according to claim 6, and a pharmaceutically acceptable carrier.

8. The pharmaceutical composition of claim 7, wherein the pharmaceutical composition further comprises an additional therapeutic agent.

9. The pharmaceutical composition of claim 8, wherein the additional therapeutic agent comprises an MCT inhibitor.

10. The pharmaceutical composition of claim 9, wherein the MCT inhibitor is an MCT1 inhibitor, an MCT3 inhibitor and / or an MCT4 inhibitor.

11. The pharmaceutical composition of any one of claims 8-10, wherein the pharmaceutical composition comprises an immune checkpoint blockade therapeutic agent.

12. The pharmaceutical composition of claim 11, wherein the immune checkpoint blockade therapeutic agent is an anti-PD1 antibody, an anti-PDL1 antibody, an anti-CTLA4 antibody, an anti-PDL2 antibody, an anti-LAG3 antibody, an anti-BTLA antibody, an anti-B7H3 antibody, an anti-B7H4 antibody, an anti-TIM3 antibody and / or an anti-KIR antibody.

13. A kit comprising the anti-CD147 antibody or antigen-binding fragment according to any one of claims 1 to 5, the polynucleotide according to claim 6, or the pharmaceutical composition according to any one of claims 7 to 10.

14. The kit of claim 13, wherein the kit comprises an MCT inhibitor and / or an immune checkpoint blockade therapeutic agent.

15. A method for preventing, diagnosing or treating a disease or condition in a subject in need thereof, the method comprising administering to the subject an anti-CD147 antibody or antigen-binding fragment, or a polynucleotide encoding the anti-CD147 antibody or antigen-binding fragment, or a pharmaceutical composition comprising the anti-CD147 antibody or antigen-binding fragment or the polynucleotide, wherein the anti-CD147 antibody or antigen-binding fragment thereof comprises a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 2, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 3, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 having the amino acid sequence of SEQ ID NO:

6.

16. The method of claim 15, wherein the heavy chain CDR1 has the amino acid sequence shown in SEQ ID NO:7; the heavy chain CDR2 has the amino acid sequence shown in SEQ ID NO:8; the heavy chain CDR3 has the amino acid sequence shown in SEQ ID NO:9; the light chain CDR1 has the amino acid sequence shown in SEQ ID NO:10; the light chain CDR2 has the amino acid sequence shown in SEQ ID NO:11; and the light chain CDR3 has the amino acid sequence shown in SEQ ID NO:

12.

17. The method of claim 15 or 16, wherein the anti-CD147 antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 13 and a light chain variable region having the amino acid sequence shown in SEQ ID NO:

14.

18. The method of any one of claims 15 to 17, wherein the anti-CD147 antibody or antigen-binding fragment thereof comprises a heavy chain constant region having the amino acid sequence shown in SEQ ID NO: 15 and a light chain constant region having the amino acid sequence shown in SEQ ID NO:

16.

19. The method of any one of claims 15 to 18, wherein the anti-CD147 antibody or antigen-binding fragment thereof comprises a heavy chain having the amino acid sequence shown in SEQ ID NO: 17 and a light chain having the amino acid sequence shown in SEQ ID NO:

18.

20. The method of any one of claims 15-20, wherein the disease or condition comprises a disease with expression of CD147, MCT1, MCT3 and / or MCT4.

21. The method of any one of claims 15-20, wherein the disease or condition comprises a disease with immune checkpoint expression.

22. The method of any one of claims 15-20, wherein the disease or condition comprises a tumor.

23. The method of claim 22, wherein the tumor comprises prostate cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, gastric cancer, lymphoma, esophageal cancer, intestinal cancer, bone cancer.

24. The method of any one of claims 15-23, further comprising administering an MCT inhibitor to the subject before, after, or simultaneously with administering the anti-CD147 antibody or antigen-binding fragment, or the polynucleotide, or the pharmaceutical composition.

25. The method of claim 25, wherein the MCT inhibitor is an MCT1 inhibitor, an MCT1 inhibitor, an MCT3 inhibitor and / or an MCT4 inhibitor.

26. The method of any one of claims 15-25, comprising administering to the subject an immune checkpoint blockade therapeutic agent before, after, or simultaneously with administering the anti-CD147 antibody or antigen-binding fragment or the polynucleotide or the pharmaceutical composition.

27. The method of claim 26, wherein the immune checkpoint blockade therapeutic agent is an anti-PD1 antibody or an anti-PDL1 antibody.

28. The method of any one of claims 15-23, wherein the subject comprises a human or a non-human animal.

29. Use of an anti-CD147 antibody or antigen-binding fragment thereof as described in any one of claims 1 to 5, or a polynucleotide as described in claim 6, or a pharmaceutical composition as described in any one of claims 7 to 12 in the manufacture of a medicament / therapeutic agent or kit for diagnosing, preventing or treating a disease or condition.

30. Use of an anti-CD147 antibody or antigen-binding fragment thereof as described in any one of claims 1-5, or a polynucleotide as described in claim 6, or a pharmaceutical composition as described in any one of claims 7-12 in combination with an MCT inhibitor in the manufacture of a medicament / therapeutic agent or kit for diagnosing, preventing or treating a disease or condition.

31. Use of an anti-CD147 antibody or antigen-binding fragment thereof as described in any one of claims 1 to 5, or a polynucleotide as described in claim 6, or a pharmaceutical composition as described in any one of claims 7 to 12 in combination with an immune checkpoint blockade therapeutic agent in the manufacture of a therapeutic agent or kit for diagnosing, preventing or treating a disease or condition.

32. The use of any one of claims 29-31, wherein the disease or condition comprises a disease with expression of CD147, MCT1, MCT3 inhibitor and / or MCT4.

33. The use of any one of claims 29-31, wherein the disease or condition comprises a disease with immune checkpoint expression.

34. The use of any one of claims 29-33, wherein the disease or disorder comprises a tumor.

35. The use according to claim 35, wherein the tumor comprises prostate cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, gastric cancer, lymphoma, esophageal cancer, intestinal cancer, bone cancer.

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