Antibodies against MIC A / B and their uses

Abstract

The present disclosure relates to an antibody against MIC A / B and its uses. Specifically, there is provided an antibody against MIC A / B or an antigen-binding fragment thereof, which comprises a heavy-chain variable region and a light-chain variable region. Among them, the heavy-chain variable region comprises: HCDR1 having the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 13 or any variant thereof, HCDR2 having the amino acid sequence shown in SEQ ID NO: 2 or any variant thereof, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 14 or any variant thereof; the light-chain variable region comprises: LCDR1 having the amino acid sequence shown in SEQ ID NO: 4 or any variant thereof, LCDR2 having the amino acid sequence shown in SEQ ID NO: 5 or any variant thereof, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 6 or any variant thereof. The antibody against MIC A / B of the present disclosure can not only bind to full-length MICA and MICB, but also bind to the α3 domain of MICA and MICB, and has good tumor killing effects both in vitro and in vivo.

Description

Technical Field

[0001] The present disclosure belongs to the field of biomedicine, and in particular, relates to an antibody against MIC A / B and its uses. Background Art

[0002] Colon cancer (CC) is one of the most common malignant tumors of the digestive tract. Among the types of cancer that cause human death, colon cancer ranks fourth. At the same time, there is no significant difference in the incidence and mortality rates between men and women, and the overall mortality rate is about 50%. The development of colon cancer mainly goes through three stages: tumor - nodule - metastasis, and the prognosis of patients mainly depends on the stage of development at the time of diagnosis and the possibility of surgically removing the tumor radically. The molecular mechanism of colon cancer occurrence is complex and cumbersome, such as defects in tumor suppressor gene SMAD4 and DNA mismatch repair genes. Although various methods for early screening of colon cancer have been developed currently, including endoscopic methods and fecal occult blood tests, etc., the endoscopic method has a risk of inducing patient contraindications, and the sensitivity of the fecal occult blood test is easily affected by the sample processing method. For the treatment of colon cancer, the widely used means are surgical resection, radiotherapy, and chemotherapy, but these treatment methods all have limitations to a certain extent: surgical resection can only be performed on some patients, and radiotherapy and chemotherapy have relatively large side effects. Therefore, there is an urgent need to find new strategies for treating colon cancer.

[0003] The Natural Killer Group 2D receptor (NKG2D receptor) is a type II transmembrane glycoprotein expressed on the surface of natural killer cells (NK), natural killer T cells (NKT), and T cells. The ligands of NKG2D include major histocompatibility complex class I - related chain A and B (MICA / B) and UL16 - binding proteins (ULBPs). The interaction between NKG2D and its ligands can activate antigen - specific cytotoxic T lymphocyte - mediated cytotoxicity, NK cell responses, and cytokine production, and play an important role in cancer treatment by recognizing ligands on the surface of tumor cells and mediating NK cell killing of tumor cells. MICA and MICB are highly homologous type I transmembrane proteins, having two extracellular domains, α1 and α2, that interact with NKG2D, and an α3 domain located near the transmembrane domain. The expression level of NKG2D ligands in healthy human tissues is relatively low. Therefore, the targeting effect based on NKG2D can be directed only against tumor tissues without affecting surrounding non - tumor tissues.

[0004] In tumor immune escape strategies, the expression of MIC A / B on the cell surface is downregulated, and under the action of enzymes such as endoplasmic reticulum protein 5 (ERp5) and matrix metalloproteinases (MMPs), MIC A / B is hydrolyzed from the surface of tumor cells including CC cells, resulting in cancer cells evading immune surveillance. Specific antibodies targeting the α3 domain of MIC A / B can inhibit the cleavage of MICA / B from the surface of tumor cells, and the MICA immune complex formed with α3-specific antibodies mediates the killing of tumor cells by NK cells, indicating that MICA / B may be a potential therapeutic target for cancer.

[0005] Therefore, there is an urgent need to develop a new MIC A / B antibody that can specifically bind to both full-length MICA and MICB and also bind to its α3 domain, providing a new idea and new therapy for tumor immunotherapy. Summary of the Invention

[0006] To solve the problems existing in the prior art, the purpose of the present disclosure is to provide an antibody against MIC A / B or its antigen-binding fragment and its pharmaceutical use.

[0007] To solve the above technical problems, the present disclosure proposes the following technical solutions:

[0008] On the one hand, the present disclosure provides an antibody against MIC A / B or its antigen-binding fragment, which comprises a heavy-chain variable region and a light-chain variable region, wherein,

[0009] The heavy-chain variable region comprises: HCDR1 having the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:13 or any variant thereof, HCDR2 having the amino acid sequence shown in SEQ ID NO:2 or any variant thereof, HCDR3 having the amino acid sequence shown in SEQ ID NO:3 or SEQ ID NO:14 or any variant thereof;

[0010] The light-chain variable region comprises: LCDR1 having the amino acid sequence shown in SEQ ID NO:4 or any variant thereof, LCDR2 having the amino acid sequence shown in SEQ ID NO:5 or any variant thereof, LCDR3 having the amino acid sequence shown in SEQ ID NO:6 or any variant thereof.

[0011] On the other hand, the present disclosure provides a polynucleotide encoding the aforementioned antibody or its antigen-binding fragment.

[0012] On the other hand, the present disclosure provides an expression vector containing the aforementioned polynucleotide.

[0013] On the other hand, the present disclosure provides a host cell into which or which contains the aforementioned polynucleotide or the aforementioned expression vector.

[0014] On the other hand, the present disclosure provides a pharmaceutical composition comprising the aforementioned antibody or its antigen-binding fragment, and a pharmaceutically acceptable excipient, diluent or carrier;

[0015] Preferably, the pharmaceutical composition further comprises a second therapeutic agent;

[0016] Preferably, the therapeutic agent is selected from antibodies, chemotherapeutic agents and small molecule drugs.

[0017] On the other hand, the present disclosure provides a conjugate comprising the aforementioned antibody or its antigen-binding fragment, and a chemical moiety conjugated thereto;

[0018] Preferably, the chemical moiety is selected from therapeutic agents, detectable moieties and immunostimulatory molecules.

[0019] On the other hand, the present disclosure provides a kit for detecting or quantifying MIC A / B in a clinical or biological sample, comprising the aforementioned antibody or its antigen-binding fragment or the aforementioned conjugate.

[0020] On the other hand, the present disclosure provides the use of the aforementioned antibody or its antigen-binding fragment, the aforementioned polynucleotide, the aforementioned expression vector, the aforementioned host cell, the aforementioned pharmaceutical composition, the aforementioned conjugate and / or the aforementioned kit in the preparation of a drug for preventing and / or treating MIC A / B-mediated diseases;

[0021] Preferably, the MIC A / B-mediated diseases are cancer, autoimmune diseases, infectious diseases or transplant rejection;

[0022] Preferably, the cancer is at least one of colon cancer, adrenocortical carcinoma, anal cancer, bladder cancer, brain tumor, breast cancer, carcinoid tumor, cancer of unknown primary origin in the gastrointestinal tract, cervical cancer, endometrial cancer, esophageal cancer, extrahepatic bile duct cancer, extracranial germ cell tumor, intraocular melanoma eye cancer, gallbladder cancer, gastric cancer, external germ cell tumor, gestational trophoblastic tumor, head and neck cancer, hypopharyngeal cancer, islet cell cancer, kidney cancer, laryngeal cancer, oral cancer, liver cancer, lung cancer, AIDS-related lymphoma, central nervous system lymphoma, cutaneous T-cell lymphoma, Hodgkin lymphoma, malignant mesothelioma, melanoma, Merkel cell carcinoma, multiple myeloma, plasmacytoma, nasopharyngeal cancer, osteoblastoma, oropharyngeal cancer, osteosarcoma, ovarian epithelial cancer, ovarian germ cell tumor, pancreatic cancer, exocrine pancreatic cancer, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pituitary cancer, prostate cancer, rhabdomyosarcoma, rectal cancer, transitional cell carcinoma of the renal pelvis and urethra, salivary gland cancer, skin cancer, small intestine cancer, soft tissue sarcoma, testicular cancer, malignant thymoma, thyroid cancer, uterine cancer.

[0023] Compared with the prior art, the present disclosure has at least the following beneficial effects:

[0024] The present disclosure prepared monoclonal antibody RDM028 by immunizing mice, and further obtained chimeric antibody RDM028hG1 and humanized antibody RDH028 through modification. Experimental results prove that the anti-MIC A / B antibodies of the present disclosure can not only bind to full-length MICA and MICB, but also bind to the α3 domain of MICA and MICB. At the same time, monoclonal antibody RDM028 has a high binding affinity for the α3 domain of MICA and MICB, and has good tumor killing effects both in vitro and in vivo. The variable region gene thereof was recombined with the constant region gene of human antibody to obtain the modified chimeric antibody RDM028hG1, which also has a relatively high binding affinity. In addition, after humanizing the antibody, it still has good affinity. Compared with the antibodies disclosed in the prior art, it has stronger affinity, and it can be foreseen that it will have better potential physiological effects in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 A design diagram showing the expression structure of MIC protein is shown.

[0026] Figure 2 Flow cytometry for sorting antigen-specific memory B cells is shown. The upper right quadrant is sorted by flow cytometry with MIC A / B protein labeled with bv421 (y-axis) and pe-cy7 (x-axis).

[0027] Figure 3 A and Figure 3 B respectively show the comparison results of the binding affinity detection of RDM028 and RDM028hG1 antibodies with MICA / B.

[0028] Figure 4A and Figure 4B respectively show the affinity detection results of RDM028 with MICA and MICB proteins.

[0029] Figure 5 The affinity detection results of RDM028hG1 antibody with MICA and its α3 domain detected by SPR are shown.

[0030] Figure 6 The affinity detection results of RDM028hG1 antibody with MICB and its α3 domain detected by SPR are shown.

[0031] Figure 7 Flow cytometry for detecting the inhibition of MICA / MICB protein shedding by RDM028 and RDM028hG1 antibodies is shown.

[0032] Figure 8Shows the detection of RDM028 and the inhibition of MICA / MICB protein shedding by the RDM028hG1 antibody using the ELISA method.

[0033] Figure 9 Shows the cytotoxicity assay for detecting the killing of colon cancer cells by RDM028 and the RDM028hG1 antibody-mediated NK cells. Data are the mean + / − SD of three replicates, and p-values were generated by unpaired t-tests, *p < 0.05, **p < 0.01.

[0034] Figure 10 Shows the experimental results of the inhibition of HCT-116 human colon cancer cell growth by the RDM028hG1 antibody in mice. Among them, Figure 10 A shows the tumor size of the mice, Figure 10 B is a schematic diagram of the dissection of the mice tumors after the treatment, Figure 10 C shows the tumor weights of the mice in the experimental group and the control group, Figure 10 D shows the body weight changes of the mice during the treatment.

[0035] Figure 11 Shows the detection results of the affinity of the RDH028 antibody with MICA and MICB proteins.

[0036] Figure 12 Shows the detection results of the affinity of anti-MICA / B antibodies in the prior art with MICA protein respectively.

[0037] Figure 13 Shows the detection results of the affinity of anti-MICA / B antibodies in the prior art with MICB protein respectively. Detailed implementation manners

[0038] To more clearly illustrate the technical solutions in the embodiments of the present disclosure or the prior art, the following will briefly introduce the terms and implementation manners used in the description of the embodiments or the prior art. Obviously, the following described implementation manners are only one implementation manner of the present disclosure. For those of ordinary skill in the art, other implementation manners can also be obtained based on these drawings.

[0039] I. Terms

[0040] To make it easier to understand the present disclosure, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present disclosure pertains.

[0041] The articles "a" and "an" used herein refer to one or more (i.e., at least one) grammatical objects referred to by the article. For example, "an element" means one element or more than one element.

[0042] As used herein, the term "about" means and encompasses a specified value and ranges that are greater than and less than that value. In certain embodiments, the term "about" can mean a variation of ±0.1%, ±0.5%, ±1%, ±2%, ±3%, ±4%, ±5%, ±6%, ±7%, ±8%, ±9%, or ±10%. In certain embodiments, where applicable, the term "about" means the specified value ± one standard deviation of that value.

[0043] The endpoints of the ranges and any values disclosed herein are not limited to the exact ranges or values, and these ranges or values should be understood to include values that are close to those ranges or values. For numerical ranges, the endpoints of the various ranges, between the endpoints of the individual ranges, between the endpoints of the individual ranges and the individual point values, and between the individual point values can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be considered to be specifically disclosed herein.

[0044] The term "consisting essentially of" or variations thereof used throughout the specification and claims means including all of the recited elements or groups of elements, and optionally including other elements of a similar or different nature, which do not significantly alter the basic or novel properties of the specified dosage regimen, method, or composition.

[0045] "Optionally" or "optionally" means that the subsequently described event or circumstance may but need not occur, and this description includes the instances where the event or circumstance occurs or does not occur.

[0046] The term "antibody" refers to any form of antibody that exhibits the desired biological or binding activity. Thus, it is used in the broadest sense and specifically includes, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, humanized, fully human antibodies, and chimeric antibodies.

[0047] Typically, the basic antibody structural unit comprises a tetramer. Each tetramer includes two pairs of identical polypeptide chains, each pair having one "light" chain (about 25 kDa) and one "heavy" chain (about 50 - 70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids that is primarily responsible for antigen recognition. The carboxyl-terminal portion of the heavy chain may define a constant region that is primarily responsible for effector functions. Typically, human light chains are divided into kappa light chains and lambda light chains. In addition, human heavy chains are generally divided into mu, delta, gamma, alpha, or epsilon, and the isotypes of antibodies are defined as IgM, IgD, IgG, IgA, and IgE, respectively. In the light and heavy chains, the variable region and the constant region are joined by a "J" region of about 12 or more amino acids, and the heavy chain also includes a "D" region of about 10 or more amino acids. See generally Fundamental Immunology Ch.7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)).

[0048] The variable regions of each light / heavy chain pair form the antibody binding site. Thus, generally speaking, a complete antibody has two binding sites. Except for bifunctional or bispecific antibodies, these two binding sites are usually identical.

[0049] Typically, both the variable regions of the heavy and light chains contain three hypervariable regions, also called complementarity-determining regions, located within relatively conserved framework regions. The CDRs are typically aligned by the framework regions so as to enable binding to a specific epitope. Generally speaking, from the N-terminus to the C-terminus, the variable domains of the light and heavy chains both include FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The amino acid assignment to each domain generally conforms to the following definitions: Sequences of Proteins of Immunological Interest, Kabat, et al.; National Institutes of Health, Bethesda, Md.; 5th ed.; NIH Publ. No. 91 - 3242 (1991); Kabat (1978) Adv. Prot. Chem. 32:1 - 75; Kabat, et al., (1977) J. Biol. Chem. 252:6609 - 6616; Chothia, et al., (1987) J Mol. Biol. 196:901 - 917 or Chothia, et al., (1989) Nature 342:878 - 883.

[0050] As used herein, "antibody fragment" or "antigen-binding fragment" refers to an antigen-binding fragment of an antibody, i.e., an antibody fragment that retains the ability to specifically bind an antigen that the full-length antibody binds to, such as a fragment that retains one or more CDR regions. Examples of antibody-binding fragments include, but are not limited to, Fab, Fab’, F(ab’)2, and Fv fragments.

[0051] As used herein, "CDR" or "CDR region" refers to the complementarity-determining region in the variable region of an immunoglobulin, typically defined using the Kabat numbering system.

[0052] As used herein, an antibody that "specifically binds" to a particular target protein is an antibody that exhibits preferential binding to that target compared to other proteins, although this specificity does not require absolute binding specificity. The binding of an antibody determines the presence of the target protein in a sample. For example, if it specifically binds to the target protein in a sample without producing unwanted results such as false positives, the antibody is considered to be "specific" for its intended target. The antibodies or their binding fragments used in the present invention will bind to the target protein with an affinity that is at least two-fold higher, preferably at least ten-fold higher, more preferably at least 20-fold higher, and most preferably at least 100-fold higher than the affinity for non-target proteins. As used herein, if an antibody binds to a polypeptide containing a given amino acid sequence, such as the amino acid sequence of the mature human PD-1 or human PD-L1 molecule, but does not bind to a protein lacking that sequence, the antibody is said to specifically bind to the polypeptide containing that sequence.

[0053] As used herein, "chimeric antibody" refers to an antibody in which a portion of the heavy chain and / or light chain is identical or homologous to the corresponding sequence in an antibody derived from a particular species (e.g., human) or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to the corresponding sequence in an antibody derived from another species (e.g., mouse) or belonging to another antibody class or subclass, as well as fragments of such antibodies, provided that they exhibit the desired biological activity.

[0054] As used herein, "human antibody" refers to an antibody that contains only human immunoglobulin sequences. If produced in a mouse, mouse cell, or hybridoma derived from a mouse cell, a human antibody may contain murine sugar chains. Similarly, "mouse antibody" or "rat antibody" refers to an antibody that contains only mouse or rat immunoglobulin sequences, respectively.

[0055] As used herein, a "humanized antibody" refers to a form of an antibody that contains sequences from non-human (e.g., murine) antibodies as well as human antibodies. Such an antibody contains a minimal sequence derived from a non-human immunoglobulin. Generally, a humanized antibody will contain substantially all of at least one (usually two) variable regions, where all or substantially all of the hypervariable loops correspond to the hypervariable loops of a non-human immunoglobulin, and all or substantially all of the FR regions are the FR regions of a human immunoglobulin sequence. A humanized antibody may optionally also contain at least a portion of the immunoglobulin constant region (Fc), typically the constant region of a human immunoglobulin. When necessary, a prefix such as "hum", "hu", or "h" is added to the antibody clone name to distinguish the humanized antibody from the parental rodent antibody. The humanized form of a rodent antibody typically contains the same CDR sequences as the parental rodent antibody, although certain amino acid substitutions may be included to improve affinity, increase the stability of the humanized antibody, or for other reasons.

[0056] As used herein, a "fully humanized antibody" refers to an antibody in which human antibody genes are transferred entirely into a genetically engineered antibody gene-deficient animal through transgenic or transchromosomal techniques, enabling the animal to express human antibodies and achieving full humanization of the antibody.

[0057] As used herein, a "conservative modified variant" or "conservative substitution" refers to the replacement of an amino acid in a protein with another amino acid having similar characteristics (such as charge, side chain size, hydrophobicity / hydrophilicity, backbone conformation, and rigidity, etc.), such that the change can be made frequently without altering the biological activity or other desired properties of the protein (such as antigen affinity and / or specificity). Those skilled in the art recognize that, generally, a single amino acid substitution in a non-essential region of a polypeptide does not significantly alter biological activity (see Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224 (4th Ed.)). Additionally, substitutions of amino acids with similar structure or function are less likely to disrupt biological activity. Exemplary conservative substitutions are listed in Table 1.

[0058] Table 1. Exemplary conservative amino acid substitutions

[0059]

[0060]

[0061] As used herein, the terms "cell", "cell line", and "cell culture" are used interchangeably and all such names include their progeny. Thus, the words "transformant" and "transformed cell" include the primary subject cell and cultures derived therefrom, regardless of the number of transfers. It should also be understood that all progeny may not be precisely identical in DNA content due to either deliberate or inadvertent mutations. Mutant progeny that have the same function or biological activity as screened in the original transformed cells are included.

[0062] As used herein, the term "pharmaceutical composition" refers to a composition for administration to a mammalian patient (preferably a human patient). In a preferred embodiment, the pharmaceutical composition comprises a composition for parenteral injection or infusion. The parenteral injection or infusion can take advantage of the reabsorption process, in the form of intradermal, subcutaneous, intramuscular, and / or intraperitoneal injection or infusion. Alternatively, the parenteral injection or infusion can bypass the reabsorption process and be in the form of intracardiac, intraarterial, intravenous, intralumbar, and / or intramembranous injection or infusion. In another preferred embodiment, the pharmaceutical composition comprises a composition for transdermal administration. An example of transdermal administration is epidermic administration, where the pharmaceutical composition is administered in the form of, for example, a solution, suspension, emulsion, foam, ointment, paste, and / or patch applied to the skin. Alternatively, administration of the pharmaceutical composition can be achieved through one or more mucous membranes. For example, the administration can be buccal, lingual, or sublingual, i.e., through the mucous membranes of the mouth and / or tongue, and the application forms can be, for example, tablets, lozenges, troches (i.e., sugar-coated pills), and / or mouthwash solutions. Alternatively, the administration can be enteral, i.e., through the mucous membranes of the stomach and / or intestine, and the application forms can be, for example, tablets, troches (i.e., sugar-coated pills), capsules, solutions, suspensions, and / or emulsions. Alternatively, the administration can be rectal, and the application forms can be, for example, suppositories, rectal capsules, and / or ointments or pastes. Alternatively, the administration can be intranasal, and the application forms can be, for example, drops, ointments or pastes, and / or sprays. Alternatively, the administration can be pulmonary, i.e., through the bronchi and / or alveoli, and the application forms can be, for example, aerosols and / or inhalants. Alternatively, the administration can be conjunctival, and the application forms can be, for example, eye drops, eye ointments, and / or eye washes. Alternatively, the administration can be achieved through the mucous membranes of the urogenital tract, such as intravaginal or intraurethral, and the application forms can be, for example, suppositories, ointments, and / or medicated pencils. It should be understood that the alternative forms of administration described above are not mutually exclusive and can be combined in any number of them to constitute an effective treatment regimen.

[0063] The pharmaceutical composition of the present disclosure may further comprise a pharmaceutically acceptable carrier. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions (such as oil / water emulsions), various wetting agents, sterile solutions, etc. Compositions containing these carriers can be formulated by conventional methods well known in the art. These pharmaceutical compositions can be administered to a subject in suitable doses. The dosage regimen can be determined by the attending physician and clinical factors. As is well known in the art of medicine, the dosage for any patient depends on many factors, including the patient's size, body surface area, age, the particular compound being administered, sex, time and route of administration, general health, and other drugs being administered concurrently. For example, parenteral dosage forms include sterile water or non-aqueous solutions, suspensions, emulsions, and liposomes. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils (such as olive oil), and injectable organic esters (such as ethyl oleate). Carriers for intravenous or intraarterial administration include fluids and nutrient supplements, electrolyte supplements (such as those based on Ringer's dextrose), etc. Preservatives and other additives may also be included, for example, antimicrobial, antioxidant, chelating agents, inert gases, etc. Additionally, the pharmaceutical composition of the present invention may contain protein carriers, such as serum albumin or immunoglobulins, preferably of human origin. It is noted that, in addition to the humanized monoclonal antibody or its fragment (as described in the present invention), the pharmaceutical composition of the present invention may also contain other bioactive agents, depending on the intended use of the pharmaceutical composition. The agent can be a drug acting on the gastrointestinal system, a drug acting as a cytostatic agent, a drug preventing hyperuricemia, a drug inhibiting the immune response (such as corticosteroids), a drug regulating the inflammatory response, a drug acting on the circulatory system, and / or an agent known in the art such as cytokines.

[0064] As used herein, the terms “cancer,” “cancerous,” or “malignant” refer to or describe a physiological condition in a mammal that is typically characterized by uncontrolled cell growth. Examples of cancers include, but are not limited to, lymphoma, leukemia, blastoma, and sarcoma. More specific examples of such cancers include, but are not limited to, squamous cell carcinoma, myeloma, small cell lung cancer, non-small cell lung cancer, glioma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, acute myeloid leukemia (AML), multiple myeloma, gastrointestinal cancer, renal cancer, ovarian cancer, liver cancer, lymphoblastic leukemia, lymphocytic leukemia, colorectal cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, melanoma, chondroma, neuroblastoma, pancreatic cancer, glioblastoma multiforme, cervical cancer, brain cancer, gastric cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, and head and neck cancer.

[0065] As used herein, the positive therapeutic effects of cancer can be measured in a variety of ways (see W.A. Weber, J. Nucl. Med. 50:1S-10S (2009)). For example, with respect to tumor growth inhibition, according to the NCI criteria, T / C ≤ 42% is the minimum level of anti-tumor activity. T / C < 10% is considered a high level of anti-tumor activity, where T / C (%) = median treated tumor volume / median control tumor volume × 100. In some embodiments, the treatment achieved by a therapeutically effective amount is any one of progression-free survival (PFS), disease-free survival (DFS), or overall survival (OS). PFS, also known as "Time to Tumor Progression", refers to the length of time during and after treatment that the cancer does not grow, including the amount of time the patient experiences a complete response or partial response, as well as the amount of time the patient experiences stable disease. DFS refers to the length of time the patient remains disease-free during and after treatment. OS refers to an extended life expectancy compared to an untreated or un-treated individual or patient. Although embodiments of the treatment methods, compositions, and uses of the present invention may not effectively achieve positive therapeutic effects in every patient, this should be done in a statistically significant number of subjects determined by any statistical test known in the art (e.g., Student's t-test, chi2-test, U-test according to Mann and Whitney, Kruskal-Wallis test (H-test), Jonckheere-Terpstra test, and Wilcoxon test).

[0066] As used herein, a "therapeutically effective amount" (or "effective amount") refers to the amount of an active ingredient (e.g., a medicament of the present invention) that is sufficient to produce a beneficial or desired result when administered to a subject or patient. The effective amount can be administered in one or more administrations, applications, or doses. The therapeutically effective amount of the compositions of the present invention can be readily determined by one of ordinary skill in the art. In the context of the present invention, a "therapeutically effective amount" is an amount that produces an objectively measurable change in one or more parameters related to the treatment of a tumor (including clinical improvement of symptoms). Of course, depending on the particular subject and disease to be treated, the weight and age of the subject, the severity of the disease symptoms, the particular compound selected, the dosing regimen to be followed, the time of administration, the mode of administration, etc., the therapeutically effective amount will vary, all of which can be readily determined by one of ordinary skill in the art.

[0067] As used herein, the term "anti-tumor effect" refers to a biological effect that can manifest as any of the following: reduction in tumor volume, decrease in the number of tumor cells, decrease in the number of metastases, increase in life expectancy, or improvement in various physiological symptoms associated with cancer. The "anti-tumor effect" can also be manifested by the ability of the peptides, polynucleotides, antibodies (or antigen-binding portions thereof), and pharmaceutical compositions of the invention to prevent the emergence of tumors at a first location.

[0068] As used herein, "administering" and "treatment" when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids refer to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with an animal, human, subject, cell, tissue, organ, or biological body fluid. "Treatment" or "treating" cancer, as used herein, refers to administering an anti-antibody or antigen-binding fragment thereof to a subject having or diagnosed with cancer, alone or in combination with a second drug, to achieve at least one positive therapeutic effect, e.g., reducing the number of cancer cells, reducing tumor size, reducing the rate of tumor cell infiltration into surrounding organs, or reducing the rate of tumor metastasis or tumor growth. "Treatment" can include one or more of the following: inducing / increasing an anti-tumor immune response, reducing the number of one or more tumor markers, halting or delaying the growth of a tumor or blood cancer, extending the survival of a patient relative to the expected survival of a similar untreated patient, and inducing a complete or partial remission of cancer symptoms or other related diseases.

[0069] The term "patient" (also referred to herein as "subject" or "individual") refers to a mammal (e.g., rat, mouse, dog, cat, rabbit) that can be treated with the methods and compositions of the invention, most preferably a human. In some embodiments, the patient is an adult patient. In other embodiments, the patient is a pediatric patient.

[0070] II. DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS

[0071] In one aspect, the present disclosure provides an anti-MIC A / B antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein,

[0072] the heavy chain variable region comprises: HCDR1 having the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:13 or any variant thereof, HCDR2 having the amino acid sequence shown in SEQ ID NO:2 or any variant thereof, HCDR3 having the amino acid sequence shown in SEQ ID NO:3 or SEQ ID NO:14 or any variant thereof;

[0073] The light chain variable region comprises: LCDR1 having the amino acid sequence shown in SEQ ID NO:4 or any variant thereof, LCDR2 having the amino acid sequence shown in SEQ ID NO:5 or any variant thereof, and LCDR3 having the amino acid sequence shown in SEQ ID NO:6 or any variant thereof.

[0074] In some embodiments, the heavy chain variable region of the anti-MIC A / B antibody or its antigen-binding fragment comprises:

[0075] HCDR1 shown in SEQ ID NO:13, HCDR2 shown in SEQ ID NO:2, and HCDR3 shown in SEQ ID NO:14;

[0076] The light chain variable region comprises:

[0077] LCDR1 shown in SEQ ID NO:4, LCDR2 shown in SEQ ID NO:5, and LCDR3 shown in SEQ ID NO:6.

[0078] In some embodiments, the heavy chain variable region of the anti-MIC A / B antibody or its antigen-binding fragment comprises:

[0079] HCDR1 shown in SEQ ID NO:1, HCDR2 shown in SEQ ID NO:2, and HCDR3 shown in SEQ ID NO:3;

[0080] The light chain variable region comprises:

[0081] LCDR1 shown in SEQ ID NO:4, LCDR2 shown in SEQ ID NO:5, and LCDR3 shown in SEQ ID NO:6.

[0082] In some embodiments, the anti-MIC A / B antibody or its antigen-binding fragment, wherein,

[0083] The heavy chain variable region comprises HCDR1 shown in SEQ ID NO:1, HCDR2 shown in SEQ ID NO:2, and HCDR3 shown in SEQ ID NO:3; the light chain variable region comprises LCDR1 shown in SEQ ID NO:4, LCDR2 shown in SEQ ID NO:5, and LCDR3 shown in SEQ ID NO:6.

[0084] In some embodiments, the antibody or its antigen-binding fragment, wherein the antibody is selected from monoclonal antibody, single-chain antibody, chimeric antibody, humanized antibody or fully humanized antibody.

[0085] In some embodiments, the antigen-binding fragment is selected from Fab, Fab’, F(ab’)2, Fv fragment, scFv, di-scFv, VHH, and / or dAb.

[0086] In some embodiments, the anti-MIC A / B antibody or its antigen-binding fragment comprises a heavy chain variable region selected from the sequences shown below, or a heavy chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity compared to the following sequences: SEQ ID NO:7 or SEQ ID NO:11; and / or,

[0087] a light chain variable region selected from the sequences shown below, or a light chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity compared to the following sequences: SEQ ID NO:8, SEQ ID NO:10, or SEQ ID NO:12.

[0088] In some embodiments, the anti-MIC A / B antibody or its antigen-binding fragment comprises:

[0089] (1) the heavy chain variable region sequence shown in SEQ ID NO:7 and the light chain variable region sequence shown in SEQ ID NO:8; or,

[0090] (2) the heavy chain variable region sequence shown in SEQ ID NO:11 and the light chain variable region sequence shown in SEQ ID NO:12.

[0091] In some embodiments, the murine anti-MIC A / B antibody RDM028 comprises the heavy chain variable region sequence shown in SEQ ID NO:7 and the light chain variable region sequence shown in SEQ ID NO:8.

[0092] In some embodiments, the humanized anti-MIC A / B antibody RDH028 comprises the heavy chain variable region sequence shown in SEQ ID NO:11 and the light chain variable region sequence shown in SEQ ID NO:12.

[0093] In some embodiments, the anti-MIC A / B antibody is the chimeric antibody RDM028hG1, which comprises the heavy chain amino acid sequence shown in SEQ ID NO:9 and the light chain amino acid sequence shown in SEQ ID NO:10.

[0094] On the other hand, the present disclosure provides a polynucleotide encoding the aforementioned antibody or its antigen-binding fragment.

[0095] On the other hand, the present disclosure provides an expression vector containing the aforementioned polynucleotide.

[0096] On the other hand, the present disclosure provides a host cell into which or which contains the foregoing polynucleotide or the foregoing expression vector.

[0097] On the other hand, the present disclosure provides a pharmaceutical composition comprising the foregoing antibody or its antigen-binding fragment, and a pharmaceutically acceptable excipient, diluent or carrier.

[0098] In some embodiments, the pharmaceutical composition further comprises a second therapeutic agent.

[0099] In some embodiments, the therapeutic agent is selected from antibodies, chemotherapeutic agents and small molecule drugs.

[0100] On the other hand, the present disclosure provides a conjugate comprising the foregoing antibody or its antigen-binding fragment, and a chemical moiety conjugated thereto.

[0101] In some embodiments, the chemical moiety is selected from therapeutic agents, detectable moieties and immunostimulatory molecules.

[0102] On the other hand, the present disclosure provides a kit for detecting or quantifying MIC A / B in a clinical or biological sample, which comprises the foregoing antibody or its antigen-binding fragment or the foregoing conjugate.

[0103] On the other hand, the present disclosure provides the use of the foregoing antibody or its antigen-binding fragment, the foregoing polynucleotide, the foregoing expression vector, the foregoing host cell, the foregoing pharmaceutical composition, the foregoing conjugate and / or the foregoing kit in the preparation of a drug for preventing and / or treating MIC A / B-mediated diseases;

[0104] In some embodiments, the MIC A / B-mediated diseases are cancer, autoimmune diseases, infectious diseases or transplant rejection.

[0105] In some embodiments, the cancer is colon cancer, adrenocortical carcinoma, anal cancer, bladder cancer, brain tumor, breast cancer, carcinoid tumor, cancer of unknown primary origin in the gastrointestinal tract, cervical cancer, endometrial cancer, esophageal cancer, extrahepatic bile duct cancer, extracranial germ cell tumor, intraocular melanoma, eye cancer, gallbladder cancer, gastric cancer, external germ cell tumor, gestational trophoblastic tumor, head and neck cancer, hypopharyngeal cancer, islet cell cancer, kidney cancer,

[0106] laryngeal cancer, oral cancer, liver cancer, lung cancer, AIDS-related lymphoma, central nervous system lymphoma, cutaneous T-cell lymphoma, Hodgkin lymphoma, malignant mesothelioma, melanoma, Merkel cell carcinoma, multiple myeloma, plasmacytoma, nasopharyngeal cancer, osteoblastoma, oropharyngeal cancer,

[0107] At least one of osteosarcoma, ovarian epithelial cancer, ovarian germ cell tumor, pancreatic cancer, exocrine pancreatic cancer, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pituitary cancer, prostate cancer, rhabdomyosarcoma, rectal cancer, transitional cell carcinoma of the renal pelvis and urethra, salivary gland cancer, skin cancer, small intestine cancer, soft tissue sarcoma, testicular cancer, malignant thymoma, thyroid cancer, uterine cancer.

[0108] In some embodiments, the cancer is colon cancer and melanoma.

[0109] On the other hand, the present disclosure provides a method for treating a MIC A / B-mediated disease by using a therapeutically effective amount of the aforementioned anti-MIC A / B antibody or its antigen-binding fragment, the aforementioned pharmaceutical composition, or the aforementioned conjugate.

[0110] Examples

[0111] Further understanding of the present disclosure can be obtained by referring to some specific examples given herein. These examples are only for illustrating the present disclosure and are not intended to limit the scope of the present disclosure in any way. Obviously, various modifications and variations can be made to the present disclosure without departing from the essence of the present disclosure. Therefore, these modifications and variations are also within the scope claimed in this application.

[0112] Example 1: Expression and purification of MICA / B protein

[0113] 1. Cell culture

[0114] HCT-116 human colon cancer cells (Wuhan Pusai) were cultured in RPMI-1640 medium (Gibco) supplemented with 10% fetal bovine serum and 1% streptomycin and penicillin in a 37 °C, 5% CO2 incubator.

[0115] 2. Expression and purification of MICA / B and MICA / Bα3 domain proteins

[0116] Design gene expression vectors encoding MICA, MICB, MICAα3 domain or MICBα3 domain proteins, the expression structures of which are shown in Figure 1 , by Figure 1As can be seen, it contains an Avi-tag (GLNDIFEAQKIEWHE) for biotinylation and a 6-His tag for N-terminal purification. Exemplary sequences of MICA can be found in NCBI accession number NP_000238.1 (allele MICA*001), and the α-3 domain sequence of MICA is the amino acid sequence from 205 to 297 in the MICA sequence with accession number NP_000238.1 in GeneBank. Exemplary sequences of MICB can be found in the sequence with accession number Q29980.1 in UniProtKB, and the MICB α-3 domain sequence is the amino acid sequence from 205 to 297 in UniProtKB accession number Q29980.1. The above proteins were de novo synthesized and cloned into the pCDNA3.1 mammalian expression plasmid (Genfvript). Plasmid DNA was purified from the culture using the PureLink HiPure Plasmid Maxiprep Kit (Thermo Fisher Scientific), and recombinant proteins were produced by transient transfection in 293F cells with polyethyleneimine (Sigma-Aldrich). Four days after cell culture, His-tagged recombinant proteins were purified from the supernatant of the transfected cell culture using Ni-Charge MagBeads (GenScript) and stored at -80 °C for later use.

[0117] Example 2: Mouse Immunization

[0118] Using QuickAntibody-Mouse2W (Biodragon) as an adjuvant, an immune mixture containing 25 μg each of MICA, MICB, MICA α3 domain, and MICB α3 domain was injected intramuscularly into three BALB / c mice. The total volume of each injection was 100 mL, and a total of three injections were given at two-week intervals. The mice were sacrificed on the 7th day after the last immunization, and the spleens and blood were collected.

[0119] Example 3: ELISA Detection of Mouse Serum

[0120] Example 1 The purified MICA / B protein was coated on an ELISA plate, with a coating dose of 100 ng per well and incubated at 4°C overnight. Blocked with 10% goat serum at 37°C for 2h, washed 3 times, and then 100 μL of immune mouse serum was added, with a starting concentration of 1:200, 2-fold gradient dilution, a total of 12 gradients, incubated at 37°C for 1h, washed 3 times, and HRP-labeled goat anti-mouse IgG-HRP (1:5 000) was added, and incubated at 37°C for 1h. After washing 5 times, 100 μL of TMB colorimetric solution was added to each well and incubated at room temperature for 5-10min, and 50 μL of 2mol H2SO4 was used to stop the color development. The absorbance (OD) value at 450nm-640nm was read. The antibody in the well with an absorbance of at least 4 times that of the negative control was determined to be positive.

[0121] Example 4: Sorting of MIC A / B antigen-specific B cells

[0122] Single-cell spleen cell suspension (BD Biosciences) was prepared by filtering the spleen cells of mice with the highest titer of serum-binding antibodies through a cell filter. Single memory B cells were sorted by fluorescent staining and flow cytometry. The spleen cells were washed once with RPMI 1640 culture medium (10% fetal bovine serum) and resuspended in 200 μL PBS containing 5% fetal bovine serum. Cell viability dye aque vital dye-AmCyan and fluorescent antibodies anti-mouse CD4-FITC, CD8a-FITC, CD45R-APC, IgD-PE, IgG-BV650, and PE-Cy7 and BV421 fluorescently labeled EqIgG1-C antigen proteins were added, and the cells were stained for 30 minutes at room temperature in the dark. The cells were washed twice with PBS (5% FBS), resuspended with 3 mL PBS (5% FBS) and filtered out cell clumps with a 200-mesh filter. After preparing the single cell suspension, single specific memory B cells were sorted using the single cell sorting mode of the AriaIII flow cytometer. The logical relationship of the flow sorting gates was: Aque Vitalneg / CD4neg / CD8aneg / CD45Rpos / IgGpos / IgDneg / Ag-PE-Cy7pos / Ag-BV421pos. Single cells were sorted into a 96-well PCR plate. Each well of the PCR plate was pre-mixed with the reverse transcription reaction system: 5 μL of 5×cDNA Buffer, 0.5 μL RNase Out, 1.25 μL DTT, 0.0625 μL Igepal and 13.25 μL ddH2O. After sorting, the PCR plate was frozen at -80°C for amplification of the variable region of the antibody gene.

[0123] The flow cytometry results are shown in Figure 2, the successfully sorted antigen - specific memory B cells can be seen in the upper right quadrant.

[0124] Example 5: Isolation of Immunoglobulin (Ig) Variable Region Genes by RT / PCR

[0125] Using Superscript IV reverse transcriptase (Thermo Fisher) and specific reverse transcription primers, cDNA was extracted from single B cells in a single well of a 96 - well plate. The mouse antibody variable region genes were amplified by two - round PCR using AmpliTaq Gold 360 Master Mix DNA polymerase. Since most mouse B - cell surface Ig genes have a κ light chain, the light primers used in this example only include primers for amplifying the κ light chain. The first - round nested PCR contains forward primers with Ig gene primer sequences from the variable region genes of the heavy chain (VH) and light chain (VL), and reverse primers from the constant regions of the heavy chain and κ light chain. The second - round PCR uses forward primers designed for a new round of PCR amplification that overlaps the sequence tags in the first - round PCR products. In the second round, reverse primers were designed to target the mouse antibody constant region CH1 / CL1 genes, and the ends of these reverse primers contain fragments of the mouse antibody gene IgG2a (CH) or Igκ (CL) constant regions. The PCR products were annotated for Ig genes by DNA sequencing and used to generate expression constructs for recombinant antibody production. Among them, the sequences of the heavy and light chain variable regions and their CDRs of the monoclonal antibody RDM028 are shown as follows:

[0126] Heavy - chain variable region of RDM028 (SEQ ID NO.7)

[0127] QVQLQQSGPELVRPGVSVKISCKGSNYTFTDYTIHWVKQSHAKTLEWIGVISTYFGNTDYNQKFKGKATMTVDKSSTTAYMELARLTSEDSAVYYCARTYYRYDGLYAMDYWGQGTSVTVSS

[0128] HCDR1: GSNYTFTDYTIH (SEQ ID NO.1)

[0129] HCDR2: VISTYFGNTDYNQKFKG (SEQ ID NO.2)

[0130] HCDR3: TYYRYDGLYAMDY (SEQ ID NO.3)

[0131] Light - chain variable region of RDM028 (SEQ ID NO.8)

[0132] DIVMTQSPSSLSVSTGEKVTMSCKSSQNLLNSGNQKNYLAWYQQKPGQPPKLLIYGASTRESGVPDRFTGSGSGSDFTLTISSVQAEDLAVYYCQNDLIYPYTFGGGTKLEIK

[0133] LCDR1: KSSQNLLNSGNQKNYLA (SEQ ID NO.4)

[0134] LCDR2: GASTRES (SEQ ID NO.5)

[0135] LCDR3: QNDLIYPYT (SEQ ID NO.6)

[0136] Example 6: Monoclonal Antibody Production

[0137] As described above, the amplified VH and VK genes were assembled into a linear expression construct by overlapping PCR to produce recombinant antibodies. The forward overlapping DNA fragment contains the CMV promoter, whose DNA sequence overlaps with the tag sequence at the 5' end of the amplified PCR products of the isolated VH and VK genes. The reverse overlapping DNA fragment includes the murine IgG2a constant region (330 aa, GenBank accession number: V00798.1) or the murine Igκ light chain constant region (107 aa, GenBank accession number: V00802.1), whose DNA sequence overlaps with the tag sequence at the 3' end of the isolated VH or VK PCR products. The linear full-length murine IgG2 and Igκ gene expression constructs generated by overlapping PCR were purified using the QIAquick PCR Purification Kit (Qiagen, Valencia, CA) and used for transfection.

[0138] To produce a small amount of recombinant antibodies for initial screening, HEK 293T cells were cultured at 80 - 90% confluence in a 12-well tissue culture plate (Becton Dickson, Franklin Lakes, NJ). The cells in the 12-well tissue culture plate were transfected with the purified Ig VH (1 μg) and VK gene linear expression vectors using PolyFect transfection reagent (Qiagen, Valencia, CA). The transfected cells were cultured in RPMI-1640 basal medium (Life Technologies, USA) supplemented with 2% fetal bovine serum (FBS) (Life Technologies), 1% streptomycin, and 1% penicillin in a humidified incubator at 37°C and 5% CO2 for 3 days. The supernatant of the transfected culture was harvested and used directly for screening MICA / B-specific monoclonal antibodies.

[0139] To produce purified full-length IgG antibodies, the isolated VH and VK genes were cloned into the pCDNA3.1+ (Invitrogen) mammalian expression vector using standard recombinant DNA techniques. This vector contains the murine IgG2a constant region gene or the murine κ light chain constant region gene, and HEK 293T cells were transfected with polyethyleneimine (Sigma-Aldrich) to produce the antibodies. After 4 days of culture, the recombinant antibodies were purified from the transfected cell culture supernatant using Protein A / G (GenScript).

[0140] Example 7: Preparation of Chimeric Antibodies

[0141] To produce a chimeric antibody with the human IgG1 heavy chain constant region, the murine IgG2a heavy chain constant region expression vector was modified to the human IgG1 heavy chain constant region expression vector, and HEK 293T cells were transfected with polyethyleneimine (Sigma-Aldrich) to produce the antibodies. After 4 days of culture, the recombinant chimeric antibody RDM028hG1 was purified from the transfected cell culture supernatant using Protein A / G (GenScript).

[0142] Among them, the sequence information of the light and heavy chains of the chimeric antibody RDM028hG1 is as follows:

[0143] The amino acid sequence of the heavy chain of the chimeric antibody RDM028hG1 (SEQ ID NO.9)

[0144] QVQLQQSGPELVRPGVSVKISCKGSNYTFTDYTIHWVKQSHAKTLEWIGVISTYFGNTDYNQKFKGKATMTVDKSSTTAYMELARLTSEDSAVYYCARTYYRYDGLYAMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0145] Light chain amino acid sequence of chimeric antibody RDM028hG1 (SEQ ID NO.10)

[0146] DIVMTQSPSSLSVSTGEKVTMSCKSSQNLLNSGNQKNYLAWYQQKPGQPPKLLIYGASTRESGVPDRFTGSGSGSDFTLTISSVQAEDLAVYYCQNDLIYPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0147] Example 8: Antibody ELISA screening

[0148] The monoclonal antibody RDM028 and chimeric antibody RDM028hG1 in the culture supernatant of transfected 293T cells were screened by ELISA method respectively to evaluate their binding ability to MICA, MICB and their respective α3 domain proteins. The antigen proteins were coated on a 96-well ELISA plate (Nunc) at a concentration of 100 ng / well and incubated overnight at 4°C. Subsequently, it was blocked with goat serum at room temperature for 2 hours, and the plate was washed 3 times with PBST. Then, it was co-incubated with the culture supernatant of transfected 293T cells at 37°C for 1 hour, and then incubated with goat anti-mouse HRP-labeled IgG (Promega) diluted to a concentration of 1:10,000 at 37°C for 1 hour. Finally, it was washed 5 times with PBST and 100 μL of TMB solvent was added. At the end of the reaction, 50 μL / well of 2 mol of H2SO4 was added to stop the reaction, and the absorbance value was measured using an enzyme-linked immunosorbent assay reader in the wavelength range of 450 - 630 nm. The half-maximal effective concentration (EC50) of the antibody was calculated by fitting the curve with GraphPad Prism software. The results are shown in Table 2 and Table 3.

[0149] Table 2 Binding affinity results of RDM028

[0150]

[0151] Table 3 Binding affinity results of RDM028hG1

[0152]

[0153] As shown in Table 2, the purified RDM028 antibody specifically binds to the full-length MIC A / B protein and its α3 domain. Among them, the EC50 values of RDM028 binding to MICA and MICB are 0.002569 and 0.0029 respectively, and the EC50 values of binding to the α3 domains of MICA and MICB are 0.04688 and 0.003711 respectively. As shown in Table 3, the EC50 values of the chimeric antibody RDM028hG1 binding to MICA and MICB are 0.003803 and 0.001679 respectively.

[0154] This indicates that both the purified RDM028 and the chimeric antibody RDM028hG1 can strongly bind to MICA and MICB and their α3 domains.

[0155] ELISA was used to compare the binding affinities of RDM028 and RDM028hG1 antibodies to MIC A / B. Figure 3 As can be seen, there is no obvious change in the binding affinities of the modified RDM028hG1 antibody and RDM028 to MICA and MICB.

[0156] Example 9: Detection of Antibody Affinity by Biacore (SPR)

[0157] In this example, the kinetic parameters of the binding of the antibody to the antigens MIC A and MIC B were detected based on surface plasmon resonance (SPR) technology on a Biacore 8K instrument (GE Healthcare, USA). The running buffer used was HBS-EP Buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% surfactant P20). The specific experimental process was as follows: First, the anti-MIC A / B antibody was injected into channel 2 at room temperature, and the antibody was captured using a commercial Protein A chip (GE Healthcare, USA). Then, MICA or MICB diluted to different concentration gradients was injected into channels 1 and 2. The binding time was 90 s, the flow rate was 40 μL / min, and the dissociation time was 5 - 10 min. The regeneration solution was 10 mM glycine (pH 1.5), the flow rate was 30 μL / min, and the regeneration time was 90 s. The experimental results are shown in Figure 4 and Table 4.

[0158] Table 4 Binding Kinetics of RDM028 to MIC A / B

[0159]

[0160] As shown in Table 4 and Figure 4, the KD values of the binding affinities of the antibody RDM028 to MIC A and MIC B are 2.14 nM and 2.84 nM respectively.

[0161] Table 5 KD values of the binding of RDM028hG1 to MICA / B and their respective α3 domains

[0162]

[0163] Similarly, from Figures 5 - 6 and Table 5, it can be seen that the KD values of the binding affinities of the RDM028hG1 antibody to MICA and MICB are 0.166 nM and 0.914 nM respectively, and the binding affinities to their respective α3 domains are 15.7 nM and 5.38 nM.

[0164] Example 10: In vitro detection of the performance of antibodies in stabilizing MICA / B on the cell surface

[0165] HCT-116 human colon cancer cells were resuspended and mixed with RPMI-1640 medium (Gibco) containing 10% fetal bovine serum, 1% streptomycin and 1% penicillin, and then plated in a 6-well plate at a density of 1.2×10 6 cells / well. At the same time, different concentration gradients of anti-MIC A / B antibodies were added to each well (2 replicates for each experimental group). Tetanus toxin antibody TRN0011 was selected as a negative control (for specific information on TRN0011, see CN108218984B). After incubation in a 37°C, 5% CO2 incubator for 24 h, the cells were digested with cell dissociation solution (Gibco). The cells were resuspended in 100 μL of sterile PBS containing 2% fetal bovine serum, and then 3 μL of Human TruStain FcX TM (Biolegend) was added and incubated on ice for 10 min to block Fc receptors on the cell surface. Then, 2 μL of APC anti-human MICA / MICB Antibody (clone number 6D4) (specifically binding to the MIC A / B α3 domain, Biolegend) was added and incubated on ice for 30 min. The mean fluorescence intensity on the cell surface was detected using a BD flow cytometer. 10,000 live cells were collected for each sample, and the flow cytometry data were analyzed using FlowJo 10.8.1 software.

[0166] The experimental results are shown in Figure 7 , and from Figure 7 it can be seen that compared with the control group, both RDN028 and RDM028hG1 significantly increased the expression of MICA and MICB on the surface of HCT-116 cells. That is, both antibodies RDM028 and RDM028hG1 can stabilize MICA / B on the cell surface.

[0167] Example 11: Verification of the performance of antibodies in inhibiting the shedding of MICA / B

[0168] The HCT-116 human colon cancer cells were resuspended and mixed with RPMI-1640 medium (Gibco) containing 10% fetal bovine serum, 1% streptomycin, and 1% penicillin, and then plated in a 6-well plate at a density of 1.2×10 6 cells / well. At the same time, different concentration gradients of anti-MICA / B antibodies were added to each well (2 replicates for each experimental group) to inhibit the shedding of MICA / B on the cell surface. The tetanus toxin antibody TRN0011 was selected as the negative control (the specific information of TRN0011 can be found in CN108218984B). After incubation in a 37°C, 5% CO2 incubator for 48 h, the supernatant was collected and centrifuged at 1000×g for 10 min. The shed MIC A was detected by the Human MICA DuoSet ELISA kit (R&D system). The specific experimental operations can be referred to the instruction manual.

[0169] The experimental results are shown in Figure 8 , and it can be seen from Figure 8 that compared with the negative control group, both the RDM028 antibody and the RDM028hG1 chimeric antibody can significantly reduce the content of MIC A in the cell culture supernatant, that is, significantly inhibit the shedding of MICA on the cell surface.

[0170] Example 12: Cytotoxicity assay

[0171] To verify the effect of anti-MICA / B antibodies on the killing ability of NK cells, the applicant further determined their performance through a cytotoxicity experiment. CytoTox Non-radioactive cytotoxicity detection kit (Promega) was used for cytotoxicity detection. First, 1.2×10 6 HCT-116 human colon cancer cells were plated in a 6-well cell culture plate together with the RDM028 antibody, the RDM028hG1 chimeric antibody, and the negative control tetanus toxin antibody TRN0011, so that the final concentration of the antibody was 20 μg / mL. Then the plate was placed in a 37°C, 5% CO2 incubator for 24 h to inhibit the shedding of MICA / B on the cell surface; then it was rinsed with pre-cooled sterile PBS and then used Digest the cells with cell dissociation solution, and plate NK cells and HCT-116 human colon cancer cells in two 96-well round bottom plates at an effector-to-target ratio of 10:1. In one plate, only the above anti-MICA / B antibody is supplemented in the corresponding cell wells to a concentration of 20 μg / mL. In the other plate, in addition to supplementing 20 μg / mL, an NKG2D blocking antibody 1D11 (from Biolegend) of the same concentration is added. At the same time, set the following control wells for calibration: maximum target cell (lactate dehydrogenase) LDH release well, spontaneous LDH release well of target cells, spontaneous LDH release well of NK cells, medium background and volume calibration control group. After plating, centrifuge at 250×g for 4 min, and place the cells in a 37°C, 5% CO2 cell culture incubator for 6 h; then, 45 min before collecting the supernatant, add 10 μL of Lysis Solution (10×) to the maximum release well of target cells and the volume calibration control well to fully lyse the target cells. During this period, pipette 12 mL and add it to a bottle of Substrate Mix, and gently invert and shake to dissolve the substrate; then, when the cell incubation is over, centrifuge the 96-well round bottom plate at 250×g for 4 min at room temperature, and then pipette 50 μL of the supernatant and transfer it to a new 96-well enzyme-linked immunosorbent assay (ELISA) plate. Add 50 μL of the newly prepared Substrate Mix to each well, and incubate at room temperature for 30 min in the dark; finally, add 50 μL of Stop Solution to each well, and measure the absorbance at a wavelength of 490 nm using a multifunctional microplate reader (Bio Tek, USA). The cytotoxicity is calculated according to the following formula:

[0172]

[0173] Experimental group: All experimental wells - medium background;

[0174] Spontaneous effector cells: Spontaneous LDH release well of NK cells - medium background;

[0175] Spontaneous target cells: Spontaneous LDH release well of target cells - medium background;

[0176] Maximum target cells: Maximum LDH release well of target cells - volume calibration control.

[0177] The experimental results are shown in Figure 9 , and it can be seen from Figure 9 Result A that RDM028 and RDM028hG1 significantly enhanced the tumor cell killing ability mediated by NK cells. At the same time, the known NKG2D blocking antibody 1D11 was used to treat NK cells to evaluate the effect of RDM028hG1 and RDM028 on tumor growth after blocking the NKG2D receptor, Figure 9The experimental results of B proved that after the antibody blockade, the tumor killing ability of RDM028 and RDM028hG1 was indeed significantly reduced. This indicates that the enhanced NK cell-mediated tumor cell killing ability is indeed caused by the increased expression of MICA / B on the cell surface, that is, the increased cytotoxic activity of NK cells against tumors is achieved through the interaction between MICA and NKG2D.

[0178] Example 13: Antitumor effect of antibody in vivo

[0179] Male Balb / c nude mice (Nude) at 4-6 weeks old were purchased from Zhuhai BestBio Technology Co., Ltd. and entrusted to it for feeding. The HCT-116 human colon cancer cells in the logarithmic growth phase were resuspended with sterile PBS to a cell suspension of 5×10 7 cell / mL, and 100 μL (5×10 6 cells) were subcutaneously inoculated into each nude mouse. On the second day after inoculation, the mice were evenly divided into an experimental group and a control group. Each mouse in the experimental group was intraperitoneally injected with 200 μg of RDM028hG1 antibody, and the control group was injected with the same volume of sterile PBS. The first day of antibody injection was recorded as D1, and then the mice were injected with antibody or sterile PBS on D3, D7, and D14 respectively. During this period, the tumor size was regularly measured with a vernier caliper, and the tumor volume (V) was calculated according to the formula: V = (L×W 2 )×0.5, where L is the length (mm) of the tumor and W is the width (mm) of the tumor. When the tumor volume approached 2000 mm 3 , the experiment was stopped. After blood collection, the mice were euthanized and the tumor cells were dissected and weighed.

[0180] The experimental results are shown in Figure 10 , and it can be seen from Figure 10 that injecting RDM028hG1 can significantly inhibit tumor growth and reduce its size and weight. Although there is no statistically significant difference in body weight change between the mice in the RDM028hG1 treatment group and the negative control group mice, it can be expected that the mice in the RDM028hG1 treatment group are healthier than the control group mice.

[0181] Example 14 Humanization of anti-MICA / B antibody

[0182] The framework region (FR) of the variable region of the murine antibody RDM028 was replaced with the human antibody framework region, and the glycosylation site and isomerization site of the variable region were subjected to point mutations to obtain the humanized antibody RDH028, and its sequence information is shown as follows:

[0183] Heavy chain variable region sequence of humanized antibody RDH028 (SEQ ID NO.11)

[0184] QVQLVQSGAEVKKPGSSVKVSCKGSGYTFTDYTIHWVRQAPGQGLEWMGVISTYFGNTDYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARTYYRYDALYAMDYWGQGTTVTVSS

[0185] HCDR1: GSGYTFTDYTIH (SEQ ID NO.13)

[0186] HCDR2: VISTYFGNTDYNQKFKG (SEQ ID NO.2)

[0187] HCDR3: TYYRYDALYAMDY (SEQ ID NO.14)

[0188] Humanized antibody RDH028 light chain variable region sequence (SEQ ID NO.12)

[0189] DIVMTQSPDSLAVSLGERATINCKSSQNLLNSGNQKNYLAWYQQKPGQPPKLLIYGASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDLIYPYTFGGGTKVEIK

[0190] LCDR1: KSSQNLLNSGNQKNYLA (SEQ ID NO.4)

[0191] LCDR2: GASTRES (SEQ ID NO.5)

[0192] LCDR3: QNDLIYPYT (SEQ ID NO.6)

[0193] Similarly, the antibody affinity of humanized antibody RDH028 was detected. The specific detection method was the same as that in Example 9, and the experimental results are shown in Figure 11 and Table 6.

[0194] Table 6 RDH028 binding kinetics with MICA / B

[0195]

[0196] As can be seen from Table 6, the humanized antibody RDH028 has good affinity for MICA and MICB, and the KD values are 4.49 nM and 2.41 nM, respectively.

[0197] Example 15 Comparative experiment

[0198] To further prove the affinity of the anti-MICA / B murine antibody RDM028 disclosed in the present invention, this example conducted repeated experiments based on the anti-MICA / B antibodies disclosed in the prior art to verify its affinity. According to the heavy and light chain variable region sequences of the antibody provided in the corresponding patent, the DNA sequences of the heavy and light chain variable regions of the antibody were respectively ligated upstream of the DNA of murine IgG 2a and Kappa constant regions to synthesize the DNA sequence of the complete antibody. The DNA sequence of the complete antibody was constructed on the pcDNA3.1 plasmid, and antibody protein expression was carried out by transient transfection of 293i suspension cells, and the antibody was purified using a Protein A affinity column.

[0199] Among them, the sample information is shown in Table 7.

[0200] Table 7 Sample Information

[0201]

[0202]

[0203] In this example, the kinetic parameter detection of the binding of the antibody to antigens MIC A and MIC B was carried out on a Biacore 8K instrument (GE Healthcare, USA) based on surface plasmon resonance technology (SPR). The running buffer used was HBS-EP Buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% surfactant P20). Different concentration gradients of MICA or MICB solutions were set, and the specific experimental parameters for capturing the antibody using the purified Protein A chip are shown in Table 8.

[0204] Table 8 Experimental Parameters of Protein A Chip

[0205]

[0206] The experimental results are shown in Figures 12 - 13 and Table 9.

[0207] Table 9 Results of Comparative Experiments

[0208] Kinetic model Capture 1 solution Analyte 1 solution ka (1 / Ms) kd (1 / s) KD (M) Rmax (RU) 1:1 binding DN MICA 9.65E+04 1.62E-04 1.68E-09 26.9 1:1 binding TG MICA 2.05E+05 1.56E-03 7.60E-09 32.2 1:1 binding ZJ - 1 MICA 8.06E+04 1.10E-03 1.37E-08 29.2 1: binding ZJ - 2 MICA 4.51E+04 6.31E-04 1.40E-08 38.2 1:1 binding DN MICB 8.80E+04 3.84E-04 4.36E-09 38.0 1:1 binding TG MICB 4.37E+04 5.60E-04 1.28E-08 92.7 1:1 binding ZJ - 1 MICB 5.43E+04 9.08E-04 1.67E-08 60.4 1:1 binding ZJ - 2 MICB 3.03E+04 7.83E-04 2.58E-08 102.2

[0209] As can be seen from the results in Table 9, the KD values of the affinity of the DN antibody with MICA and MICB are 1.68 nM and 4.36 nM respectively, the KD values of the affinity of the TG antibody with MICA and MICB are 7.60 nM and 12.8 nM respectively, the KD values of the affinity of the ZJ-1 antibody with MICA and MICB are 13.7 nM and 16.7 nM respectively, and the KD values of the affinity of the ZJ-2 antibody with MICA and MICB are 14.0 nM and 25.8 nM respectively. Under the same experimental conditions, the murine antibody of the present application has better affinity, and its affinity with MICA and MICB is better, with KD values of 2.14 nM and 2.84 nM respectively (see Table 4 and Figure 4). Therefore, compared with the anti-MICA / B antibodies in the prior art, the antibody prepared in the present application has better affinity, and it can be predicted that it will have better potential physiological effects in the future.

[0210] The above are only embodiments of the present invention, and do not impose any formal or substantial limitations on the present invention. It should be noted that for those of ordinary skill in the art in this technical field, without departing from the method of the present invention, several improvements and supplements can still be made, and these improvements and supplements should also be regarded as the protection scope of the present invention. For those skilled in the art, without departing from the spirit and scope of the present invention, any equivalent changes made by using the technical content disclosed above, such as slight modifications, decorations, and evolutions, are equivalent embodiments of the present invention; at the same time, any equivalent changes made to the above embodiments based on the essential technology of the present invention, such as modifications, decorations, and evolutions, still fall within the scope of the technical solution of the present invention.

Claims

1. An anti-MIC A / B antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein: The heavy chain variable region comprises HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2, and HCDR3 as shown in SEQ ID NO: 3; the light chain variable region comprises LCDR1 as shown in SEQ ID NO: 4, LCDR2 as shown in SEQ ID NO: 5, and LCDR3 as shown in SEQ ID NO: 6; or The heavy chain variable region comprises HCDR1 as shown in SEQ ID NO:13, HCDR2 as shown in SEQ ID NO:2, and HCDR3 as shown in SEQ ID NO:14; the light chain variable region comprises LCDR1 as shown in SEQ ID NO:4, LCDR2 as shown in SEQ ID NO:5, and LCDR3 as shown in SEQ ID NO:

6.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody is selected from a monoclonal antibody, a single-chain antibody, a chimeric antibody, a humanized antibody or a fully humanized antibody.

3. The antibody or antigen-binding fragment thereof according to claim 1, wherein The antigen binding fragment is selected from Fab, Fab', F(ab')2, Fv fragment, scFv or di-scFv.

4. The antibody or antigen-binding fragment thereof according to claim 1, wherein The anti-MIC A / B antibody or antigen-binding fragment thereof comprises a heavy chain variable region selected from the following sequence, or a heavy chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity compared to the following sequence: SEQ ID NO:7 or SEQ ID NO:11; and / or, Selected from the light chain variable region set forth in the following sequence, or a light chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity compared to the following sequence: SEQ ID NO:8 or SEQ ID NO:

12.

5. The antibody or antigen-binding fragment thereof according to claim 4, wherein The anti-MIC A / B antibody or antigen-binding fragment thereof comprises: (1) the heavy chain variable region sequence shown in SEQ ID NO: 7 and the light chain variable region sequence shown in SEQ ID NO: 8; or, (2) The heavy chain variable region sequence shown in SEQ ID NO: 11 and the light chain variable region sequence shown in SEQ ID NO:

12.

6. The antibody or antigen-binding fragment thereof according to claim 2, wherein: The anti-MIC A / B antibody is a chimeric antibody, which comprises a heavy chain amino acid sequence shown in SEQ ID NO:9 and a light chain amino acid sequence shown in SEQ ID NO:

10.

7. A polynucleotide encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6. An expression vector comprising the polynucleotide according to claim 7.

9. A host cell introduced with or containing the polynucleotide according to claim 7 or the expression vector according to claim 8.

10. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, and a pharmaceutically acceptable excipient, diluent or carrier.

11. The pharmaceutical composition according to claim 10, wherein The pharmaceutical composition further comprises a second therapeutic agent.

12. The pharmaceutical composition according to claim 11, wherein The therapeutic agent is selected from the group consisting of an antibody, a chemotherapeutic agent, and a small molecule drug.

13. A kit for detecting or quantifying MIC A / B in a biological sample, comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6.

14. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, the polynucleotide according to claim 7, the expression vector according to claim 8, the host cell according to claim 9, the pharmaceutical composition according to any one of claims 10 to 12, and / or the kit according to claim 13 in the preparation of a medicament for treating a disease mediated by MIC A / B, wherein the disease mediated by MIC A / B is cancer, and the cancer is colon cancer, breast cancer, lung cancer or melanoma.

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