An antibody or antigen-binding fragment thereof, and methods of making and uses thereof

Abstract

The present application relates to the technical field of biotechnology, and particularly relates to an antibody or antigen binding fragment thereof and a preparation method and application thereof. The antibody or antigen binding fragment thereof provided by the present application can be combined with CD147, wherein the antigen epitope combined by the antibody or antigen binding fragment thereof includes Ala149, glycosylation modification of Asp136 or Asp136, Tyr140 or glycosylation modification of Tyr140, Gln195 or glycosylation modification of Gln195, Asp194 or glycosylation modification of Asp194, Pro104, Asp147 or glycosylation modification of Asp147, Ser193 or glycosylation modification of Ser193, Lys191 or glycosylation modification of Lys191, Gly192 of CD147. The antibody or antigen binding fragment thereof provided by the present application has high affinity for MCT1 / CD147.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to an antibody or antigen-binding fragment thereof and a preparation method and use thereof. BACKGROUND

[0002] Tumor metabolism is heterogeneous and has Warburg effect. In the hypoxic environment far away from the perfusion blood vessels, tumor cells rely on glycolysis to maintain survival and proliferation. The supply of ATP and intermediate metabolites depends on the high utilization of glucose, and the release of metabolic product lactic acid is completed by MCT4. In contrast, although oxygen-sufficient tumor cells also express glucose transporters (GLUT), oxygen-sufficient tumor cells have a higher metabolic preference for pyruvate. MCT1 transports lactic acid released by hypoxic tumor cells into oxygen-sufficient tumor cells, and lactic acid is oxidized to pyruvate by lactate dehydrogenase and participates in the tricarboxylic acid (TCA) cycle. The metabolic preference of oxygenated tumor cells for lactic acid allows hypoxic tumor cells to obtain more glucose. This metabolic synergy is critical for tumor cell survival under hypoxic conditions in vivo. Therefore, MCT1 and MCT4 are coupled tumor therapy targets.

[0003] In various cancer tissues, CD147 (extracellular matrix metalloproteinase inducer) can not only assist the expression of MCT1 / MCT4 and GLUT1 on the plasma membrane of cancer cells, but also act as a co-receptor of vascular endothelial growth factor 2 in endothelial cells, enhance vascular endothelial growth factor-mediated signaling, and promote angiogenesis in cancer tissues. Current research has found that MCT1 / 4-mediated lactic acid transfer plays an important role in tumor growth, and small molecule inhibitors AZD3965 and 7ACC2 targeting MCT1 / 4 are in clinical trials and preclinical experiments. However, AZD3965 and 7ACC2 can only efficiently target MCT1 and inefficiently inhibit MCT4, so their efficacy is limited. In addition, some normal tissues and cells, although not highly expressing CD147, highly express MCT1 or MCT4. These tissues and cells will be damaged due to the inhibition of MCT1 or MCT4 activity and function by AZD3965 and 7ACC2. As a molecular chaperone of MCT1 and MCT4, CD147 promotes the plasma membrane localization of MCT1 and MCT4, and plays a key role in tumor growth, invasion, and angiogenesis, so inhibitory antibodies targeting CD147 have the potential to develop into drugs for treating extracranial solid tumors.

[0004] However, it has been known from the structure that CD147 is composed of a single transmembrane domain and an extracellular domain composed of two or three IgG domains, which is not conducive to the development of small molecule inhibitors. SUMMARY

[0005] In view of the above-mentioned shortcomings of the prior art small molecule drug technology, the present application aims to provide an antibody or antigen-binding fragment thereof, and a preparation method and use thereof, for solving the problems in the prior art.

[0006] To achieve the above-mentioned objects and other related objects, one aspect of the present application provides an antibody or antigen-binding fragment thereof, which can bind to CD147, wherein the antigen epitope bound by the antibody or antigen-binding fragment thereof comprises Ala149, glycosylation modification of Asp136 or Asp136, Tyr140 or glycosylation modification of Tyr140, Gln195 or glycosylation modification of Gln195, Asp194 or glycosylation modification of Asp194, Pro104, Asp147 or glycosylation modification of Asp147, Ser193 or glycosylation modification of Ser193, Lys191 or glycosylation modification of Lys191, Gly192 of CD147.

[0007] Another aspect of the present application provides an isolated polynucleotide encoding the above-mentioned antibody or antigen-binding fragment thereof.

[0008] Another aspect of the present application provides a construct containing the above-mentioned isolated polynucleotide.

[0009] Another aspect of the present application provides an expression system of the antibody, which contains the above-mentioned construct or the genome into which the above-mentioned polynucleotide is integrated as a foreign gene.

[0010] Another aspect of the present application provides a preparation method of the above-mentioned antibody or antigen-binding fragment thereof, which comprises the following steps: culturing the above-mentioned expression system of the antibody under conditions suitable for expression of the antibody, thereby expressing the antibody or antigen-binding fragment thereof, and purifying and isolating the antibody or antigen-binding fragment thereof.

[0011] Another aspect of the present application provides a use of the above-mentioned antibody or antigen-binding fragment thereof, or the culture of the above-mentioned expression system of the antibody, in the preparation of a medicament.

[0012] Another aspect of the present application provides a pharmaceutical composition comprising the above-mentioned antibody or antigen-binding fragment thereof, or the culture of the above-mentioned expression system of the antibody. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 A schematic diagram showing the mechanism of tumor metabolism Warburg effect.

[0014] Figure 2 A schematic diagram showing the three-dimensional atomic structure between the antibody HAbLJ001 (6E5F4) provided by the present application and human CD147.

[0015] Figure 3 Figure 7 shows a schematic diagram of the two-dimensional structure of the interaction between the antibody HAbLJ001 (6E5F4) provided by the present application and human CD147.

[0016] Figure 4 Figure 8 shows a schematic diagram of the results of the change in fluorescence intensity demonstrating the in vitro activity of the antibody in Example 3 of the present application.

[0017] Figure 5 Figure 9 shows a schematic diagram of the D-value of fluorescence intensity demonstrating the in vitro activity of the antibody in Example 3 of the present application.

[0018] Figure 6 Figure 10 shows a schematic diagram of the tumor growth in mice in Example 4 of the present application. DETAILED DESCRIPTION

[0019] In order to make the inventive purpose, technical scheme and beneficial technical effects of the present application clearer, the present application will be further described in detail below in conjunction with examples, and other advantages and effects of the present application can be easily understood by those skilled in the art from the contents disclosed in the present description.

[0020] In the present application, the terms "extracellular matrix metalloproteinase inducer", "CD147", "extracellular matrix metalloproteinase inducer", "Emmprin" are used interchangeably and include variants, isoforms, species homologues of human CD147, or CD147 of other species and analogues having at least one common epitope of CD147.

[0021] In the present application, "antibody" generally refers to a binding agent belonging to a polypeptide comprising at least a light chain or heavy chain immunoglobulin variable region or a fragment thereof that specifically recognizes and binds to one or more epitopes of an antigen, such as a peptide, a lipid, a polysaccharide or a nucleic acid containing an antigenic determinant, such as those recognized by immune cells.

[0022] In the present application, "epitope" or "antigenic determinant" generally refers to a region of an antigen that is bound by a binding agent. Epitopes can be formed by contiguous amino acids or non-contiguous amino acids brought into proximity by the tertiary folding of a protein. Epitopes formed by contiguous amino acids are generally retained when exposed to denaturing solvents, while epitopes formed by tertiary folding are generally lost when treated with denaturing solvents. In a unique spatial conformation, an epitope generally comprises at least 3 and more commonly at least 5, about 9 or about 8-10 amino acids.

[0023] In the present application, "isolated antibody or antigen-binding fragment thereof" refers to an antibody or antigen-binding fragment thereof that has been identified and separated and / or recovered from a component of its natural environment.

[0024] Through extensive practical research, the inventors of this invention have provided a high-affinity antibody HABLJ001 targeting MCT1 / CD147 and / or MCT4 / CD147. Furthermore, they have resolved the three-dimensional atomic structures of antibody HABLJ001 and CD147, thereby revealing the mechanism of antibody function at the molecular level and providing the corresponding antibody or its antigen-binding fragment. Based on this, the present invention was completed.

[0025] The first aspect of the present invention provides an antibody or an antigen-binding fragment thereof, the antibody or the antigen-binding fragment thereof being capable of binding to CD147, wherein the binding antigenic epitope of the antibody or the antigen-binding fragment thereof includes Ala149, Asp136 or a glycosylation modification of Asp136, Tyr140 or a glycosylation modification of Tyr140, Gln195 or a glycosylation modification of Gln195, Asp194 or a glycosylation modification of Asp194, Pro104, Asp147 or a glycosylation modification of Asp147, Ser193 or a glycosylation modification of Ser193, Lys191 or a glycosylation modification of Lys191, and Gly192. As described above, the inventors of this application have thoroughly analyzed the three-dimensional atomic structure of high-affinity antibodies against MCT1 / CD147 and / or MCT4 / CD147 and mouse CD147, and provided the antigenic epitope corresponding to CD147 and the high-affinity antibody against the antigenic epitope based on the amino acid sequence comparison results of mouse and human CD147.

[0026] The antibody or its antigen-binding fragment provided in this application can generally inhibit CD147 binding to MCT1 and / or MCT4, thereby inhibiting the interaction between CD147 and MCT1 and / or MCT4, and inhibiting the expression and / or function of the MCT1 / CD147 complex and / or MCT4 / CD147 complex. For example, the inhibition of MCT1 / CD147 and / or MCT4 / CD147 can be partial inhibition, i.e., reducing the expression and / or function of MCT1 / CD147 and / or MCT4 / CD147, or it can be complete inhibition, i.e., completely eliminating the expression and / or function of MCT1 / CD147 and / or MCT4 / CD147. The aforementioned expression can be at the cytoplasmic membrane level, and the aforementioned function can be a substrate transport function. As another example, the inhibition of MCT1 / CD147 and / or MCT4 / CD147 can be an inhibition at the protein molecular level.

[0027] The antibody or antigen-binding fragment provided in this application can specifically bind to CD147 and also has a high affinity for CD147. For example, if the antibody or antigen-binding fragment can bind to CD147 at an affinity of about ≥10...5 M -1 , 10 6 M -1 , 10 7 M -1 , 10 8 M -1 , 10 9 M -1 , 10 10 M -1 , 10 11 M -1 , 10 12 M -1 , or 10 13 M -1 , an antibody or antigen-binding fragment thereof "binds specifically to" CD147. By way of further example, "high affinity" generally refers to an antibody or antigen-binding fragment thereof having a Kaof 10 7 M -1 , 10 8 M -1 , 10 9 M -1 , 10 10 M -1 , 10 11 M -1 , 10 12 M -1 , or 10 13 M -1 or greater. Methods for detecting the affinity of a suitable antibody or antigen-binding fragment thereof will be known to those of skill in the art. For example, affinity can be determined by competitive ELISA, or by binding association, or by displacement assays using labeled ligands, or using surface plasmon resonance devices such as the Biacore T100 available from Biacore, Inc., Piscataway, NJ, or optical biosensor technology such as the EPIC system or EnSpire available from Corning and Perkin Elmer, respectively (see also, e.g., Scatchard et al., (1949) Ann. N.Y. Acad. Sci 51 :660; and U.S. Pat. Nos. 5,283,173; 5,468,614; or equivalents).

[0028] The antibody or antigen-binding fragment thereof provided herein can be a monoclonal antibody. A "monoclonal antibody" generally refers to an antibody produced by a single clone of B lymphocytes or by a cell into which the light and heavy chain genes of a single antibody have been transfected. Monoclonal antibodies are produced by methods known to those skilled in the art, for example, by forming hybrid antibody-forming cells according to the fusion of myeloma cells with immunized spleen cells. Monoclonal antibodies can include humanized monoclonal antibodies.

[0029] The antibody or antigen-binding fragment thereof provided herein can include a heavy chain variable region including CDR-H1 having an amino acid sequence as set forth in SEQ ID No. 1, CDR-H2 having an amino acid sequence as set forth in SEQ ID No. 2, and CDR-H3 having an amino acid sequence as set forth in SEQ ID No. 3, and a light chain variable region including CDR-L1 having an amino acid sequence as set forth in SEQ ID No. 8, CDR-L2 having an amino acid sequence as set forth in SEQ ID No. 9, and CDR-L3 having an amino acid sequence as set forth in SEQ ID No. 10.

[0030] The antibody or antigen-binding fragment thereof provided herein is generally derived from Mus musculus, for example, can be obtained by murine hybridoma cells.

[0031] The antibody or antigen-binding fragment thereof provided herein can be humanized. A humanized antibody is an immunoglobulin that comprises a human framework region and one or more CDRs from a non-human (e.g., mouse, rat, or synthetic) immunoglobulin. The non-human immunoglobulin providing the CDRs is referred to as the "donor," and the human immunoglobulin providing the framework is referred to as the "acceptor." For example, all of the CDRs can be from the donor immunoglobulin in the humanized immunoglobulin, and for another example, a constant region need not be present, but if present, it generally needs to be essentially identical to a human immunoglobulin constant region, i.e., at least about 85-90% identical, such as about 95% or more identical. Thus, apart from the possible CDRs, all of the parts of the humanized immunoglobulin are essentially identical to corresponding parts of natural human immunoglobulin sequences. Humanized or other monoclonal antibodies can have additional conservative amino acid substitutions that have essentially no effect on antigen binding or other immunoglobulin functions. Humanized antibodies can be constructed by genetic engineering (see, e.g., U.S. Patent No. 5,585,089).

[0032] The antibody or antigen-binding fragment thereof provided in this application, wherein the heavy chain variable region includes FR1 as shown in SEQ ID No. 4, FR2 as shown in SEQ ID No. 5, FR3 as shown in SEQ ID No. 6, and FR4 as shown in SEQ ID No. 7, and the light chain variable region includes FR11 as shown in SEQ ID No. 8, FR2 as shown in SEQ ID No. 12, FR3 as shown in SEQ ID No. 13, and FR4 as shown in SEQ ID No. 14.

[0033] Table 1

[0034]

[0035] In a specific embodiment of the present invention, the amino acid sequence of the heavy chain variable region of the antibody or its antigen-binding fragment may include:

[0036] a) an amino acid sequence as shown in SEQ ID No. 15; or b) an amino acid sequence having more than 80% sequence identity with the amino acid sequence shown in SEQ ID No. 15 and having the function of the amino acid sequence defined in a). Specifically, the amino acid sequence in b) refers to: a polypeptide fragment obtained by substituting, deleting, or adding one or more (specifically, 1-50, 1-30, 1-20, 1-10, 1-5, or 1-3) amino acids to the amino acid sequence shown in SEQ ID No. 15, or by adding one or more (specifically, 1-50, 1-30, 1-20, 1-10, 1-5, or 1-3) amino acids to the N-terminus and / or C-terminus, and having the function of the polypeptide fragment shown in SEQ ID No. 15. For example, it could be the specific binding ability to CD147, the inhibition of CD147 binding to MCT1 and / or MCT4, the inhibition of the interaction between CD147 and MCT1 and / or MCT4, or the inhibition of the expression and / or function of the MCT1 / CD147 complex and / or the MCT4 / CD147 complex. The amino acid sequence in b) may have 80%, 85%, 90%, 93%, 95%, 97%, or 99% or more of identity with SEQ ID No. 15.

[0037] In this paper, sequence identity refers to the percentage of identical residues in sequences being compared. Sequence identity of two or more entries can be calculated using computational software known in the art, such as NCBI.

[0038] In another specific embodiment of the present application, the amino acid sequence of the heavy chain variable region of the antibody or antigen-binding fragment thereof can comprise:

[0039] c) the amino acid sequence as shown in SEQ ID No. 16; or, d) an amino acid sequence having more than 80% sequence identity to the amino acid sequence as shown in SEQ ID No. 16, and having the function of the amino acid sequence defined in c). Specifically, the amino acid sequence in d) specifically refers to a polypeptide fragment obtained by substituting, deleting or adding one or more (specifically, 1-50, 1-30, 1-20, 1-10, 1-5, or 1-3) amino acids to the amino acid sequence as shown in SEQ ID No. 16, or adding one or more (specifically, 1-50, 1-30, 1-20, 1-10, 1-5, or 1-3) amino acids to the N-terminal and / or C-terminal end, and having the function of the polypeptide fragment of the amino acid sequence as shown in SEQ ID No. 16. For example, it can be the specific binding ability to CD147, it can be the inhibition of CD147 binding to MCT1 and / or MCT4, it can be the inhibition of the interaction between CD147 and MCT1 and / or MCT4, or it can be the inhibition of the expression and / or function of MCT1 / CD147 complex and / or MCT4 / CD147 complex. The amino acid sequence in d) can have more than 80%, 85%, 90%, 93%, 95%, 97%, or 99% identity to SEQ ID No. 16.

[0040] The antibody or antigen-binding fragment thereof provided in the present application can be, for example, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a bispecific Fab dimer (Fab2), a trispecific Fab trimer (Fab3), an Fv, a single-chain Fv protein ("scFv"), a bis-scFv, (scFv)2, a minibody, a diabody, a triabody, a tetrabody, a disulfide stabilized Fv protein ("dsFv"), or a single-domain antibody (sdAb, nanobody), etc. and other various full-length antibodies that can be responsible for antigen binding.

[0041] The second aspect of the present application provides an isolated polynucleotide encoding the antibody or antigen-binding fragment thereof provided in the first aspect of the present application.

[0042] The third aspect of the present application provides a construct comprising the isolated polynucleotide of the second aspect of the present application. The construct can be constructed by inserting the above-mentioned isolated polynucleotide into a suitable vector, and the skilled person can select a suitable expression vector. For example, the type of the vector can be, but is not limited to, a plasmid, a phagemid, a phage derivative, an animal virus, a cosmid, and the like. For another example, the vector can be an expression vector, or a cloning vector.

[0043] The fourth aspect of the present application provides an expression system of the antibody, which comprises the construct of the third aspect of the present application or the polynucleotide of the second aspect of the present application integrated into the genome of the host cell, so as to express the above-mentioned antibody or antigen-binding fragment thereof. The expression system can be a host cell, which can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; a filamentous fungal cell, or a higher eukaryotic cell, such as a mammalian cell. Representative examples include: E. coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast, filamentous fungi, plant cells; insect cells of Drosophila S2 or Sf9; animal cells of CHO, COS, 293, or Bowes melanoma cells, and the like. The method for introducing the construct into the host cell should be known to the skilled person, for example, microinjection, gene gun, electroporation, virus-mediated transformation, electron bombardment, calcium phosphate precipitation, and the like.

[0044] The fifth aspect of the present application provides a method for preparing the antibody or antigen-binding fragment thereof of the first aspect of the present application, which comprises the following steps: culturing the expression system of the fourth aspect of the present application under conditions suitable for expressing the antibody, so as to express the above-mentioned antibody or antigen-binding fragment thereof, and purifying and isolating the above-mentioned antibody or antigen-binding fragment thereof.

[0045] The sixth aspect of the present application provides use of the culture of the expression system of the antibody or antigen-binding fragment thereof provided by the first aspect of the present application, or the antibody provided by the fourth aspect of the present application in the preparation of a medicament. As described above, the above-mentioned antibody or antigen-binding fragment thereof can generally inhibit the binding of CD147 to MCT1 and / or MCT4, and thus can be used as a CD147 antagonist, and can also be used as an MCT1 / CD147 inhibitor and / or MCT4 / CD147 inhibitor. By inhibiting the expression and / or function of the MCT1 / CD147 complex and / or the MCT4 / CD147 complex, these medicaments can be used to treat diseases related to MCT1 / CD147 or MCT4 / CD147-mediated lactic acid transmission. For example, the related diseases can be diseases in which MCT1 / CD147 or MCT4 / CD147 is highly expressed, and more specifically, tumors, infectious diseases, etc. The tumors can be specifically prostate cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, gastric cancer, lymphoma, esophageal cancer, intestinal cancer, bone cancer, etc. The infectious diseases can be specifically inflammatory bowel disease, COVID-19, enteritis caused by E. coli and Salmonella, measles, mononucleosis, and malignant malaria, etc.

[0046] In the use provided by the present application, the above-mentioned substances can be single pharmaceutical active ingredients, or can be combined with other active ingredients for the treatment of related indications.

[0047] The seventh aspect of the present application provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof provided by the first aspect of the present application or the culture of the expression system provided by the fourth aspect of the present application. The pharmaceutical composition provided by the present application can further comprise a pharmaceutically acceptable carrier. The above-mentioned carrier can comprise various excipients and diluents, which are not essential active ingredients themselves and have no excessive toxicity after administration. Suitable carriers should be well known to those skilled in the art, and a full discussion of pharmaceutically acceptable carriers can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J., 1991).

[0048] The eighth aspect of the present application provides a treatment method, comprising: administering a therapeutically effective amount of the fusion protein provided by the first aspect of the present application, the culture of the expression system provided by the fourth aspect of the present application, or the pharmaceutical composition provided by the seventh aspect of the present application to an individual.

[0049] In the present application, the term "treatment" includes prophylactic, curative or palliative treatment that can result in a desired pharmacological and / or physiological effect. The effect preferably means that one or more symptoms of a disease are reduced medically or the disease is completely eliminated, or the occurrence of the disease is blocked, delayed and / or the risk of development or aggravation of the disease is reduced.

[0050] In the present application, "individual" generally includes humans, non-human primates, or other mammals (e.g., dogs, cats, horses, sheep, pigs, cows, etc.) that can benefit from treatment with the formulations, kits or combinations described herein.

[0051] In the present application, "therapeutically effective amount" generally refers to an amount that, after an appropriate period of administration, is capable of achieving a therapeutic effect on the diseases listed above. The selection of a preferred therapeutically effective amount can be determined by a person of ordinary skill in the art (e.g., through clinical trials) according to various factors, for example, when the above-mentioned substance is used for an individual to be administered, the growth, proliferation, recurrence and / or metastasis of a tumor can be inhibited, more specifically, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% of the growth, proliferation, recurrence and / or metastasis of a tumor is inhibited.

[0052] Tumor cells consume a large amount of glucose and produce ATP and substrates required for cell growth and division through glycolysis as the main pathway, and a large amount of lactic acid is mainly discharged through the MCT4 / CD147 complex. These lactic acids not only change the tumor tissue microenvironment, but also can be absorbed and utilized by adjacent cells through the MCT1 / CD147 complex (see Figure 1 ). The antibody or antigen-binding fragment thereof provided in the present application has high affinity for MCT1 / CD147, can inhibit the transport of substrates by the MCT1 or MCT4 / CD147 complex, and can inhibit the growth of MCT1 / CD147 and MCT4 / CD147 highly-expressed tumors. By analyzing the three-dimensional atomic structure of the antibody and CD147, the present application further provides the corresponding epitope and paratope of CD147.

[0053] The present application is further illustrated by the following examples, but the scope of the present application is not limited thereto.

[0054] Unless otherwise indicated, the experimental methods, detection methods, preparation methods disclosed in the present application all employ conventional techniques in the fields of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology and related fields. These techniques are well described in the existing literature, see, for example, Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001; Ausubel et al. CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987 and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS IN ENZYMOLOGY, Vol. 304, Chromatin (P. M. Wassarman and A. P. Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol. 119, Chromatin Protocols (P. B. Becker, ed.) Humana Press, Totowa, 1999, etc.

[0055] Example 1

[0056] Screening of hybridoma cells

[0057] Purification of mouse MCT1 / CD147 complex: Compared with prokaryotic protein expression, eukaryotic protein expression has more complex regulation, such as removal of signal peptide, post-transcriptional modification, etc. In order to obtain active MCT1 / CD147 complex, the purpose protein was expressed by baculovirus expression vector system which has been maturely applied. (Preparation method refers to Toshimitsu, Kawate, and, Eric, & Gouaux. (2006). Fluorescence-detection size-exclusion chromatography for precrystallization screening of integral membrane proteins. Structure, 14(4), 673-681.)

[0058] The purified mouse MCT1 / CD147 complex was injected into the abdominal cavity of mice, and the supernatant of the polyclonal hybridoma cell strain with high affinity to the complex was screened by Elisa detection. 20 monoclonal hybridoma cell strains were obtained by sorting the cell strain, and 5 monoclonal hybridoma cell strains with the highest affinity to the complex were obtained by Elisa detection screening.

[0059] Culture of hybridoma cells

[0060] The monoclonal hybridoma cells screened were resuscitated in high glucose DMEM (Life) culture medium containing 10% serum (Gibco) and 1% glutaMA (Life), and the cell concentration was not higher than 5×10 5 individuals / mL, and the liquid was changed after 12 hours. When the cells were about to cover the bottom of the T25 flask, they needed to be passaged. When the cells were passaged to the fifth generation, they were diluted to 5×10 4 individuals / mL and divided into T75 flasks. After 21 days of culture at 37 degrees and 5% CO2, the supernatant was collected. During the culture, sufficient CO2 supply was ensured, and high glucose medium was prone to contamination, so sterile operation was paid attention to.

[0061] Purification of monoclonal antibodies

[0062] The collected hybridoma cell supernatant was centrifuged at 8000 rpm for 40 minutes, and the precipitate was removed. 20 uL of clear and transparent supernatant was taken for western blotting experiment to estimate the total amount of antibodies in the collected supernatant. According to the method, 1 mL of dry volume of protein A beads (1 mL of protein A beads can theoretically bind 10 mg of antibodies) was added per 5 mg of antibodies, and 1 / 10 volume of 200 mM Hepes pH 7.0, 1500 mM NaCl solution was added, and the mixture was incubated at 4 degrees for 24 hours.

[0063] Collect the protein A beads after incubation in a gravity column, rinse with 20 column volumes of 20mM Hepes pH 7.0, 150mM NaCl solution at low speed, then elute with 5 column volumes of 0.1M Glycin pH 3.0 solution in total. The elution solution should be mixed well when added, and 1 / 10 volume of 1M Tris pH 8.5 should be added immediately after the elution solution flows out.

[0064] The collected elution solution should be left on ice overnight. After overnight, centrifuge at 8000rpm for 20 minutes to remove the precipitate. The purity of the antibody can be tested by SDS PAGE (loading buffer does not contain reducing agents such as β-mercaptoethanol). Generally, a single band is observed on SDS-PAGE. The obtained antibody solution is concentrated and replaced in PBS buffer.

[0065] Obtaining of Fab fragments

[0066] Take 10mg of the obtained relatively pure monoclonal antibody, and add papain at a mass ratio of 1:100. Add 5ml of 40mM EDTA Ph 8.0, 40mM L-cysteine solution, and dilute with PBS to a final antibody concentration of 1mg / mL. Incubate in a 37°C incubator at 60rpm for 5 hours.

[0067] Add 1mL of 1M iodoacetamide to the solution after incubation, mix well, then add 2mL of protein A beads and incubate at room temperature for 2 hours. Collect and concentrate the flow-through, and collect 0.5mL of the eluate from the size exclusion chromatography column. Identify the eluate by SDS-PAGE. The high-purity Fab solution is quickly frozen with liquid nitrogen and stored at -80°C.

[0068] V region sequencing and humanization

[0069] Take 1x10 6The V region sequencing was performed by CRO commercial company and the antibody affinity maturation and humanization were performed by phage display technology. The MCT1 / CD147 used in phage display was provided by the experiment. The VL sequence obtained by antibody affinity maturation by phage display technology was integrated into the pFuse2ss-CLIg plasmid containing human constant region CL, and the VH sequence was integrated into the pFuse2ss-CHIg plasmid containing human constant region CH. Then the plasmid was large-scale extracted and transiently transfected into 293F cells. After one week, the supernatant was collected and enriched. The collected supernatant was buffer exchanged with 20mM Hepes pH7.0, 150mM NaCl solution. Then the humanized HAbLJ001(6E5F4) Fab fragment was obtained according to the above antibody purification method (protein A was replaced by protein L). The variable region sequence of the humanized monoclonal antibody obtained is shown in Table 1 above.

[0070] Example 2

[0071] Research on spatial information of antigen-antibody binding site

[0072] The mouse MCT1 / CD147 complex was wrapped in Nanodisc formed by POPE:POPC, and Fab was added at a molar ratio of 1:1.3 and incubated for 1 hour. After molecular exclusion chromatography, the MCT1 / CD147-Fab component was collected, the protein concentration was 0.5mg / mL, and the sample was prepared by freezing. The sample with good thin particle uniformity in ice layer was selected for data collection. The three-dimensional structure of the antigen and the antibody obtained by software analysis of the collected data is shown in Figure 2 , in which the upper blue area represents the extracellular IgG2 domain of CD147, and the lower four areas represent the Fab fragments of the antibody HAbLJ001(6E5F4). The two-dimensional structure of the interaction between the antigen and the antibody is shown in Figure 3 , in which the amino acid residues on the human CD147(A) are above the dotted line, and the amino acid residues of the heavy chain(H) and the light chain(L) of the antibody HAbLJ001(6E5F4) are below the dotted line.

[0073] Example 3

[0074] Verification of in vitro activity of antibody

[0075] 1. Preparation of mixed lipids

[0076] Take 0.6 mL POPC phospholipid (25 mg / mL), 0.2 mL POPE phospholipid (25 mg / mL), 0.2 mL POPG phospholipid (25 mg / mL), weigh 5.0 mg cholesterol into the phospholipid solution, after complete mixing, use a rotary evaporator to spin dry at low speed. While spinning, make sure the nitrogen flow rate is appropriate, too fast nitrogen flow will cause the lipid solution to splash; too small gas flow, the lipid mixture is not uniform, may remain more chloroform solvent. After blowing dry mixed fat, use a high-pressure freezer to pump overnight. A small amount of mixed fat is added to the prepared Tris pH 8.5 solution (20 mM Tris pH 8.5, 150 mM NaCl, mass ratio 5.76% OM) several times, water bath ultrasonic 15 minutes, ice water bath 15 minutes, until the lipid solution is transparent, the final concentration is 12.5 mg / mL. 200 uL per branch, frozen at -80℃.

[0077] 2. Preparation of artificial liposomes

[0078] Add 0.1 mg of mouse MCT1 / CD147 complex to the above 200 ul liposome mixture, supplement 20 mM Hepes pH 8.5, 150 mM NaCl solution to a total volume of 625 uL. After 4 hours of low-speed mixing at 4℃, add 90 mg / mL Bio-Beads SM-2 to the above mixture and mix at 4℃ for 4 hours. After 4 hours, replace the new Bio-Beads SM-2 at a concentration of 120 mg / mL and mix overnight to remove the detergents DDM and OM. Empty liposomes do not add mouse MCT1 / CD147 complex. The above mixture is placed on ice, and the supernatant is carefully aspirated, the supernatant volume is measured, and 100 uM HPTS proton fluorescence indicator is added. After ice bath ultrasonic for 10 minutes, stand for half an hour, promote HPTS into the liposome. Then use a 0.2 um porous filter membrane to prepare uniform size liposomes in an Avanti liposome preparation device. Liposomes pass through the membrane 21 times.

[0079] HPTS is a water-soluble, membrane-impermeable pH indicator. Since MCT1 is a proton-coupled monocarboxylate transporter, the transport activity of MCT1 / CD147 complex to the substrate can be characterized by the change of intramembrane pH.

[0080] The mixed liposome solution was loaded into a desalting column PD10 (GE-Healthcare) which had been equilibrated with 20 mM Hepes pH 8.5, 150 mM NaCl solution. Then 20 mM Hepes pH 8.5, 150 mM NaCl solution was slowly added, 0.4 mL of effluent was collected in a tube, and the concentration was measured. The highest concentration of liposome solution was taken, and diluted to 2 mg / mL. The artificial liposome of this subject needs to be made on demand, and the liposome stored in the freezer has low transport activity.

[0081] 10 uL of liposome was taken, 90 uL of extracellular solution was added, and a pH gradient was established to test the transport activity of the mouse MCT1 / CD147 complex. After 10 minutes of standing, the liposome solution was measured every 15 seconds by a microplate reader (Molecular Device) at an emission wavelength of 511 nm (excitation wavelength of 450 nm) Fs. After two minutes of measurement, 5 uL of transport substrate (20 mM Hepes pH 7.0, 150 mM NaCl, 0.5 M Sodium Pyruvate) was added to the liposome, and the emission signal F was measured every 15 seconds. Generally, after 5 minutes, the emission signal was stable. After 15 minutes, the liposome was ultrasonically broken, and the emission signal Fd of the sample was recorded again. Each measurement value was normalized as F normalized = (F - Fd) / (Fs - Fd).

[0082] 3. Verification of the in vitro activity of the antibody

[0083] The empty liposome was taken as a negative control (empty liposome, none in the figure), and the MCT1 / CD147-containing liposome was also taken and divided into two parts, one part was added with PBS solution (mMCT1mCD147), and the other part was added with monoclonal antibody HAbLJ001 (6E5F4) (mMCT1mCD147_6E5F4) at a final concentration of 0.36 mg / mL and incubated for half an hour. Then the change in fluorescence intensity was measured according to the above method, and the results are shown in Figure 4 The D-value corresponding to the reaction endpoint (time 520 s) was calculated, and the results are shown in Figure 5

[0084] From the results of Figure 4 and Figure 5 , it can be found that compared with the MCT1 / CD147 complex liposome, the change in fluorescence intensity is smaller after the addition of monoclonal antibody HAbLJ001 (6E5F4). The smaller change in fluorescence intensity indicates that less substrate is transported into the liposome. This indicates that monoclonal antibody HAbLJ001 (6E5F4) can inhibit the substrate transport activity of the MCT1 / CD147 complex.​

[0085] Example 4

[0086] Experimental animals and methods

[0087] Animal experiments were conducted in accordance with the ethical guidelines and were approved by the Ethics Committee of the Affiliated Tumor Hospital of Guangxi Medical University. SPF-grade BALB / c nude mice were purchased from the Experimental Animal Center of Guangxi Medical University, and the mice were 4-6 weeks old. Lung cancer A549 cells were cultured in RPMI-1640 complete medium (10% fetal bovine serum, 100 U / ml penicillin and 100 μg / ml streptomycin). 100 μL of A549 cell suspension (1×10 6 cells) was subcutaneously injected into the flank area of the nude mice. After the cells formed tumors, the mouse was injected intraperitoneally with the mouse anti-CD147 antibody (HAbLJ001 (6E5F4), 5 mg / kg), and the same antibody subtype IgG was set as the control group. The growth of the tumor was measured every other day with a vernier caliper, and the volume of the tumor was calculated as length x width x width / 2. The body weight of the nude mice was also measured, and the experiment was stopped and the mice were disposed when the tumor volume reached 1,500 mm 3 Figure 6

[0088] From the change in body weight, the control group and the anti-CD147 antibody group had no significant effect on the change in body weight of the mice, indicating that the anti-CD147 antibody had no obvious side effects. From the tumor growth, the anti-CD147 antibody group could inhibit the growth of A549 tumors, indicating that the blocking of cell surface CD147 with specific anti-CD147 antibody could have an inhibitory effect on the proliferation of lung adenocarcinoma cell lines.

[0089] In summary, the present application effectively overcomes the shortcomings in the prior art and has a high industrial utilization value.

[0090] The above examples are only illustrative of the principles and effects of the present application, and are not intended to limit the present application. Any person skilled in the art can modify or change the above examples without departing from the spirit and scope of the present application. Therefore, all equivalent modifications or changes made by those skilled in the art without departing from the spirit and technical thought disclosed by the present application should be covered by the claims of the present application. Sequence listing <110> Shanghai University of Science and Technology <120> An antibody or antigen-binding fragment thereof and preparation method and use thereof <160> 16 <170> SIPOSequenceListing 1.0 <210> 1 <211> 8​​ <212> PRT <213> Artificial Sequence <400> 1 Gly Tyr Thr Phe Thr Asn Tyr Trp 1 5 <210> 2 <211> 8 <212> PRT <213> Artificial Sequence <400> 2 Ile His Pro Asn Ser Gly Thr Ile 1 5 <210> 3 <211> 10 <212> PRT <213> Artificial Sequence <400> 3 Ala Arg Val Gly Thr Gly Ser Leu Asp Tyr 1 5 10 <210> 4 <211> 25 <212> PRT <213> Artificial Sequence <400> 4 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Asn Leu Ser Cys Lys Ala Ser 20 25 <210> 5 <211> 17 <212> PRT <213> Artificial Sequence <400> 5 Ile His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly 1 5 10 15 Met <210> 6 <211> 38 <212> PRT <213> Artificial Sequence <400> 6 Asn Tyr Asn Glu Lys Phe Lys Thr Lys Ala Thr Leu Thr Val Asp Lys 1 5 10 15 Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp 20 25 30 Ser Ala Val Tyr Tyr Cys 35 <210> 7 <211> 11 <212> PRT <213> Artificial Sequence <400> 7 Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser 1 5 10 <210> 8 <211> 6 <212> PRT <213> Artificial Sequence <400> 8 Gln Ser Ile Ser Asp Tyr 1 5 <210> 9 <211> 3 <212> PRT <213> Artificial Sequence <400> 9 Tyr Val Ser 1 <210> 10 <211> 9 <212> PRT <213> Artificial Sequence <400> 10 Gln Asn Gly His Arg Phe Pro Tyr Thr 1 5 <210> 11 <211> 26 <212> PRT <213> Artificial Sequence <400> 11 Asp Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Thr Pro Gly 1 5 10 15 Asp Arg Val Ser Leu Ser Cys Arg Ala Ser 20 25 <210> 12 <211> 17 <212> PRT <213> Artificial Sequence <400> 12 Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile 1 5 10 15 Lys <210> 13 <211> 36 <212> PRT <213> Artificial Sequence <400> 13 Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly 1 5 10 15 Ser Tyr Phe Thr Leu Ser Ile Asp Ser Val Glu Pro Glu Asp Val Gly 20 25 30 Val Tyr Tyr Cys 35 <210> 14 <211> 10 <212> PRT <213> Artificial Sequence (Artificial Sequence) <400> 14 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 1 5 10 <210> 15 <211> 117 <212> PRT <213> Artificial Sequence (Artificial Sequence) <400> 15 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Asn Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Trp Ile His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Met Ile His Pro Asn Ser Gly Thr Ile Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Thr Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gin Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Val Gly Thr Gly Ser Leu Asp Tyr Trp Gly Gin Gly Thr Ser 100 105 110 Val Thr Val Ser Ser 115 <210> 16 <211> 107 <212> PRT <213> Artificial Sequence (Artificial Sequence) <400> 16 Asp He Val Met Thr Gin Ser Pro Ala Thr Leu Ser Val Thr Pro Gly 1 5 10 15 Asp Arg Val Ser Leu Ser Cys Arg Ala Ser Gin Ser He Ser Asp Tyr 20 25 30 Leu His Trp Tyr Gin Gin Lys Ser His Glu Ser Pro Arg Leu Leu He 35 40 45 Lys Tyr Val Ser Gin Ser He Ser Gly He Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Ser Tyr Phe Thr Leu Ser He Asp Ser Val Glu Pro 65 70 75 80 Glu Asp Val Gly Val Tyr Tyr Cys Gin Asn Gly His Arg Phe Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105

Claims

1. An antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment thereof being capable of binding to CD147, wherein, The binding antigenic epitopes of the antibody or its antigen-binding fragment include Ala149, Asp136 or glycosylation of Asp136, Tyr140 or glycosylation of Tyr140, Gln195 or glycosylation of Gln195, Asp194 or glycosylation of Asp194, Pro104, Asp147 or glycosylation of Asp147, Ser193 or glycosylation of Ser193, Lys191 or glycosylation of Lys191, and Gly192; The antibody or its antigen-binding fragment includes a heavy chain variable region and a light chain variable region. The heavy chain variable region includes amino acid sequences CDR-H1 as shown in SEQ ID No. 1, CDR-H2 as shown in SEQ ID No. 2, and CDR-H3 as shown in SEQ ID No.

3. The light chain variable region includes amino acid sequences CDR-L1 as shown in SEQ ID No. 8, CDR-L2 as shown in SEQ ID No. 9, and CDR-L3 as shown in SEQ ID No.

10.

2. The antibody or its antigen-binding fragment as described in claim 1, characterized in that, The antibody or its antigen-binding fragment inhibits CD147 binding to MCT1 and / or MCT4; And / or, the antibody or its antigen-binding fragment inhibits the expression and / or function of the MCT1 / CD147 complex and / or the MCT4 / CD147 complex.

3. The antibody or its antigen-binding fragment as described in claim 1, characterized in that, The antibody or its antigen-binding fragment can specifically bind to CD147; And / or, the CD147 is a human CD147; And / or, the antibody or its antigen-binding fragment is a monoclonal antibody.

4. The antibody or its antigen-binding fragment as described in claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the antibody or its antigen-binding fragment includes: a) The amino acid sequence as shown in SEQ ID No. 15; or, b) An amino acid sequence that has more than 80% sequence identity with the amino acid sequence shown in SEQ ID No. 15 and has the function of the amino acid sequence defined in a).

5. The antibody or its antigen-binding fragment as described in claim 1, characterized in that, The amino acid sequence of the light chain variable region of the antibody or its antigen-binding fragment includes: c) The amino acid sequence as shown in SEQ ID No. 16; or, d) An amino acid sequence that has more than 80% sequence identity with the amino acid sequence shown in SEQ ID No. 16 and has the function of the amino acid sequence defined in c).

6. The antibody or its antigen-binding fragment as described in claim 1, characterized in that, The antibody or its antigen-binding fragment is derived from mice; And / or, the antibody or its antigen-binding fragment is humanized.

7. An isolated polynucleotide encoding an antibody or an antigen-binding fragment thereof as claimed in any one of claims 1-6.

8. A construct comprising the isolated polynucleotide as described in claim 7.

9. An antibody expression system comprising an exogenous polynucleotide as described in claim 7 integrated into a construct or genome as described in claim 8.

10. A method for preparing the antibody or its antigen-binding fragment according to any one of claims 1-6, comprising the following steps: culturing the expression system of the antibody according to claim 9 under suitable conditions for expressing the antibody, thereby expressing the antibody or its antigen-binding fragment, and purifying and separating the antibody or its antigen-binding fragment.

11. Use of the antibody or antigen-binding fragment thereof as claimed in any one of claims 1-6, or the expression system of the antibody as claimed in claim 9, in the preparation of an antitumor drug, wherein the tumor is selected from lung cancer.

12. A pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof as claimed in any one of claims 1-6, or an expression system for an antibody as claimed in claim 9.

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