Anti-MICA / B antibodies and their applications

Abstract

This invention proposes an anti-MICA / B antibody and its applications, belonging to the field of biomedicine. Specifically, it discloses a MICA / B-specific antibody or its antigen-binding fragment comprising: heavy chain variable regions CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO: 1, 2, and 3; and light chain variable regions CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO: 4, 5, and 6. This antibody exhibits excellent binding affinity to MICA / B, significant endocytosis, and the ability to inhibit MICA / B shedding.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to an anti-MICA / B antibody and its use in the preparation of medicaments for the treatment and / or prevention of tumors. Background Technology

[0002] NKG2D (Natural Killer Group 2D) is a homodimer C-type lectin-like receptor expressed on the surface of almost all human cytotoxic lymphocytes (NK cells, CD8+ T cells, and γδ T cells), and serves as the major activation receptor for NK cells. MICA / B (MHC class I chain-associated sequence A / B) is the most well-defined ligand for NKG2D. The MICA / B gene is located in the MHC class I region of human chromosome 6, encoding a protein composed of three extracellular domains (α1, α2, and α3) as well as transmembrane and intracellular regions. Studies have shown that under normal physiological conditions, MICA / B glycoproteins are usually not expressed in healthy tissues, but various stress conditions (such as viral infection and malignant transformation) can induce MICA / B expression on the cell surface. Although the interaction between MICA / B and NKG2D promotes the recognition and lysis of stressed cells expressing MICA / B by immune cells, tumor cells often cleave MICA molecules from the cell surface using various metalloproteinases, thereby escaping NKG2D-mediated immune recognition. Elevated serum levels of soluble MICA / B (sMICA / B) in cancer patients (due to MICA / B shedding) are negatively correlated with overall survival, and sMICA / B is considered a cancer biomarker. These characteristics make MICA / B shedding an attractive target in cancer research. To date, only one MICA / B monoclonal antibody drug (CLN-619) has entered clinical trials. Therefore, developing monoclonal antibody drugs targeting MICA / B offers a new approach to cancer treatment and is of great significance for cancer therapy. Summary of the Invention

[0003] This invention relates to an anti-MICA / B antibody or its antigen-binding fragment and its applications. The antibody of this invention possesses high specificity and high sensitivity; it mediates endocytosis and inhibits MICA / B shedding, reduces the level of soluble MICA / B in the tumor microenvironment, increases the abundance of MICA / B on the surface of tumor cells, and restores the tumor recognition and killing capabilities of immune cells such as NK cells.

[0004] This invention relates in one aspect to an antibody or its antigen-binding fragment, 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; and the antibody or its antigen-binding fragment specifically binds to MICA / B.

[0005] In some implementations, the antibody is a monoclonal antibody, a single-chain antibody, a chimeric antibody, a humanized antibody, or a human antibody.

[0006] In some implementations, the antibody is a monoclonal antibody.

[0007] In some implementations, the antibody is a chimeric antibody.

[0008] In some embodiments, the antigen-binding fragment is selected from Fab, Fab', F(ab')2, Fv fragment, scFv, or di-scFv.

[0009] In some embodiments, the heavy chain variable region comprises an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:7.

[0010] In some embodiments, the heavy chain variable region comprises an amino acid sequence having 90%, 95%, or 99% sequence identity with SEQ ID NO:7.

[0011] In some embodiments, the heavy chain variable region comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO:7.

[0012] In some embodiments, the light chain variable region comprises an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:8.

[0013] In some embodiments, the light chain variable region comprises an amino acid sequence having 90%, 95%, or 99% sequence identity with SEQ ID NO:8.

[0014] In some embodiments, the light chain variable region comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO:8.

[0015] In some embodiments, the heavy chain variable region comprises an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:7; and the light chain variable region comprises an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:8.

[0016] In some embodiments, the heavy chain variable region comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO:7; and the light chain variable region comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO:8.

[0017] In some embodiments, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:7.

[0018] In some embodiments, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:8.

[0019] In some embodiments, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:7; and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:8.

[0020] In some embodiments, the antibody or its antigen-binding fragment contains a heavy chain constant region derived from human IgG1, IgG2, IgG3 or IgG4.

[0021] In some embodiments, the antibody or its antigen-binding fragment comprises the IgG1 heavy chain constant region shown in SEQ ID NO:9.

[0022] In some embodiments, the antibody or its antigen-binding fragment comprises a light chain constant region derived from the human κ chain and λ chain.

[0023] In some embodiments, the antibody or its antigen-binding fragment comprises the light chain constant region of the human κ chain shown in SEQ ID NO:10.

[0024] In some embodiments, the antibody comprises the heavy chain amino acid sequence shown in SEQ ID NO:11 and the light chain amino acid sequence shown in SEQ ID NO:12.

[0025] In some embodiments, the antibody or its antigen-binding fragment mediates endocytosis.

[0026] In some embodiments, the antibody or its antigen-binding fragment has an inhibitory effect on preventing MICA / B shedding.

[0027] Another aspect of the present invention relates to biological materials associated with the aforementioned antibodies or their antigen-binding fragments, as follows (i), (ii), or (iii):

[0028] (i) Nucleic acid molecules that encode the aforementioned antibodies or their antigen-binding fragments;

[0029] (ii) an expression vector, which includes the nucleic acid molecule in (i);

[0030] (iii) Host cells, including the nucleic acid molecules in (i) and / or the expression vectors in (ii).

[0031] Another aspect of the present invention relates to a pharmaceutical composition comprising the aforementioned antibody or antigen-binding fragment thereof and / or the aforementioned biological material, and optionally a pharmaceutically acceptable carrier.

[0032] Another aspect of the present invention relates to an immune conjugate comprising the aforementioned antibody or its antigen-binding fragment, said antibody or its antigen-binding fragment being conjugated to a therapeutic or diagnostic agent.

[0033] In some embodiments, the antibody described in this invention is conjugated to a heterologous molecule to obtain an immunoconjugate.

[0034] In some implementations, the heterologous molecule may be, for example, a therapeutic or diagnostic agent, such as a cytotoxic agent or a chemotherapeutic agent.

[0035] In some embodiments, the cytotoxic agent includes any agent that is harmful to cells.

[0036] In some implementations, the different entities of the immunoconjugate are covalently linked using a connector.

[0037] In some implementations, the connector includes a chemical connector or a peptide connector.

[0038] In some embodiments, the antibody described in this invention is conjugated with a diagnostic agent or a detectable agent, and the resulting conjugate can be used as part of a detection method to detect or predict the occurrence, formation, progression, and / or severity of a disease or condition.

[0039] In some embodiments, the detectable agent is conjugated with the antibody of the present invention.

[0040] In some implementations, the detectable agent includes, but is not limited to, a variety of enzymes, such as, but not limited to, horseradish peroxidase, fluorescent substances, luminescent substances, and radioactive substances.

[0041] In some implementations, the immune conjugate is used for the prevention or treatment of cancer.

[0042] Another aspect of the present invention relates to a kit containing the aforementioned antibody or its antigen-binding fragment, the aforementioned biological material, and / or the aforementioned pharmaceutical composition.

[0043] In some implementations, the kit is used to detect MICA / B in biological samples.

[0044] Another aspect of the present invention relates to the use of the aforementioned antibodies or antigen-binding fragments thereof, the aforementioned biological materials and / or the aforementioned pharmaceutical compositions in the preparation of a kit for detecting MICA / B in biological samples.

[0045] Another aspect of the present invention relates to the use of the aforementioned antibodies or antigen-binding fragments thereof, the aforementioned biological materials and / or the aforementioned pharmaceutical compositions in the preparation of pharmaceuticals.

[0046] In some implementations, the drug is used to treat or prevent MICA / B-mediated diseases.

[0047] In some implementations, the MICA / B-mediated disease is cancer.

[0048] In some implementations, the cancer is colon cancer.

[0049] Another aspect of the present invention relates to a medicament comprising the aforementioned antibody or antigen-binding fragment thereof, the aforementioned biological material and / or the aforementioned pharmaceutical composition; the medicament is used to treat or prevent MICA / B-mediated diseases.

[0050] In some implementations, the disease is a cancer expressing MICA / B.

[0051] In some implementations, the cancer is colon cancer. Attached Figure Description

[0052] Figure 1 Image showing the result of antibody M9 inhibiting the shedding of MICA / B from HCT-116 cells. Detailed Implementation

[0053] The embodiments described are provided to better illustrate the present invention, but are not intended to limit the scope of the invention to the embodiments described. Therefore, non-essential improvements and adjustments made to the embodiments by those skilled in the art based on the above description are still within the scope of protection of the present invention.

[0054] Unless otherwise stated, the following terms and phrases as used herein are intended to have the following meanings. When a trademark name is used herein, unless the context otherwise indicates, the trademark name includes the product formulation, generic medicine, and active ingredient of the product for which the trademark name is used.

[0055] Certain terms in the invention

[0056] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit this disclosure. Singular expressions include plural expressions unless they have a distinct meaning in the context. As used herein, it should be understood that terms such as “comprising,” “having,” “including,” are intended to indicate the presence of a feature, number, operation, material, or combination thereof. The terminology of the invention is disclosed in the specification and is not intended to exclude the possibility that one or more other features, numbers, operations, materials, or combinations thereof may be present or added. As used herein, “ / ” may be interpreted as “and” or “or,” depending on the context.

[0057] The term "MICA / B" refers to MICA protein, MICB protein, or both MICA and MICB proteins, including their variants, isotypes, and species homologs of human MICA / B.

[0058] The term "CDR" refers to the complementarity-determining region within the variable sequence of an antibody. For each variable region, there are three CDRs in each variable region of the heavy and light chains, which are called CDR1, CDR2, and CDR3. The exact boundaries of these CDRs are defined differently depending on the system. The system described by Kabat et al. (Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides a definitive residue numbering system applicable to antibody variable regions but also provides residue boundaries that define the three CDRs. These CDRs can be called Kabat CDRs. Each complementarity-determining region can contain amino acid residues from what is defined as a "complementarity-determining region" as by Kabat. Chothia et al. (Chothia & Lesk, J. Mol. Biol, 196:901-917 (1987) and Chothia et al., Nature 342:877-883 (-1989)) found that Kabat Certain sub-regions within a CDR adopt nearly identical peptide skeletons, despite significant diversity at the amino acid sequence level. These sub-regions are referred to as L1, L2, and L3, or H1, H2, and H3, where "L" and "H" denote the light and heavy chain regions, respectively. These regions may be termed Chothia CDRs, with boundaries overlapping with the Kabat CDR. Other CDR boundary definitions may not strictly adhere to one of the aforementioned systems but will still overlap with the Kabat CDR. The methods used herein can utilize CDRs defined according to any of these systems, although the preferred embodiment uses CDRs defined by Kabat or Chothia.

[0059] The term "antibody" refers to immunoglobulin, a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. The amino acid composition and sequence of the constant region of the immunoglobulin heavy chain differ, thus their antigenicity also differs. Based on this, immunoglobulins can be classified into five classes, or different types of immunoglobulins: IgM, IgD, IgG, IgA, and IgE, with their corresponding constant regions of the heavy chain designated α, δ, ε, γ, and μ, respectively. IgG represents the most important class of immunoglobulins, and due to differences in chemical structure and biological function, it can be further divided into four subclasses: IgG1, IgG2, IgG3, and IgG4. The light chains are classified as κ or λ chains based on differences in their constant regions. The subunit structure and three-dimensional conformation of different immunoglobulin classes are well-known to those skilled in the art. The term "chimeric antibody" refers to an antibody containing sequences derived from two different antibodies, typically from different species. For example, a chimeric antibody contains fragments of human and rodent antibodies, typically the human constant region and the mouse variable region. Methods for generating chimeric antibodies include conventional recombinant DNA and gene transfection techniques known to those skilled in the art.

[0060] The term "antigen-binding fragment" refers to an antigen-binding fragment of an antibody and antibody analogues, which typically includes at least a portion of the antigen-binding region or variable region (e.g., one or more CDRs) of the parent antibody. The antibody fragment retains at least some of the binding specificity of the parent antibody. Typically, when activity is expressed on a molar basis, the antibody fragment retains at least 10% of the parent antibody's binding activity. Preferably, the antibody fragment retains at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or more of the parent antibody's binding affinity to the target. Examples of antigen-binding fragments include, but are not limited to: Fab, Fab', F(ab')2, Fv fragments, linear antibodies, single-chain antibodies, nanobodies, domain antibodies, and multispecific antibodies.

[0061] The term "specific binding" refers to an antibody that preferentially binds to a specific target protein compared to other proteins, but this specificity does not require absolute binding specificity. Antibody binding depends on the presence of the target protein in the sample; for example, if an antibody specifically binds to the target protein in the sample without producing undesirable results such as false positives, then the antibody is considered "specific" to its intended target. The antibodies or their binding fragments used in this invention will bind to the target protein with an affinity at least two times, preferably at least ten times, more preferably at least 20 times, and most preferably at least 100 times higher than that with non-target proteins.

[0062] The term "humanized antibody," also known as a CDR-grafted antibody, refers to an antibody generated by grafting a mouse CDR sequence into the variable region framework of a human antibody. Humanized antibodies overcome the drawback of chimeric antibodies, which, due to carrying a large amount of mouse protein components, induce a strong immune response. To avoid a decrease in activity along with a decrease in immunogenicity, minimal reverse mutations can be performed on the variable region of the human antibody to maintain its activity.

[0063] The term "chimeric antibody" refers to an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody. It can mitigate the immune response induced by murine antibodies. To create a chimeric antibody, a hybridoma that secretes murine-specific monoclonal antibodies is selected. The variable region gene is then cloned from mouse hybridoma cells, followed by the desired human antibody constant region gene. The mouse variable region gene and the human constant region gene are linked to form a chimeric gene, which is then inserted into a human vector. Finally, the chimeric antibody molecule is expressed in a eukaryotic or prokaryotic industrial system. The constant region of the human antibody can be selected from the heavy chain constant region of human IgG1, IgG2, IgG3, or IgG4 or their variants, preferably containing the heavy chain constant region of human IgG1, IgG2, or IgG4. Alternatively, an IgG1 heavy chain constant region that has been mutated to enhance ADCC (antibody-dependent cell-mediated cytotoxicity) can be used.

[0064] The term "human antibody" includes antibodies having variable and constant regions of human germline immunoglobulin sequences. Human antibodies of the present invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo). However, the term "human antibody" does not include antibodies in which a CDR sequence derived from another mammalian species (such as a mouse) has been grafted onto a human backbone sequence (i.e., "humanized antibody").

[0065] The term "identity" refers to the sequence similarity between two polynucleotide sequences or two polypeptides. When positions in two compared sequences are occupied by the same base or amino acid monomer subunit—for example, if every position in two DNA molecules is occupied by adenine—then the molecules are homologous at that position. The percentage of identity between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared, multiplied by 100%. For example, at optimal sequence alignment, if six out of ten positions in two sequences match or are homologous, then the two sequences are 60% homologous. Generally, comparisons are made when the highest percentage of identity is obtained by aligning the two sequences.

[0066] The term "expression vector" or "vector" refers to a delivery system that allows for the operative insertion of polynucleotides or nucleic acids encoding a protein, thereby enabling the expression of that protein. Vectors can be used to transform, transduce, or transfect host cells, allowing the genetic material they carry to be expressed within the host cells.

[0067] The term "host cell" refers to a cell into which a foreign polynucleotide or nucleic acid and / or vector has been introduced. Host cells include "transformers" and "transformed cells," which include the initially transformed cell and its progeny, regardless of the number of passages. Progeny cells may not be identical to parental cells in their nucleic acid content and may contain mutations. This application includes screening or selecting mutant progeny with the same function or biological activity from the initially transformed cells.

[0068] The term "immunoconjugate" refers to an antibody conjugated with one or more other substances, including but not limited to cytotoxic agents, chemotherapeutic agents, diagnostic agents, or detectable agents.

[0069] The term “treatment” refers to alleviating a disease or symptom, slowing the onset or development of a disease or symptom, reducing the risk of developing a disease or symptom, or delaying the development of symptoms associated with a disease or symptom, reducing or terminating symptoms associated with a disease or symptom, producing a complete or partial reversal of a disease or symptom, curing a disease or symptom, or a combination of the above.

[0070] The term "prevention" includes the suppression of the occurrence or development of a disease or condition, or symptoms of a particular disease or condition. In some implementations, subjects with a family history of the disease are candidates for preventative programs. Generally, the term "prevention" refers to the administration of a drug before the onset of symptoms or signs, particularly in subjects at risk.

[0071] The term "pharmaceutical composition" herein refers to a composition in which an active substance is formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, the composition is suitable for administration to human or animal subjects. In some embodiments, the active substance is present in a unit dose that is suitable for administration in a treatment regimen and for demonstrating a statistically significant probability of achieving the intended therapeutic effect when used in the relevant population.

[0072] The term “and / or” should be understood to mean any one of the options or any combination of two or more of the options.

[0073] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Test methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise stated, all percentages, proportions, ratios, or parts are by weight.

[0074] Example 1: Preparation and purification of anti-MICA / B antibody

[0075] 1.1 Preparation and purification of antigens

[0076] The antigen expression plasmid was transfected into 293F cells using PEI transfection. After transfection, cells were cultured with shaking for 16-20 hours. 5% (v / v) feed was added on days 1, 3, and 5 post-transfection. The cell supernatant was harvested, centrifuged, filtered, and purified to obtain the following antigens: MICA*002-α3-his (amino acid sequence as shown in SEQ ID NO:15), MICA*008-α3-his (amino acid sequence as shown in SEQ ID NO:16), MICB-α3-his (amino acid sequence as shown in SEQ ID NO:17), MICA*002-ECD-his (amino acid sequence as shown in SEQ ID NO:18), MICA*008-ECD-his (amino acid sequence as shown in SEQ ID NO:19), MICB*004-ECD-his (amino acid sequence as shown in SEQ ID NO:20), and MICB*005-ECD-his (amino acid sequence as shown in SEQ ID NO:21).

[0077] 1.2 Construction of overexpression cell lines

[0078] HEK293T cells were processed at a rate of 5 × 10 5Cells were seeded into six-well plates and cultured overnight. Following the instructions of the transfection reagent (Thermo, L3000015), the pLVX-IRES-puro or pLVX-IRES-neo vector containing the target gene was co-transfected with the backbone vectors pMD2G and psPAX2 into 293T cells. Viral supernatant was obtained.

[0079] The nucleotide sequences of the target gene are SEQ ID NO:22-25, which encode MICA*002, MICA*008, MICB*004, and MICB*005, respectively.

[0080] Add all viral supernatant to a solution containing 3×10 5 In 6-well plates containing CHO-K1 cells, a final concentration of 5 μg / mL of polybrene (Sigma) was added. The supernatant was then discarded, and fresh puromycin-containing or G418 complete F12K medium was added for further culture. Subsequently, single clones were selected for amplification, resulting in CHO-K1-MICA*002 cells, CHO-K1-MICA*008 cells, CHO-K1-MICB*004 cells, and CHO-K1-MICB*005 cells, which respectively express MICA*002 protein (amino acid sequence as shown in SEQ ID NO:26), MICA*008 protein (amino acid sequence as shown in SEQ ID NO:27), MICB*004 protein (amino acid sequence as shown in SEQ ID NO:28), and MICB*005 protein (amino acid sequence as shown in SEQ ID NO:29).

[0081] 1.3 Obtaining Monoclonal Antibodies

[0082] FVB, KM, and CD1 / ICR mice (6 weeks old) were immunized with MICA*002-α3-his, MICA*008-α3-his, MICB-α3-his, MICA*002-ECD-his, MICA*008-ECD-his, MICB*004-ECD-his, and MICB*005-ECD-his. The binding ability of serially diluted tail blood to MICA / B protein and MICA / B overexpressing cells was monitored. Mice with the best ELISA titers and FACS positivity were selected for sprint immunization.

[0083] Mouse spleen cells were obtained under aseptic conditions and ground to obtain single cells, which were then mixed with myeloma cells and electrofused. After culture, hybridoma monoclonal cells were screened using ELISA and FACS. The heavy chain and light chain nucleotide sequences of the candidate molecule were obtained by sequencing (SEQ ID NO: 30 and SEQ ID NO: 31). After expression and purification, a monoclonal antibody named M9 was obtained. The amino acid sequence of the heavy chain variable region of the M9 monoclonal antibody is shown in SEQ ID NO: 7, the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 8, the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO: 9, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO: 10; the CDR region sequences (HCDR1-3, LCDR1-3, Kabat numbering system) are shown in Table 1.

[0084]

[0085] 1.4 Preparation of control antibodies

[0086] According to the method described in patent CN119930819A, a control antibody (heavy chain amino acid sequence SEQ ID NO:13 and light chain amino acid sequence SEQ ID NO:14) was obtained.

[0087] Example 2 Antibody and Antigen Binding Experiment

[0088] Following the preparation and purification of antigens in Example 1.1, antigens MICA*002-ECD-his, MICA*008-ECD-his, MICB*004-ECD-his, and MICB*005-ECD-his were obtained and coated into 96-well plates respectively. The intensity of the signal after the antibody was added was used to determine the binding characteristics of the antibody and the responding antigen.

[0089] The specific experimental procedure is as follows: The protein was diluted to 1 μg / mL with coating buffer and added to a 96-well plate at a volume of 100 μL / well, and incubated overnight. The coating buffer in the 96-well plate was aspirated, and PBS / 2% BSA was added and incubated for 2 h, after which the blocking buffer was removed. The candidate antibody was serially diluted with 2% BSA, and 100 μL / well was added to the microplate and incubated for 1 h. The reaction system was removed, the plate was washed with PBST, and HRP-goat anti-human IgG was diluted 1 / 8000 with blocking buffer and incubated. After incubation for 30 minutes, the plate was washed and 100 μL of TMB chromogenic solution was used for color development. After color development, 50 μL of stop solution was added to each well to terminate the reaction. Finally, the OD value was measured at 450 nm using a microplate reader. The results are shown in Table 2.

[0090]

[0091] The results showed that antibody M9 could bind to antigens MICA*002, MICA*008, MICB*004, and MICB*005, and the binding activity was comparable to that of the control antibody.

[0092] Example 3: Affinity detection of antibody with MICA / B

[0093] The affinity between the antibody and the proteins MICA*002 (A2ECD), MICA*008 (A8ECD), MICB*004 (B4ECD), and MICB*005 (B5ECD) was detected using the Biacore T200.

[0094] The specific experimental procedure is as follows: After activating the CM9 chip, the anti-his antibody was immobilized and blocked. A2ECD, A8ECD, B4ECD, and B5ECD were captured onto the chip surface, respectively. Then, the signal values ​​of the interaction between different concentrations of antibody (0~100nM) and MICA / B-ECD were detected. The flow rate of the flow cell was 30 μL / min, the binding time was 200 s, the dissociation time was 300 s, and regeneration with Glycine 1.5 (Cytiva# BR100354) was performed for 30 s, after which the baseline stabilized. The data were kinetically fitted using Biacore Evaluation Software according to a 1:1 binding model. The results are shown in Table 3.

[0095]

[0096] The results show that the affinity of antibody M9 for MICA / B proteins (such as MICA*002 (A2ECD), MICA*008 (A8ECD), MICB*004 (B4ECD), and MICB*005 (B5ECD)) is comparable to that of the control antibody.

[0097] Example 4: Detection of antibody endocytic activity

[0098] HCT-116 cells (purchased from Nanjing Kebai / Shanghai Cell Center) suspension was collected by digestion, washed once with PBS, and the cell density was adjusted to 4E6 cells / mL. 50 μL / well was added to a 96-well U-plate and labeled Plate A. Then, 50 μL / well of 2× anti-MICA antibody diluted to a final concentration of 10 μg / mL was added to the above 96-well plates. After shaking and incubation for 1 hour, the cells were washed with 1×PBS buffer, and 100 μL / well was added to complete culture medium to resuspend the cells. 50 μL of cell suspension was aspirated from each well and placed into a new 96-well U-plate and labeled Plate B. Plate B remained on ice, while plate A was incubated at 37 °C. After 3 hours, plates A and B were washed once each with pre-chilled FACS buffer (1 mM EDTA, 0.02% NaN3, 0.5% BSA in 1X PBS). 20 μL / well of PE anti-human IgG Fc Recombinant Antibody was added, and the plates were incubated on ice for 30 minutes. Cells were then washed three times with FACS buffer, and the cell pellet was resuspended in FACS buffer. PE fluorescence signal was detected using a flow cytometer (Agilent). The endocytosis rate was calculated based on the MFI (Mean Functional Index). Endocytosis rate % = (MFI 4 °C - MFI 37 °C) / MFI 4 °C × 100%. The results are shown in Table 4.

[0099]

[0100] The results show that antibody M9 has endocytic activity.

[0101] Example 5: Antibody inhibits MICA / B exfoliation from HCT-116 cells

[0102] HCT-116 cell suspension was collected by digestion, counted, and resuspended in McCoys 5A + 10% FBS complete medium to a concentration of 1E6 cells / ml. 50 μL / well was added to each well of a 96-well plate and incubated overnight. The next day, serially diluted anti-MICA / B antibody (50 μL / well) was added to the corresponding wells, mixed, and incubated for 24 hours. The plate was removed, the supernatant was discarded, 100 μL / well of PBS was added, and after standing, the PBS was removed. 50 μL of digestion buffer was added to each well, and after digestion, 100 μL of McCoys 5A + 10% FBS complete medium was added to each well to stop the digestion. The entire cell suspension was transferred to a 96-well U-shaped plate. After centrifugation and discarding the supernatant, the cells were washed twice with pre-chilled FACS buffer, and 2 μg / mL of PE anti-human MICA / MICB Antibody (clone 6D4, biolegend#320906) was added. The plate was incubated on ice for 60 minutes. The cells were then washed three times with FACS buffer and the cell pellet was resuspended in FACS buffer. PE fluorescence signal was detected using a flow cytometer (Agilent). Results are as follows: Figure 1 As shown.

[0103] The results show that antibody M9 can inhibit the shedding of MICA / B from the surface of HCT-116 cells, and its inhibitory activity is comparable to or slightly stronger than that of the control antibody.

[0104] The above description is merely an embodiment of the present invention and is not intended to limit the present invention in any form or substance. It should be noted that those skilled in the art can make various improvements and additions without departing from the method of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention. Any modifications, alterations, and equivalent changes made by those skilled in the art based on the above-disclosed technical content without departing from the spirit and scope of the present invention are equivalent embodiments of the present invention. Furthermore, any modifications, alterations, and evolutions made to the above embodiments based on the essential technology of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. An 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 includes 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 includes 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; the antibody or its antigen-binding fragment specifically binds to MICA / B.

2. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody is a monoclonal antibody, single-chain antibody, chimeric antibody, humanized antibody, or human antibody.

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

4. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:7; the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

8.

5. The antibody or its antigen-binding fragment according to claim 2, characterized in that, The antibody comprises the heavy chain amino acid sequence shown in SEQ ID NO:11 and the light chain amino acid sequence shown in SEQ ID NO:

12.

6. The biological material relating to the antibody or antigen-binding fragment thereof according to any one of claims 1-5 is (i), (ii), or (iii) as follows: (i) A nucleic acid molecule encoding an antibody or an antigen-binding fragment thereof as described in any one of claims 1-5; (ii) an expression vector, which includes the nucleic acid molecules in (i); (iii) Host cells, including the nucleic acid molecules in (i) or the expression vectors in (ii).

7. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1-5 and / or the biological material according to claim 6, and optionally a pharmaceutically acceptable carrier.

8. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-5, the biological material of claim 6, or the pharmaceutical composition of claim 7 in the preparation of a kit for detecting MICA / B in biological samples.

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