Anti-mica / b antibodies and uses thereof

Abstract

The present application relates to an anti-MICA / B antibody and application thereof, and belongs to the field of biological medicine. Specifically, specific binding MICA / B antibody or antigen binding fragment thereof, nucleic acid molecule, expression vector, method for preparing the antibody or antigen binding fragment thereof, recombinant cell, composition, and particularly use in preparing a medicament for treating and / or preventing tumors are disclosed. Meanwhile, the provided antibody is specific, has high affinity, inhibits the shedding of MICA / B, and mediates intracellular endocytosis.

Description

Technical Field

[0001] This disclosure pertains to the field of biomedicine. Specifically, it relates to anti-MICA / B antibodies or antigen-binding fragments thereof, nucleic acid molecules, expression vectors, methods for preparing antibodies or antigen-binding fragments thereof, recombinant cells, compositions, and their use in the preparation of medicaments for the treatment and / or prevention of tumors. Background Technology

[0002] MICA (MHC class I polypeptide-related sequence A) and MICB (MHC class I polypeptide-related sequence B) are stress-inducible molecules located in the MHC-I region of human chromosome 6. Whalen KA et al., in their research on tumor immunity targeting NK cells, discovered that the α chain of the MICA / B protein is composed of three subunits: α1, α2, and α3. NK cells achieve their immune objectives by binding to the α1 and α2 subunits. Tumor cells utilize the α3 region of MICA / B to hydrolyze and detach MICA / B from the tumor cell surface using various proteases, resulting in soluble MICA / B. This soluble MICA / B binds to NKG2D, leading to NKG2D receptor endocytosis and degradation, thereby disrupting NKG2D's tumor immune surveillance function. This impairs NK cells' recognition and killing of tumor cells, allowing tumor cells to evade NK cell immune attacks. Because the MICA / B shedding process involves multiple proteases, small molecule inhibitors are difficult to specifically block it. Therefore, researchers hope to design a biomolecule that can inhibit MICA / B shedding without affecting the binding of NKG2D to MICA / B, thus achieving therapeutic goals. The MICA / B antibody drug CLN-619 can prevent the release of MICA / B proteolytically from tumor cells, enhance the binding of MICA / B to NKG2D, and alleviate the inhibition of MICA / B shedding.

[0003] Given the important role of MICA / B antibodies in the disease, there is a need to develop more therapeutic agents that target MICA / B. Summary of the Invention

[0004] This invention relates to anti-MICA / B antibodies, their antigen-binding fragments, and their applications.

[0005] The present invention provides an anti-MICA / B antibody or its antigen-binding fragment thereof, which comprises a heavy chain variable region and a light chain variable region, wherein: the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6.

[0006] In some embodiments, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:7.

[0007] In some embodiments, the heavy chain variable region comprises an amino acid sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:7.

[0008] In some embodiments, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:8.

[0009] In some embodiments, the light chain variable region comprises an amino acid sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:8.

[0010] In some embodiments, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:8.

[0011] In some embodiments, the antibody or its antigen-binding fragment is a Fab fragment, a Fab' fragment, an F(ab')2 fragment, or a single-chain Fv fragment (scFv).

[0012] In some implementations, the antibody is a murine antibody, a chimeric antibody, a human antibody, or a humanized antibody.

[0013] In some implementations, the antibody is a murine antibody.

[0014] In some implementations, the antibody is a chimeric antibody.

[0015] In some implementations, the antibody is a human antibody.

[0016] In some implementations, the antibody is a humanized antibody.

[0017] In some embodiments, the antibody or its antigen-binding fragment contains a heavy chain constant region derived from human IgG1, IgG2, IgG3 or IgG4.

[0018] In some embodiments, the antibody or its antigen-binding fragment comprises human IgG1.

[0019] In some embodiments, the antibody or its antigen-binding fragment comprises the IgG1 heavy chain constant region shown in SEQ ID NO:9.

[0020] 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.

[0021] In some embodiments, the antibody or its antigen-binding fragment comprises a light chain constant region derived from the human λ chain.

[0022] In some embodiments, the antibody or its antigen-binding fragment comprises a light chain constant region derived from the human κ 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 or its antigen-binding fragment has a heavy chain as shown in SEQ ID NO:11 and a light chain as 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] In another aspect, the present invention provides a nucleic acid molecule encoding an anti-MICA / B antibody or an antigen-binding fragment thereof as described in any one of the present invention.

[0028] Another aspect of the present invention provides a carrier comprising the nucleic acid molecule described herein.

[0029] In some embodiments, the vector includes bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors.

[0030] Another aspect of the present invention provides a host cell comprising the nucleic acid molecules described in the present invention, or the vector described in the present invention.

[0031] Another aspect of the present invention provides a method for preparing the antibody or antigen-binding fragment thereof described in the present invention, the method comprising culturing the cells described in the present invention under conditions that cause the expression of the antibody or antigen-binding fragment thereof that specifically binds to MICA / B as described in the present invention.

[0032] Another aspect of the present invention provides a pharmaceutical composition comprising: the antibody or antigen-binding fragment thereof described in the present invention, the nucleic acid molecule described therein, the carrier described therein and / or the cell described therein, and optionally a pharmaceutically acceptable carrier.

[0033] Another aspect of the present invention provides the use of the antibody or antigen-binding fragment thereof described in any one of the present invention, the nucleic acid molecule, the carrier, the cell, and / or the composition described herein in the preparation of a medicament for the prevention and / or treatment of diseases.

[0034] In some implementations, the disease is a MICA / B-mediated disease.

[0035] In some implementations, the disease is cancer, transplant rejection, autoimmune disease, or infectious disease.

[0036] In some implementations, the disease is cancer.

[0037] In some implementations, the disease is human colon cancer.

[0038] Another aspect of the present invention provides the use of the antibody or antigen-binding fragment thereof described in any one of the present invention, the nucleic acid molecule, the vector, the cell, and / or the composition described herein in the preparation of a diagnostic kit for detecting anti-MICA / B antibodies in biological samples.

[0039] The present invention provides an antibody that specifically binds to MICA / B and its preparation method. The antibody provided has high specificity and high affinity; the antibody provided has an inhibitory effect on preventing MICA / B shedding; at the same time, the antibody provided has the ability to mediate endocytosis with an endocytosis efficiency of 50.30%. Attached Figure Description

[0040] Figure 1 The image shows the result of antibody M8 inhibiting the shedding of MICA / B from HCT-116 cells. Detailed Implementation

[0041] 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.

[0042] 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.

[0043] Certain terms in this invention

[0044] 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.

[0045] 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.

[0046] 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.

[0047] 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 (scFv), linear antibodies, single-chain antibodies, nanobodies, domain antibodies, and multispecific antibodies.

[0048] In this invention, the term "mouse antibody" refers to a monoclonal antibody against human MICA / B prepared according to the knowledge and skills in the art. Preparation involves injecting the test subject with the MICA / B antigen, followed by isolating a hybridoma expressing an antibody with the desired sequence or functional characteristics. In a preferred embodiment of the invention, the mouse MICA / B antibody or its antigen-binding fragment may further comprise a light chain constant region of a mouse κ, λ chain or a variant thereof, or further comprise a heavy chain constant region of a mouse IgG1, IgG2, IgG3, or IgG4 or a variant thereof.

[0049] 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").

[0050] 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.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] The term “and / or” should be understood to mean any one of the options or any combination of two or more of the options.

[0059] 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.

[0060] Example 1: Preparation and purification of anti-MICA / B antibody

[0061] 1.1 Preparation and purification of antigens

[0062] 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).

[0063] 1.2 Construction of overexpression cell lines

[0064] HEK293T cells were processed at a rate of 5 × 10 5 Cells 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.

[0065] 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.

[0066] Add all viral supernatant to a solution containing 3×105 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).

[0067] 1.3 Obtaining monoclonal antibodies

[0068] 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.

[0069] 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 M8 was obtained. The amino acid sequence of the heavy chain variable region of the M8 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.

[0070] Table 1. Heavy chain variable region, light chain variable region, and CDR sequence of M8 monoclonal antibody

[0071]

[0072] 1.4 Preparation of control antibodies

[0073] 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.

[0074] Example 2 Antibody and Antigen Binding Experiment

[0075] 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.

[0076] 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 ELISA plate 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 an ELISA reader. The results are shown in Table 2.

[0077] Table 2. Binding of control antibody, M8, and MICA / MICB proteins to EC 50 value

[0078]

[0079] The results showed that antibody M8 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.

[0080] Example 3: Affinity detection of antibody with MICA / B

[0081] 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.

[0082] The specific experimental procedure is as follows: After activating the CM5 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.

[0083] Table 3. Affinity of antibodies to MICA and MICB proteins

[0084]

[0085] The results show that the affinity of antibody M8 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.

[0086] Example 4: Detection of antibody endocytic activity

[0087] 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. Endocytosis rate % = (MFI 4 °C - MFI 37 °C) / MFI 4 °C × 100%. The results are shown in Table 4.

[0088] Table 4. Antibody endocytosis

[0089]

[0090] The results show that antibody M8 has endocytic activity, and its endocytic activity is superior to that of the control antibody.

[0091] Example 5: Antibody inhibits MICA / B exfoliation from HCT-116 cells

[0092] First, HCT-116 cell suspension was collected by digestion and counted. The cell suspension was resuspended in McCoys 5A + 10% FBS complete medium to a concentration of 1E6 cells / ml, and 50 μL / well was added to 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 discarded. 50 μL of digestion solution 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 and incubated on ice for 60 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 Advanteon). Results are as follows: Figure 1 As shown.

[0093] The results show that antibody M8 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.

[0094] This invention has been illustrated through various specific embodiments. However, those skilled in the art will understand that this invention is not limited to the specific embodiments, and various modifications or variations can be made within the scope of this invention. Furthermore, the various technical features mentioned throughout this specification can be combined with each other without departing from the spirit and scope of this invention. Such modifications and variations are all within the scope of this invention.

Claims

1. An anti-MICA / B antibody or antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein: the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as set forth, in sequence, in SEQ ID NO: 1, SEQ ID NO 2, SEQ ID NO: 3, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as set forth, in sequence, in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO:

6.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein, The antibody is a murine antibody, a chimeric antibody, a human antibody or a humanized antibody.

3. The antibody or antigen-binding fragment thereof of claim 1, wherein, The heavy chain variable region amino acid sequence is set forth in SEQ ID NO: 7, and the light chain variable region amino acid sequence is set forth in SEQ ID NO:

8.

4. The antibody or antigen-binding fragment thereof of claim 1, wherein, The antibody or antigen-binding fragment thereof comprises a heavy chain constant region of IgGl, IgG2, IgG3 or IgG4 and a light chain constant region of kappa chain or lambda chain.

5. The antibody or antigen-binding fragment thereof of claim 1, wherein, The antibody or antigen-binding fragment thereof has a heavy chain set forth in SEQ ID NO: 11, and a light chain set forth in SEQ ID NO:

12.

6. A nucleic acid molecule encoding the anti-MICA / B antibody or antigen-binding fragment thereof according to any one of claims 1-5.

7. A vector comprising the nucleic acid molecule according to claim 6.

8. A host cell comprising the nucleic acid molecule of claim 6, or the vector of claim 7.

9. A pharmaceutical composition comprising: The antibody or antigen-binding fragment thereof according to any one of claims 1-5, the nucleic acid molecule of claim 6, the vector of claim 7 and / or the cell of claim 8, and optionally a pharmaceutically acceptable carrier.

10. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1-5, the nucleic acid molecule of claim 6, the vector of claim 7, the cell of claim 8 or the composition of claim 9 for the manufacture of a diagnostic kit for detecting MICA / B in a biological sample.

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