CN114369161A

CN114369161A - MICA antibody and application thereof

Info

Publication number: CN114369161A
Application number: CN202111626576.5A
Authority: CN
Inventors: 田志刚; 曹国帅; 肖卫华; 孙汭; 孙昊昱
Original assignee: Hefei Tiangang Immune Drugs Co ltd
Current assignee: Hefei Tiangang Immune Drugs Co ltd
Priority date: 2021-12-28
Filing date: 2021-12-28
Publication date: 2022-04-19
Anticipated expiration: 2041-12-28
Also published as: CN114369161B

Abstract

The invention provides a MICA antibody and application thereof, wherein the antibody or antigen binding fragment contains CDR sequences selected from at least one of the following sequences: heavy chain variable region CDR sequences: 1-3 of SEQ ID NO; light chain variable region CDR sequences: 4-6 of SEQ ID NO. The antibody prepared by the invention can be effectively combined with the alpha 3 domain of MICA, further, the NKG2D is promoted to be combined with MICA, and the antibody can be used for effectively treating or preventing MICA-mediated related diseases, such as promoting NK cells to kill tumors.

Description

MICA antibody and application thereof

Technical Field

The invention belongs to the field of biomedicine, and particularly relates to an MICA antibody and application thereof, and more particularly relates to an antibody or an antigen-binding fragment, an immunoconjugate, a composition, a kit for detecting MICA, application of the antibody or the antigen-binding fragment in preparation of the kit, a medicament, application of the antibody or the antigen-binding fragment and/or the immunoconjugate and/or the composition in preparation of the medicament, a nucleic acid, a recombinant vector or a transformant.

Background

NK cells are an important kind of inherent lymphocytes in the body, and play an important role in resisting viruses and tumors. NK cells express a variety of activating receptors on their surface, including NKG2D, NKp30, and the like. NK cells bind through these activating receptors to ligands expressed on the surface of tumor cells. These ligands are encoded by self genes and are expressed on the cell surface in the case of malignant transformation (tumor) of cells, etc.

NKG2D forms homodimers in NK cells and hexamers with four DAP10 molecules. After the ligand such as MICA, MICB and the like is combined, the NKG2D hexamer transmits an activation signal to NK cells, promotes the NK cells to secrete cytokines such as IFN-gamma and the like, and kills target cells expressing the ligand.

MICA and MICB are proteins encoded by self genes and are not expressed in normal tissues and cells; when the cells are maliciously transformed into tumor cells, MICA and MICB express on the surface of the tumor cells. NK cells recognize and kill tumors through NKG2D-MICA/B interaction.

Tumors then down-regulate cell surface MICA/B expression by a variety of mechanisms, including matrix metalloproteinase-mediated MICA/B shedding. MICA/B is a type I transmembrane protein, the extracellular domain of which comprises three domains of alpha 1, alpha 2 and alpha 3, and is combined with NKG2D through the domains of alpha 1 and alpha 2. Most of the extracellular domain amino acids of MICA/B (including a portion of the α 1, α 2 and α 3 domains) are shed from the surface of tumor cells by matrix metalloproteinases. NKG2D is unable to bind the part of the α 3 domain remaining on the cell surface, and thus tumors are able to escape immune surveillance by NK cells.

Research shows that the MICA antibody combined with the alpha 3 structural domain can inhibit the shedding of MICA from the surface of a tumor, further promote NKG2D-MICA/B mediated NK cell recognition and inhibit tumor immune escape, so that the development of the MICA antibody has important significance for treating the tumor.

Disclosure of Invention

The present application is based on the discovery and recognition by the inventors of the following facts and problems:

since MICA is only expressed in tumor cells, the production of specific antibodies targeting MICA is a potential means of treating tumors. In the application, the inventor takes MICA extracellular region Fc fusion protein (MICA-Fc) as an antigen, after immunizing a mouse, the mouse is screened for a plurality of times to obtain a mouse containing anti-human MICA immunoglobulin, and the mouse is subjected to subclone screening to obtain a target MICA antibody, and through detection, the MICA antibody can be combined with MICA, and further, the combination of the MICA and an NK cell activating receptor NKG2D is promoted, and the NK cell killing tumor is promoted.

Accordingly, in a first aspect of the invention, the invention provides an antibody or antigen-binding fragment. According to an embodiment of the invention, the CDR sequences comprise at least one of the following: heavy chain variable region CDR sequences: 1-3 of SEQ ID NO; light chain variable region CDR sequences: 4-6 of SEQ ID NO. The antibody or antigen-binding fragment according to the embodiment of the present invention can effectively bind to the α 3 domain of MICA, and further, promotes NKG2D binding to MICA, and has a good effect of preventing and/or treating MICA-mediated diseases, such as promoting NK cell killing of tumors.

GYTFTDYG(SEQ ID NO:1)。

INTYTGEP(SEQ ID NO:2)。

TRLSTVAVGAWFEY(SEQ ID NO:3)。

QNIVHSNGNTY(SEQ ID NO:4)。

KVS(SEQ ID NO:5)。

FQGSHVPT(SEQ ID NO:6)。

In a second aspect of the invention, the invention provides an immunoconjugate. According to an embodiment of the invention, there is included an antibody or antigen-binding fragment according to the first aspect. As described above, the antibody or antigen-binding fragment of the present embodiment can effectively bind to the α 3 domain of MICA protein, and further, promotes NKG2D binding to MICA, and has a good effect of preventing and/or treating MICA-mediated diseases, such as promoting NK cell killing of tumors.

In a third aspect of the invention, a composition is provided. According to an embodiment of the invention, an antibody according to the first aspect and/or an immunoconjugate according to the second aspect is comprised. As described above, the antibody or antigen-binding fragment and the immunoconjugate according to the embodiments of the present invention can effectively bind to the α 3 domain of MICA, and thus, the composition comprising the same has the same effect, and further, the composition can effectively promote the binding of NKG2D to MICA, and the antibody or antigen-binding fragment has a good effect of preventing and/or treating MICA-mediated diseases, such as promoting NK cell killing of tumors.

In a fourth aspect of the invention, the invention features a kit for detecting MICA. According to an embodiment of the invention, the kit comprises: the antibody or antigen-binding fragment of the first aspect. As described above, the antibody or antigen-binding fragment of the present embodiment can effectively bind to the α 3 domain of MICA, and thus, the antibody or antigen-binding fragment can be used for detecting MICA, and further, can be used for preparing a kit for detecting MICA, which can effectively detect MICA, and which can be used for scientific research, such as qualitative or quantitative detection of MICA protein molecules in a biological sample.

In a fifth aspect of the invention, the invention provides the use of an antibody or antigen-binding fragment of the first aspect in the preparation of a kit. According to an embodiment of the invention, the kit is for detecting MICA. As described above, the antibody or antigen-binding fragment of the present embodiment can effectively bind to the α 3 domain of MICA, and thus, the antibody or antigen-binding fragment can be used for detecting MICA, and further, can be used for preparing a kit for detecting MICA.

In a sixth aspect of the invention, a medicament is presented. According to an embodiment of the invention, comprising: the antibody or antigen-binding fragment of the first aspect and/or the immunoconjugate of the second aspect and/or the composition of the third aspect. As described above, the antibody or antigen-binding fragment, immunoconjugate, and composition according to the embodiment of the present invention each have a substance capable of effectively binding to the α 3 domain of MICA, and thus, a drug comprising the above-mentioned substance can effectively bind to the α 3 domain of MICA, further, promote NKG2D to bind to MICA, and further promote NK cell action, such as killing tumor cells, and thus the drug has a significant effect of preventing and/or treating MICA-mediated diseases.

In a seventh aspect of the invention, the invention provides the use of an antibody or antigen-binding fragment of the first aspect and/or an immunoconjugate of the second aspect and/or a composition of the third aspect in the manufacture of a medicament. According to an embodiment of the present invention, the medicament is for preventing and/or treating a MICA-mediated disease. As described above, the antibody or antigen-binding fragment, immunoconjugate, and composition according to the embodiment of the present invention each have a substance capable of effectively binding to the α 3 domain of MICA, and thus, a drug comprising the above-mentioned substance can effectively bind to the α 3 domain of MICA, further, promote NKG2D to bind to MICA, and further promote NK cell action, such as killing tumor cells, and thus the drug has a significant effect of preventing and/or treating MICA-mediated diseases.

In an eighth aspect of the invention, a nucleic acid is provided. According to an embodiment of the invention, the nucleic acid encodes the antibody or antigen-binding fragment of the first aspect. The antibody or antigen-binding fragment encoded by the nucleic acid according to the embodiment of the present invention can effectively bind to the alpha 3 domain of MICA, and further, promotes NKG2D to bind to MICA, and the protein encoded by the nucleic acid has a good effect of preventing and/or treating MICA-mediated diseases.

In a ninth aspect of the present invention, the present invention provides a recombinant vector or transformant. According to an embodiment of the present invention, the nucleic acid according to the eighth aspect is contained. The expression vector may include optional control sequences operably linked to the nucleic acid molecule. Wherein the control sequence is one or more control sequences that direct the expression of the nucleic acid molecule in a host. The recombinant vector provided by the embodiment of the invention can efficiently express the antibody or the antigen-binding fragment in a suitable host cell, the antibody or the antigen-binding fragment can be effectively combined with the alpha 3 domain of the MICA, and the antibody or the antigen-binding fragment has good effects of preventing and/or treating MICA-mediated diseases, such as promoting NK cell killing of tumors.

In a tenth aspect of the invention, a recombinant cell is provided. According to an embodiment of the invention, the recombinant cell carries the nucleic acid of the eighth aspect, the recombinant vector or transformant of the ninth aspect, or the antibody or antigen-binding fragment of the first aspect. The recombinant cell is obtained by transfection or transformation of the expression vector. According to the embodiment of the present invention, the recombinant cell can efficiently express the above antibody under suitable conditions, the antibody can effectively bind to the alpha 3 domain of MICA, and further, the antibody or the antigen-binding fragment has a good effect of preventing and/or treating MICA-mediated diseases, such as promotion of NK cell killing of tumors.

Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.

Drawings

The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:

FIG. 1 is a graph of the results of an ELISA assay for binding of MICA antibodies to human MICA according to an embodiment of the present invention;

FIG. 2 is a graph of the results of an ELISA assay for binding of MICA antibody to MICA α 3 domain according to an embodiment of the present invention;

FIG. 3 is a graph showing the results of detection of MICA antibody binding to CHO-K1-MICA cells according to an embodiment of the present invention;

FIG. 4 is a graph showing the results of detection of MICA antibody binding to CHO-K1-MICB cells according to an embodiment of the present invention;

FIG. 5 is a graph showing the results of detection of MICA antibody binding to CHO-K1-Rhesuus MIC1 cells according to an embodiment of the present invention;

FIG. 6 is a graph showing the results of detection of MICA antibody binding to CHO-K1-Rhesus MICB cells according to an embodiment of the present invention;

FIG. 7 is a graph showing the results of MICA antibody binding to lung cancer NCI-H1299 cells in accordance with an embodiment of the present invention;

FIG. 8 is a graph showing the results of using MICA antibodies for Western blot detection according to an embodiment of the present invention;

FIG. 9 is a graph of the results of MICA antibodies promoting the binding of NKG2D-Fc to CHO-K1-MICA cells according to an embodiment of the present invention; and

figure 10 is a graph showing the results of MICA antibodies promoting NK92 cell killing of a375 cells according to an embodiment of the present invention.

Detailed Description

Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention.

Furthermore, the terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. In the description of the present invention, "a plurality" means at least two, e.g., two, three, etc., unless specifically limited otherwise.

The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.

In order that the invention may be more readily understood, certain technical and scientific terms are specifically defined below. Unless clearly defined otherwise herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The abbreviations for amino acid residues are standard 3-letter and/or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.

Herein, certain regions in the variable region have a higher degree of variation in amino acid composition and arrangement order, and are called Hypervariable regions (HVRs), which are the positions where antigens and antibodies bind, and thus are also called complementarity-determining regions (CDRs). The heavy chain variable region and the light chain variable region both have three CDR regions.

Herein, "antibody" as previously described, an antibody of the invention may be full-length (e.g., an IgG1 or IgG4 antibody) or comprise only an antigen-binding portion (e.g., a Fab, F (ab') 2, or scFv fragment), or may be modified to affect function. The invention includes anti-MICA antibodies with modified glycosylation patterns. In some applications, it may be useful to modify to remove undesired glycosylation sites, or antibodies that do not have a fucose moiety on the oligosaccharide chain, for example, to enhance antibody-dependent cellular cytotoxicity (ADCC) function. In other applications, galactosylation modifications can be made to alter Complement Dependent Cytotoxicity (CDC).

The term "antigen-binding fragment" as used herein refers in particular to a partial fragment of an antibody such as Fv, scFv (sc refers to single chain), Fab, F (ab ') 2, Fab', scFv-Fc fragment or diabody (diabody), or any fragment that should be able to increase half-life by chemical modification, e.g. addition of a poly (alkylene) glycol such as polyethylene glycol ("pegylation, pegylation") (pegylated fragments known as Fv-PEG, scFv-PEG, Fab-PEG, F (ab ') 2-PEG or Fab' -PEG) ("PEG" being polyethylene glycol), or by incorporation into liposomes, said fragment having MICA binding activity. Preferably, the antigen-binding fragment will consist of or comprise a partial sequence of the light and heavy chain variable regions of the antibody from which it is derived, sufficient to retain the same binding specificity and sufficient affinity as the antibody from which it is derived, preferably 1/100 being at least equal to the affinity of the antibody from which it is derived, and more preferably 1/10 in the case of MICA. Such antigen-binding fragments will comprise a minimum of 5 amino acids, preferably 10, 15, 25, 50 and 100 consecutive amino acids of the antibody sequence from which they are derived.

Herein, to further improve the bioavailability of the antibody, the antibody may also be humanized, i.e., the antibody is a chimeric or humanized antibody. The term "chimeric antibody" refers to a recombinant antibody obtained by replacing the amino acid sequence of a constant region of a monoclonal antibody from one species (e.g., mouse) with the amino acid sequence of a constant region of an antibody from another species (e.g., human) using recombinant DNA techniques. The term "humanized antibody" refers to a recombinant antibody obtained by replacing all of the amino acid sequences of the non-CDR (Fv Framework Region (FR)) of the constant region and variable region of a monoclonal antibody from one species (e.g., mouse) with the amino acid sequences of the non-CDR of the constant region and variable region of an antibody from another species (e.g., human) using recombinant DNA techniques. That is, the constant region of an antibody is called a chimeric antibody when it is humanized, and the non-CDR amino acid sequences of the constant region and the variable region are all humanized and called a humanized antibody. The humanization method can be performed by referring to conventional antibody engineering techniques, and will not be described herein.

Homology, the present invention, in order to compare two or more nucleotide sequences, the percentage of "sequence homology" between a first sequence and a second sequence can be calculated by dividing [ the number of nucleotides in the first sequence that are identical to the nucleotide at the corresponding position ]. Nucleotide in the second sequence ] minus [ the total number of nucleotides in the first sequence ], and then multiplied by [ 100% ], wherein the deletion, insertion, substitution, or addition of each nucleotide in the second nucleotide sequence-relative to the first nucleotide sequence-is considered to be a difference in a single nucleotide (position).

Alternatively, the degree of sequence identity between two or more nucleotide sequences can be calculated using standard settings using known computer algorithms for sequence alignment, such as NCBI Blast v 2.0.

Some other techniques, computer algorithms and settings for determining the degree of sequence identity are for example described in WO 04/037999, EP 0967284, EP 1085089, WO 00/55318, WO 00/78972, WO 98/49185 and GB 2357768-A.

The term "(substantial) homology" with respect to polypeptides means that two polypeptides or designated sequences thereof are at least about 80% amino acids, usually at least about 90% to 95%, and more preferably at least about 98% to 99.5% amino acids identical when optimally aligned and compared (with appropriate insertion or deletion of nucleotides).

The% homology between two sequences varies with the number of identical positions shared by the sequences when optimally aligning the sequences (i.e.,% homology is the number of identical positions/total number of positions x 100), wherein the optimal alignment is determined taking into account the number of empty positions that need to be introduced to achieve optimal alignment of the two sequences and the length of each empty position. Sequence comparison and percent identity determination between two sequences can be accomplished using mathematical algorithms, as described in the non-limiting examples below.

One skilled in the art can substitute, add and/or delete one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more) amino acids to the sequences of the invention to obtain variants of the sequences of the antibodies or functional fragments thereof, without substantially affecting the activity of the antibodies (retaining at least 95% of the activity). All of which are considered to be included within the scope of the present invention. Such as the substitution of amino acids with similar properties in the variable region. The sequence of a variant of the invention may have at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity (or homology) to a reference sequence. The sequence identity described in the present invention can be measured using sequence analysis software. For example the computer program BLAST, in particular BLASTP or TBLASTN, using default parameters. The amino acid sequences mentioned in the present invention are shown from N-terminus to C-terminus.

Herein, "conservatively modified forms of an amino acid sequence" refers to amino acid modifications that do not significantly affect or alter the binding characteristics of an antibody comprising the amino acid sequence, including amino acid substitutions, additions and deletions. Modifications can be introduced into the antibodies of the invention by standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are those in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been identified in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine tryptophan, histidine). Thus, one or more amino acid residues in a CDR region of an antibody of the invention can be replaced with other amino acid residues from the same side chain family, and the altered antibody can be tested for retained function using the functional assay methods described herein. Preferably, the conservative modifications do not exceed 1 or 2 in number.

"operably linked" herein refers to the attachment of a foreign gene to a vector such that control elements within the vector, such as transcriptional and translational control sequences and the like, are capable of performing their intended function of regulating the transcription and translation of the foreign gene.

In one aspect of the invention, the invention provides an antibody or antigen-binding fragment comprising a CDR sequence selected from at least one of: heavy chain variable region CDR sequences: 1-3 or a conservatively modified form thereof; light chain variable region CDR sequences: 4-6 or conservative modified form thereof. After conservative modification is carried out on any one of the amino acid sequences shown in SEQ ID NO. 1-6, the binding property of an antibody or an antigen binding fragment containing the amino acid sequence is not influenced or changed, so that at least one antibody or antigen binding fragment containing at least one of the amino acid sequences shown in SEQ ID NO. 1-6 and/or at least one antibody or antigen binding fragment containing the amino acid sequence obtained after conservative modification is carried out on the amino acid sequences shown in SEQ ID NO. 1-6 according to the embodiment of the invention can be effectively bound with an alpha 3 domain of MICA, further, NKG2D is promoted to be bound with MICA, and the MICA-mediated related diseases can be effectively treated or prevented, such as NK cell killing tumor promotion.

GYTFTDYG(SEQ ID NO:1)。

INTYTGEP(SEQ ID NO:2)。

TRLSTVAVGAWFEY(SEQ ID NO:3)。

QNIVHSNGNTY(SEQ ID NO:4)。

KVS(SEQ ID NO:5)。

FQGSHVPT(SEQ ID NO:6)。

According to some specific embodiments of the present invention, the antibody or antigen-binding fragment may further comprise at least one of the following additional technical features:

heavy chain CDR1 comprises the amino acid sequence shown in SEQ ID NO. 1 or a conservatively modified form thereof,

heavy chain CDR2 comprises the amino acid sequence shown in SEQ ID NO. 2 or an amino acid sequence of a conservatively modified version thereof,

heavy chain CDR3 comprises the amino acid sequence shown in SEQ ID NO. 3 or a conservatively modified form thereof,

light chain CDR1 includes the amino acid sequence set forth in SEQ ID NO. 4 or a conservatively modified version thereof,

light chain CDR2 includes the amino acid sequence set forth in SEQ ID NO. 5 or a conservatively modified version thereof,

light chain CDR3 includes the amino acid sequence set forth in SEQ ID NO. 6 or a conservatively modified version thereof.

According to some embodiments of the invention, the gene encoding the CDR of the heavy chain variable region has at least one of the nucleotide sequences shown in SEQ ID NO. 15-17.

GGTTATACTTTTACTGACTACGGG(SEQ ID NO:15)。

ATAAACACATACACTGGCGAGCCC(SEQ ID NO:16)。

ACCCGCTTGTCTACCGTGGCCGTTGGGGCTTGGTTCGAGTAC(SEQ ID NO:17)。

According to some embodiments of the invention, the gene encoding the CDR of the light chain variable region has at least one of the nucleotide sequences shown in SEQ ID NO 18-20.

CAGAACATCGTGCACTCCAATGGGAACACTTAC(SEQ ID NO:18)。

AAAGTCTCT(SEQ ID NO:19)。

TTTCAGGGCTCACACGTGCCAACC(SEQ ID NO:20)。

According to some specific embodiments of the invention, the heavy chain variable region comprises an amino acid sequence that is at least 80% homologous to at least one of the amino acid sequence set forth in SEQ ID No. 7 and conservatively modified versions thereof; and/or, (ii) the light chain variable region comprises an amino acid sequence having at least 80% homology with at least one of the amino acid sequence shown in SEQ ID NO. 8 and the amino acid sequence of the warranted modified form thereof.

QIQLVQSGPELKKPGESVKISCKASGYTFTDYGINWVKQAPGKGLKWVGWINTYTGEPINADDFKGRFALSLETSANTAYLQINNLKNEDMATYFCTRLSTVAVGAWFEYWGQGTLVTVSA(SEQ ID NO:7)。

DVLMTQTPLSLPVSLGDQASISCRSSQNIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPTFGGGTKLEIK(SEQ ID NO:8)。

According to some embodiments of the invention, the gene encoding the heavy chain variable region has the nucleotide sequence shown in SEQ ID NO 21.

CAGATCCAGTTGGTGCAATCTGGACCTGAACTGAAAAAACCCGGCGAGTCCGTGAAAATCAGCTGTAAAGCATCTGGTTATACTTTTACTGACTACGGGATTAATTGGGTTAAGCAGGCACCCGGCAAAGGTTTGAAGTGGGTGGGGTGGATAAACACATACACTGGCGAGCCCATCAACGCTGATGATTTCAAGGGCAGGTTCGCCCTGTCCCTGGAGACCTCAGCCAATACTGCTTACCTGCAGATTAATAATTTGAAAAACGAAGACATGGCCACCTATTTCTGCACCCGCTTGTCTACCGTGGCCGTTGGGGCTTGGTTCGAGTACTGGGGCCAAGGCACCCTGGTGACTGTAAGTGCC(SEQ ID NO:21)。

According to some embodiments of the invention, the gene encoding the light chain variable region has the nucleotide sequence shown in SEQ ID NO. 22.

GACGTGCTCATGACACAAACTCCACTTTCCTTGCCTGTGTCCCTCGGAGACCAGGCCAGCATCTCTTGCAGGTCATCTCAGAACATCGTGCACTCCAATGGGAACACTTACCTGGAATGGTACCTGCAGAAACCAGGACAGTCTCCTAAACTGTTGATTTACAAAGTCTCTAACCGCTTTTCCGGCGTGCCAGATCGTTTTTCAGGCAGTGGGTCCGGAACTGATTTTACCCTGAAAATTTCAAGGGTTGAAGCTGAGGATCTGGGAGTGTACTATTGCTTTCAGGGCTCACACGTGCCAACCTTTGGGGGCGGGACAAAGCTGGAGATTAAG(SEQ ID NO:22)。

According to some specific embodiments of the invention, the heavy chain variable region comprises an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% homologous to a heavy chain variable region selected from (i); (iii) the light chain variable region comprises an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% homologous to the light chain variable region selected from (ii).

According to some specific embodiments of the invention, further comprising an Fc region, at least a portion of which is derived from at least one of a murine antibody, a human antibody, a primate antibody, or a mutant thereof;

according to some specific embodiments of the invention, at least a portion of the Fc region is from a murine, human, primate IgG antibody or mutant thereof.

According to some specific embodiments of the invention, at least a portion of the Fc region is from a murine IgG2a antibody, a human IgG1 antibody, or a mutant thereof.

According to some specific embodiments of the invention, at least a portion of the Fc region is from a murine IgG2a antibody or a mutant thereof.

According to some specific embodiments of the invention, the Fc region has an amino acid sequence as set forth in SEQ ID NO. 14.

AKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK(SEQ ID NO:14)。

According to some specific embodiments of the invention, the antibody or antigen-binding fragment comprises: a heavy chain comprising the amino acid sequence shown in SEQ ID NO. 9 and a light chain comprising the amino acid sequence shown in SEQ ID NO. 10.

QIQLVQSGPELKKPGESVKISCKASGYTFTDYGINWVKQAPGKGLKWVGWINTYTGEPINADDFKGRFALSLETSANTAYLQINNLKNEDMATYFCTRLSTVAVGAWFEYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK(SEQ ID NO:9)

DVLMTQTPLSLPVSLGDQASISCRSSQNIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC(SEQ ID NO:10)

According to some specific embodiments of the invention, the antibody or antigen-binding fragment is IgG1, IgG2, or IgG 4.

According to some specific embodiments of the invention, the antibody is a monoclonal antibody, a murine antibody, a chimeric antibody, a humanized antibody, a human antibody, Fv, a single chain antibody (scFv), Fab '-SH, or F (ab') 2.

According to some specific embodiments of the invention, the antibody or antigen-binding fragment thereof can bind to the amino acid sequence shown in SEQ ID NO. 11.

MGLGPVFLLLAGIFPFAPPGAAAEPHSLRYNLTVLSWDGSVQSGFLTEVHLDGQPFLRCDRQKCRAKPQGQWAEDVLGNKTWDRETRDLTGNGKDLRMTLAHIKDQKEGLHSLQEIRVCEIHEDNSTRSSQHFYYDGELFLSQNLETKEWTMPQSSRAQTLAMNVRNFLKEDAMKTKTHYHAMHADCLQELRRYLKSGVVLRRTVPPMVNVTRSEASEGNITVTCRASGFYPWNITLSWRQDGVSLSHDTQQWGDVLPDGNGTYQTWVATRICQGEEQRFTCYMEHSGNHSTHPVPSGKVLVLQSHWQTFHVSAVAAAAIFVIIIFYVRCCKKKTSAAEGPELVSLQVLDQHPVGTSDHRDATQLGFQPLMSDLGSTGSTEGA(SEQ ID NO:11)。

In another aspect of the invention, the invention provides an immunoconjugate comprising a therapeutic agent and the above antibody or antigen-binding fragment coupled to the therapeutic agent. As described above, the antibody or antigen-binding fragment of the present embodiment, which is capable of effectively binding to the α 3 domain of MICA protein and further promoting NKG2D to bind to MICA, has a good effect of preventing and/or treating MICA-mediated diseases, such as promoting NK cell killing of tumor, and also has the above-mentioned function as an immunoconjugate obtained by coupling the antibody or antigen-binding fragment with a therapeutic agent.

In a further aspect of the invention, the invention provides a composition comprising an antibody or antigen-binding fragment thereof as hereinbefore described and/or an immunoconjugate as hereinbefore described. As described above, the antibody or antigen-binding fragment, and the immunoconjugate according to the embodiments of the present invention can effectively bind to the α 3 domain of MICA, and thus, the composition comprising the same has the same effect as a food composition or a pharmaceutical composition, and further, the composition can effectively promote NKG2D to bind to MICA, and has a good effect of preventing and/or treating MICA-mediated diseases, such as promoting NK cell killing of tumors.

According to a specific embodiment of the present invention, the composition further comprises a pharmaceutically acceptable carrier.

It is to be noted that the compositions include temporally and/or spatially separated combinations as long as they can work together to achieve the objects of the present invention. For example, the ingredients contained in the composition may be administered to the subject in bulk, or separately. When the ingredients contained in the composition are administered separately to a subject, the individual ingredients may be administered to the subject simultaneously or sequentially.

In one aspect of the invention, the invention provides a kit for detecting MICA, wherein the kit comprises the antibody or antigen-binding fragment described above. As described above, the antibody or antigen-binding fragment of the present embodiment can effectively bind to the α 3 domain of MICA, and thus, the antibody or antigen-binding fragment can be used for detecting MICA, and further, can be used for preparing a kit for detecting MICA, which can effectively detect MICA, which can be used for scientific research, such as qualitative or quantitative detection of MICA protein molecules in a biological sample, and more particularly, can be used for immunoblotting, immunoprecipitation, etc., involving a kit for detecting using specific binding properties of MICA and an antibody, etc. These kits may comprise any one or more of the following: an antagonist, a MICA antibody or a drug reference material; a protein purification column; an immunoglobulin affinity purification buffer; assay diluent for cells. MICA antibodies can be used in different types of diagnostic tests, for example to detect a wide variety of diseases or the presence of drugs, toxins or other proteins, etc., in vitro or in vivo. For example, the MICA-associated disease can be tested by testing the serum or blood of the subject.

In another aspect of the invention, the invention features the use of an antibody or antigen-binding fragment thereof as described above in the preparation of a kit for the detection of MICA. As described above, the antibody or antigen-binding fragment of the present embodiment can effectively bind to the α 3 domain of MICA, and thus, the antibody can be used for detecting MICA, and further, can be used for preparing a kit for detecting MICA, which can effectively detect MICA, which can be used for scientific research, such as qualitative or quantitative detection of MICA protein molecules in a biological sample, more specifically, for immunoblotting, immunoprecipitation, etc., involving a kit for detection using the specific binding property of MICA and the antibody or binding fragment thereof, etc. These kits may comprise any one or more of the following: an antagonist, a MICA antibody or a drug reference material; a protein purification column; an immunoglobulin affinity purification buffer; assay diluent for cells. MICA antibodies or antigen fragments can be used in different types of diagnostic tests, for example to detect a wide variety of diseases or the presence of drugs, toxins or other proteins, etc., in vitro or in vivo. For example, the MICA-associated disease can be tested by testing the serum or blood of the subject.

In yet another aspect of the invention, the invention provides a medicament comprising: an antibody or antigen-binding fragment as hereinbefore described and/or a conjugate as hereinbefore described and/or a composition as hereinbefore described for use in the prophylaxis and/or treatment of MICA mediated diseases. As described above, the antibody or antigen-binding fragment, immunoconjugate, and composition according to the embodiment of the present invention each have a substance capable of effectively binding to the α 3 domain of MICA, and thus, a drug comprising the above-mentioned substance can effectively bind to the α 3 domain of MICA, and further, can promote the binding of NKG2D to MICA, and the drug has a significant effect of preventing and/or treating MICA-mediated diseases.

According to some embodiments of the present invention, the above-mentioned medicament may further comprise at least one of the following additional technical features:

according to some embodiments of the invention, the MICA-mediated disease is cancer or transplant rejection, an autoimmune disease, an infectious disease.

According to some embodiments of the invention, the cancer is at least one of lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, stomach cancer, esophageal cancer, oral squamous cell carcinoma and head and neck cancer.

According to some embodiments of the invention, a pharmaceutically acceptable carrier and an effective amount of the antibody active ingredient are included.

As used herein, the term "effective amount" or "effective dose" refers to an amount that produces a function or activity in, and is acceptable to, a human and/or an animal.

As used herein, a "pharmaceutically acceptable" component is one that is suitable for use in humans and/or mammals without undue adverse side effects (such as toxicity, irritation, and allergic response), i.e., at a reasonable benefit/risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, including various excipients and diluents.

The medicament of the invention contains a safe and effective amount of the active ingredient of the invention and a pharmaceutically acceptable carrier. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The medicament preparation is matched with the administration mode generally, and the medicament of the invention is in the dosage forms of injection, oral preparation (tablet, capsule, oral liquid), transdermal agent and sustained release agent. For example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. The medicament is preferably manufactured under sterile conditions.

The effective amount of the active ingredient of the present invention may vary depending on the mode of administration and the severity of the disease to be treated, etc. The selection of a preferred effective amount can be determined by one of ordinary skill in the art based on a variety of factors (e.g., by clinical trials). Such factors include, but are not limited to: pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the weight of the patient, the immune status of the patient, the route of administration, and the like. For example, divided doses may be administered several times per day, or the dose may be proportionally reduced, as may be required by the urgency of the condition being treated.

The pharmaceutically acceptable carrier of the present invention includes (but is not limited to): water, saline, liposomes, lipids, proteins, protein-antibody conjugates, peptidic substances, cellulose, nanogels, or combinations thereof. The choice of carrier should be matched with the mode of administration, which is well known to those skilled in the art.

In one aspect of the invention, the invention provides a nucleic acid comprising a sequence encoding an antibody or antigen-binding fragment thereof as described above. The antibody or antigen-binding fragment encoded by the nucleic acid according to the embodiment of the present invention can effectively bind to the alpha 3 domain of MICA, and further, promotes NKG2D to bind to MICA, and the protein encoded by the nucleic acid has a good effect of preventing and/or treating MICA-mediated diseases.

According to a particular embodiment of the invention, the nucleic acid has the nucleotide sequence shown in SEQ ID NO 12, 13.

The gene encoding the heavy chain of the antibody or antigen-binding fragment has the nucleotide sequence shown below:

CAGATCCAGTTGGTGCAATCTGGACCTGAACTGAAAAAACCCGGCGAGTCCGTGAAAATCAGCTGTAAAGCATCTGGTTATACTTTTACTGACTACGGGATTAATTGGGTTAAGCAGGCACCCGGCAAAGGTTTGAAGTGGGTGGGGTGGATAAACACATACACTGGCGAGCCCATCAACGCTGATGATTTCAAGGGCAGGTTCGCCCTGTCCCTGGAGACCTCAGCCAATACTGCTTACCTGCAGATTAATAATTTGAAAAACGAAGACATGGCCACCTATTTCTGCACCCGCTTGTCTACCGTGGCCGTTGGGGCTTGGTTCGAGTACTGGGGCCAAGGCACCCTGGTGACTGTAAGTGCCGCCAAAACAACTGCACCATCTGTTTATCCACTGGCCCCCGTTTGCGGGGATACCACTGGTAGCTCTGTCACCCTCGGCTGTCTGGTCAAAGGATATTTCCCCGAGCCTGTGACACTTACCTGGAATTCAGGCAGCTTGTCCTCCGGCGTGCATACTTTCCCTGCAGTCCTGCAGTCAGATCTGTACACCCTCAGCTCATCTGTGACTGTCACAAGTTCTACCTGGCCATCCCAGAGTATTACATGCAACGTGGCCCACCCAGCAAGTTCAACAAAAGTTGACAAGAAGATTGAGCCAAGAGGCCCTACTATCAAGCCCTGTCCCCCCTGTAAGTGTCCTGCACCCAATCTGCTGGGGGGCCCATCTGTGTTTATTTTCCCCCCAAAGATTAAGGACGTCCTCATGATTAGCCTGTCCCCAATCGTGACATGTGTGGTGGTGGATGTTTCTGAGGACGACCCAGACGTACAGATCTCTTGGTTCGTGAACAATGTCGAGGTGCATACAGCCCAGACCCAGACCCATCGGGAAGACTACAACTCTACATTGAGAGTGGTGTCCGCTTTGCCCATCCAGCATCAGGACTGGATGTCCGGCAAGGAGTTTAAATGTAAGGTCAACAACAAGGACCTGCCCGCTCCAATAGAGAGAACTATCTCAAAGCCTAAAGGTAGTGTTCGAGCCCCCCAGGTATACGTACTGCCACCCCCTGAGGAGGAGATGACCAAGAAGCAGGTGACACTGACCTGCATGGTGACAGACTTCATGCCAGAAGACATTTATGTCGAATGGACTAATAATGGCAAGACCGAACTGAATTATAAAAATACCGAACCAGTGCTGGACTCCGACGGGTCCTATTTCATGTACTCTAAGCTCCGTGTCGAAAAGAAGAACTGGGTGGAACGAAACTCTTACTCCTGCAGTGTTGTGCACGAGGGGCTTCACAACCATCATACAACCAAGTCCTTCTCCAGGACACCTGGGAAG(SEQ ID NO:12)。

the gene encoding the light chain of the antibody or antigen-binding fragment has the nucleotide sequence shown below:

GACGTGCTCATGACACAAACTCCACTTTCCTTGCCTGTGTCCCTCGGAGACCAGGCCAGCATCTCTTGCAGGTCATCTCAGAACATCGTGCACTCCAATGGGAACACTTACCTGGAATGGTACCTGCAGAAACCAGGACAGTCTCCTAAACTGTTGATTTACAAAGTCTCTAACCGCTTTTCCGGCGTGCCAGATCGTTTTTCAGGCAGTGGGTCCGGAACTGATTTTACCCTGAAAATTTCAAGGGTTGAAGCTGAGGATCTGGGAGTGTACTATTGCTTTCAGGGCTCACACGTGCCAACCTTTGGGGGCGGGACAAAGCTGGAGATTAAGCGAGCTGATGCTGCCCCCACTGTCTCTATTTTTCCTCCTAGTAGTGAGCAGCTGACATCTGGAGGCGCCTCCGTGGTGTGCTTCCTGAATAACTTTTACCCTAAGGACATCAATGTGAAGTGGAAGATCGACGGGAGTGAGAGGCAGAATGGCGTACTGAATTCCTGGACTGATCAGGACTCTAAAGATAGCACCTATAGCATGTCCTCCACCCTCACACTGACAAAGGACGAATACGAACGGCATAACTCCTATACATGTGAGGCTACCCACAAGACATCCACCTCACCAATCGTCAAGAGCTTCAACCGGAATGAGTGT(SEQ ID NO:13)。

it is to be noted that, with respect to the nucleic acids mentioned in the present specification and claims, those skilled in the art will understand that any one or two of the complementary double strands are actually included. For convenience, in the present specification and claims, although only one strand is given in most cases, the other strand complementary thereto is actually disclosed. In addition, the nucleic acid sequences in the present application include DNA forms or RNA forms, one of which is disclosed, meaning that the other is also disclosed.

In another aspect of the present invention, the present invention provides a recombinant vector or transformant containing the nucleic acid as described above. The recombinant vector may include optional control sequences operably linked to the nucleic acid molecule. Wherein the control sequence is one or more control sequences that direct the expression of the nucleic acid molecule in a host. The recombinant vector provided by the embodiment of the invention can efficiently express the antibody or the antigen-binding fragment in a suitable host cell, the antibody or the antigen-binding fragment can be effectively combined with the alpha 3 domain of the MICA, and the antibody or the antigen-binding fragment has good effects of preventing and/or treating MICA-mediated diseases, such as promoting NK cell killing of tumors.

The recombinant vector may be a cloning vector or an expression vector, and may be obtained by operably linking the nucleic acid with a commercially available vector (e.g., a plasmid or a viral vector), and the recombinant vector in the present invention is not particularly limited, and any commonly used plasmid may be used, such as pSeTag2, PEE14, pMH3, and the like.

In a further aspect of the invention, the invention provides a recombinant cell carrying a nucleic acid, a recombinant vector or transformant, or an antibody or antigen-binding fragment as described above. The recombinant cell is obtained by transfection or transformation of the expression vector. According to the embodiment of the present invention, the recombinant cell can efficiently express the above antibody under suitable conditions, the antibody can effectively bind to the alpha 3 domain of MICA, and further, the antibody or the antigen-binding fragment has a good effect of preventing and/or treating MICA-mediated diseases, such as promotion of NK cell killing of tumors.

It is to be noted that the recombinant cell of the present invention is not particularly limited, and may be a prokaryotic cell, a eukaryotic cell or a phage. The prokaryotic cell can be escherichia coli, bacillus subtilis, streptomyces or proteus mirabilis and the like. The eukaryotic cell can be fungi such as Pichia pastoris, saccharomyces cerevisiae, schizosaccharomyces and trichoderma, insect cells such as meadow armyworm, plant cells such as tobacco, and mammalian cells such as BHK cells, CHO cells, COS cells and myeloma cells. In some embodiments, the recombinant cells of the invention are preferably mammalian cells, including BHK cells, CHO cells, NSO cells, or COS cells, and do not include animal germ cells, fertilized eggs, or embryonic stem cells.

The term "suitable conditions" as used herein means conditions suitable for the expression of the recombinant antibody as described herein. It will be readily understood by those skilled in the art that suitable conditions for recombinant antibody expression include, but are not limited to, suitable transformation or transfection means, suitable transformation or transfection conditions, healthy host cell status, suitable host cell density, suitable cell culture environment, and suitable cell culture time. The "suitable conditions" are not particularly limited, and those skilled in the art can optimize the conditions for the expression of the recombinant antibody optimally according to the specific circumstances in the laboratory.

The embodiments will be described in detail below. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

EXAMPLE 1 preparation of antibodies

The embodiment mainly includes the following contents:

balb/c mice (9 weeks old, purchased from Shanghai Rice, approximately 20g in weight) were immunized by generating a murine monoclonal antibody against human MICA and then using as antigen a purified recombinant MICA extracellular domain Fc fusion protein (MICA-Fc) (recombinant MICA extracellular domain Fc fusion protein, amino acid sequence shown in SEQ ID NO: 23). The immunized mice were immunized 3 times with purified antigen and Freund's complete adjuvant, and the immunized mice were bled from the tail vein and tested by ELISA and flow cytometry to obtain mice bearing anti-human MICA immunoglobulin.

EPHSLRYNLTVLSWDGSVQSGFLTEVHLDGQPFLRCDRQKCRAKPQGQWAEDVLGNKTWDRETRDLTGNGKDLRMTLAHIKDQKEGLHSLQEIRVCEIHEDNSTRSSQHFYYDGELFLSQNLETKEWTMPQSSRAQTLAMNVRNFLKEDAMKTKTHYHAMHADCLQELRRYLKSGVVLRRTVPPMVNVTRSEASEGNITVTCRASGFYPWNITLSWRQDGVSLSHDTQQWGDVLPDGNGTYQTWVATRICQGEEQRFTCYMEHSGNHSTHPVPSGKVLVLQSHWPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:23)。

The specific experimental operation process of obtaining hybridoma cells by taking splenocytes from the mice with the highest anti-MICA immunoglobulin and fusing with mouse myeloma cells SP2/0 cells (ATCC number CRL-1581) is routine. And screening the antibody of the fused hybridoma cell to obtain the mouse monoclonal antibody.

Culturing the total number of candidate hybridoma cells to 10⁶The cells were collected by centrifugation at 800rpm for 10 minutes, and total RNA of hybridoma cells was extracted using Trizol kit (Invitrogen); the total RNA is used as a template to synthesize a cDNA library (Invitrogen) through reverse transcription, and the cDNA is used as a template to amplify the variable region nucleic acid sequence corresponding to the hybridoma cells through PCR. The primer sequences used in the PCR amplification reaction are complementary to the first framework region of the antibody variable region or the signal peptide region and the constant region (Larrick, J.W., et al., (1990) Scand.J.Immunol., 32, 121-. mu.L of cDNA, 5. mu.L of 10 XPCR buffer, 2. mu.L (5. mu. mol) of upstream and downstream primers, 2. mu.L of dNTP, 1. mu.L of Taq enzyme (Takara,ex Taq), H2O 38 μ L; pre-denaturation at 95 ℃ for 5min, and entering temperature cycle for PCR amplification. The reaction conditions are as follows: denaturation at 94 ℃ for 30S, annealing at 58 ℃ for 45S, and extension at 72 ℃ for 50S for 32 cycles, followed by extension at 72 ℃ for 7 min. After sequencing the amplified product, the nucleotide sequences of the heavy chain variable region (SEQ ID NO:21) and the light chain variable region (SEQ ID NO:22) of the encoding murine monoclonal antibody are obtained, and the specific sequences are shown. The heavy chain variable region (SEQ ID NO:7) and the light chain variable region (SEQ ID NO:8) amino acid sequences of murine mAb.

Example 2 MICA antibody ELISA binding assay

An ELISA assay was used to detect the binding properties of the MICA antibodies obtained in example 1. The MICA extracellular domain Fc fusion protein (MICA-Fc) was coated on a 96-well plate, and the signal intensity after antibody addition was used to determine the binding properties of the antibody to MICA.

The MICA-Fc fusion protein (amino acid sequence shown in SEQ ID NO: 23) was diluted to 1. mu.g/mL with PBS buffer, added to a 96-well plate at a volume of 100. mu.L/well, and left overnight at 4 ℃. The 96-well plate was aspirated off PBS buffer, and after washing the plate 6 times with PBST (pH7.2 PBS containing 0.1% Tween 20) buffer, 200. mu.L/well PBS/10% BSA was added and incubated at 37 ℃ for 2h for blocking. The blocking solution was removed, and after washing the plate 6 times with PBST, 100. mu.L/well of the MICA antibody to be tested diluted to an appropriate concentration with PBST/0.05% BSA was added, followed by incubation at 37 ℃ for 1 h. The reaction was removed and after washing the plate 6 times with PBST, HRP (horseradish peroxidase) -labeled anti-mouse antibody secondary antibody was diluted with PBST/0.05% BSA at 100. mu.L/well and incubated at 37 ℃ for 1 h. After washing the plate 6 times with PBST, 80. mu.L/well of TMB (tetramethylbenzidine) was added, the plate was incubated at room temperature for 3min, and the reaction was stopped by adding 80. mu.L/well of 4M sulfuric acid. The absorbance was read at 450mm using a microplate reader. The results of the particular experiment are shown in FIG. 1, indicating that the antibodies of the invention are capable of binding MICA.

Example 3 MICA antibody ELISA binding assay

ELISA assays were used to detect the binding properties of MICA antibodies. The aforementioned MICA extracellular region alpha 3 domain Fc fusion protein (MICA alpha 3-Fc) was coated on a 96-well plate, and the intensity of the signal after antibody addition was used to determine the binding properties of the antibody to MICA.

MICA α 3-Fc fusion protein (amino acid sequence shown in SEQ ID NO: 24) was diluted to 1. mu.g/mL with PBS buffer, added to a 96-well plate at a volume of 100. mu.L/well, and left overnight at 4 ℃. The 96-well plate was aspirated off PBS buffer, and after washing the plate 6 times with PBST (pH7.2 PBS containing 0.1% Tween 20) buffer, 200. mu.L/well PBS/10% BSA was added and incubated at 37 ℃ for 2h for blocking. The blocking solution was removed, and after washing the plate 6 times with PBST, 100. mu.L/well of the MICA antibody to be tested diluted to an appropriate concentration with PBST/0.05% BSA was added, followed by incubation at 37 ℃ for 1 h. The reaction was removed and the plate was washed 6 times with PBST, then a secondary HRP (horseradish peroxidase) -labeled anti-mouse antibody (Boshide, BA1050) was diluted with PBST/0.05% BSA at 100. mu.L/well and incubated at 37 ℃ for 1 h. After washing the plate 6 times with PBST, 80. mu.L/well of TMB (tetramethylbenzidine) was added, the plate was incubated at room temperature for 3min, and the reaction was stopped by adding 80. mu.L/well of 4M sulfuric acid. The absorbance was read at 450mm using a microplate reader. Specific experimental results are shown in figure 2, indicating that the antibodies of the invention are capable of binding to the MICA α 3 domain.

VLRRTVPPMVNVTRSEASEGNITVTCRASGFYPWNITLSWRQDGVSLSHDTQQWGDVLPDGNGTYQTWVATRICQGEEQRFTCYMEHSGNHSTHPVPSGKVLVLQSHWPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:24)。

Example 4 CHO-K1 overexpression cell construction

HEK293T cells at 5X 10⁵Cells/well were plated in six-well plates and incubated overnight in DMEM medium without double antibody. Media was discarded before transfection and 1mL of fresh DMEM media without double antibody was added. The vector pLVX-EF1a-IRES-puro containing the target gene (pLVX-EF1a-IRES-pruo vector has an EcoRI and BamHI cleavage site inserted between the EcoRI and BamHI sites to encode MICA protein (SEQ ID NO:25) MICB (SEQ ID NO:26) rhesus MIC1(SEQ ID NO:27) rhesus MICB (SEQ ID NO:28) the nucleotide sequence (synthesized by Jinzhi, Suzhou), pMD2G and psPAX2 vector (total 3 mug) are added into 200 muL serum-free DMEM medium according to the ratio of 2:1:1, 12 μ g of polyetherimide (PEI, Polysciences Co., Ltd.) was added, mixed well and then allowed to stand for 16min, then adding all the liquid into a six-hole plate paved with HEK293T cells, culturing for 6h, after 48 hours of transfection, the cell culture supernatant was collected and filtered through a 0.45 μm filter (Millipore), to obtain a virus supernatant, and the whole virus supernatant was added to a medium containing 1X 10.⁴To a 6-well plate of CHO-K1 cells, polybrene (Sigma) was added at a final concentration of 4. mu.g/mL, and the cells were cultured for 12 hours. The supernatant was then discarded and fresh complete DMEM medium was added. The resulting cellsNamely CHO-K1-MICA, CHO-K1-MICB, CHO-K1-Rhesuus MICB 1 and CHO-K1-Rhesuus MICB cells, and can express MICA (SEQ ID NO:11), MICB (SEQ ID NO:29), Rhesuus MIC1(SEQ ID NO:30) and Rhesuus MICB (SEQ ID NO:31) proteins.

ATGGGCCTTGGCCCTGTGTTTCTGTTGCTTGCTGGGATTTTTCCATTCGCACCTCCCGGTGCCGCCGCAGAGCCCCACAGCTTGCGGTACAATTTGACCGTGCTGTCCTGGGATGGCAGTGTACAAAGCGGCTTTCTGACCGAGGTTCATCTGGATGGACAACCATTCCTGCGCTGCGACAGACAGAAGTGTCGAGCAAAACCCCAGGGTCAGTGGGCAGAAGATGTACTCGGCAATAAGACATGGGATCGAGAAACAAGAGACCTCACAGGAAATGGTAAAGATCTCCGCATGACCCTTGCACACATTAAGGATCAGAAAGAAGGACTGCACTCCCTCCAGGAGATTCGAGTGTGTGAGATTCACGAGGATAACAGCACAAGAAGCTCACAGCATTTTTATTACGATGGAGAACTGTTTCTCAGCCAAAACCTGGAGACCAAAGAGTGGACTATGCCACAGTCCTCCAGAGCTCAGACCCTGGCTATGAATGTCCGTAATTTTTTGAAAGAGGACGCAATGAAGACCAAGACTCACTACCATGCCATGCACGCCGACTGCCTGCAGGAGTTGAGACGATACCTCAAATCCGGGGTTGTGCTCAGGCGGACAGTGCCCCCAATGGTAAACGTGACTCGGTCTGAAGCTTCTGAAGGAAACATCACAGTCACCTGCAGAGCTAGCGGTTTTTACCCCTGGAACATCACTCTCTCCTGGAGACAGGACGGTGTGTCCCTCAGCCATGATACCCAGCAGTGGGGAGACGTGCTGCCCGATGGCAACGGCACTTATCAGACTTGGGTGGCTACTCGAATTTGTCAAGGAGAGGAGCAGCGATTCACATGTTACATGGAGCACTCCGGGAATCATTCAACTCACCCAGTACCCAGCGGAAAGGTCCTTGTGCTGCAGAGTCATTGGCAGACATTTCACGTATCCGCTGTGGCAGCCGCCGCAATTTTCGTCATCATCATTTTCTATGTTCGCTGTTGTAAAAAAAAGACAAGCGCTGCCGAGGGGCCCGAACTGGTGAGCCTCCAGGTGCTGGACCAACATCCCGTGGGCACCAGCGACCATCGCGATGCCACCCAGCTTGGCTTCCAGCCATTGATGTCAGACCTCGGCAGCACAGGCAGCACTGAGGGCGCT(SEQ ID NO:25)。

ATGGGATTGGGTAGGGTTTTGCTTTTTCTCGCTGTGGCTTTCCCCTTTGCACCTCCTGCTGCAGCCGCAGAACCCCATAGCCTTAGGTATAACCTGATGGTGCTCAGTCAAGATGAATCCGTGCAGTCCGGATTTCTTGCCGAGGGCCATCTGGATGGACAACCTTTCTTGCGTTACGATAGGCAGAAACGACGAGCTAAACCACAGGGTCAGTGGGCAGAGGACGTGCTGGGAGCTAAGACTTGGGACACCGAGACCGAGGATCTGACTGAGAACGGGCAGGACCTGAGAAGGACTCTGACACACATCAAGGACCAGAAGGGAGGGTTGCACTCCCTGCAGGAAATCCGCGTGTGTGAGATTCACGAGGACTCATCAACCCGAGGCAGTAGGCACTTTTACTACGATGGAGAGCTGTTTCTGAGTCAGAATCTGGAGACACAAGAGAGCACTGTGCCACAGTCATCTCGGGCTCAGACTCTGGCTATGAACGTTACCAATTTCTGGAAAGAAGATGCTATGAAAACAAAGACCCATTATAGAGCTATGCAGGCCGATTGCCTGCAGAAGTTGCAGCGTTATTTGAAGTCTGGTGTTGCTATCAGGCGGACCGTTCCCCCCATGGTGAATGTGACTTGCTCCGAGGTTTCCGAAGGTAACATTACTGTTACCTGTCGAGCTAGCTCCTTCTACCCACGGAACATTACATTGACATGGAGGCAGGATGGCGTTAGTCTGAGCCATAATACTCAGCAGTGGGGAGACGTTCTGCCTGACGGAAATGGCACATACCAGACCTGGGTTGCCACCAGAATCCGACAGGGCGAAGAGCAGAGGTTTACCTGTTATATGGAGCACAGCGGAAATCATGGGACTCATCCCGTGCCTAGTGGGAAGGTGCTCGTTCTCCAGTCTCAGCGCACTGACTTTCCTTACGTGTCTGCCGCTATGCCATGCTTCGTCATCATTATCATCCTCTGTGTGCCATGCTGCAAGAAGAAAACATCTGCAGCCGAGGGGCCTGAACTTGTGTCCCTGCAGGTTCTGGACCAGCACCCTGTTGGCACCGGAGATCACCGAGACGCAGCCCAACTGGGCTTCCAACCTCTGATGAGCGCCACTGGATCTACTGGTTCTACCGAGGGAGCC(SEQ ID NO:26)。

ATGGGCCCTGGGAGGGTTTTGCTCTTTCTGACCTCTATTTTTCCCTTCGCTAGACCAGGCGCTCCAACAGAGCTCCACAGCTTGCGCTACAACGTGACAGTTCTCAGCAGGGATGGGTCCGTGCAGTCCGATTTCCTCGCTGAGGGCCATCTTGATAGCCAACTCTTCGTCCGCTACGACCGCGAGACACGGCGGGCAAGGCCACAAGGACAGTGGGCCGAGGCTGTGCTGGGCGCAAAAACATGGGACACTGAAACTGGCGATCTCACAGAGAATGGGAAGGACCTGCGGCGTACCCTGACTCACATTGAGGGACAAAAGGGTGGCCTCCACAGCTTGCAGGATATACAGAATTGTGAGATTTACGAGGACGGCTCTACTGGAGGTTCCAGGCATTTTTACTATGATGGGGAGAGATTTCTGTCCCTTAATCTGGCCACTCAGGAGTGGACAGTAGCACAGTCTAGCCGAGCACAGACTCTGGCCATGAACTTCTGGAAGGAAGATACTATGAAAACAAATACACACTACCACGCAATGCAGGCCGATTGCCTGAAAAAGCTGAGACGCTACCAAAAGTCCAGAGTGGCTGTACGACGCACCGCCCCTCCTATGGTGAATGTGACACATTCCGAGGCTAGTGAGGGTAATATTACAGTGACCTGCCGCGCTTCTGGTTTCTACCCCAGAAATATAGCCTTGACCTGGCGACAGGACGGTGTTTCATTGAACCACGACGCCCAGCAGTGGGGCGGCATACTGCCAGACCAGAATGGCACTTACCAGACTTGGGTGGCCACCAGGATTCGGCAGGGCGAGGAACAGCGTTTCGCCTGCTATATGGAGCATAGCGGTAATCACTCTACCCATCCCGTGCCTAGCGGAAAGGTCCTGGTCTTTCAATCTCAGTGGCTGGATATTCCATACGTGCTGGGTGTAGCCGCTGCCGCCGTGGCCGCCGCTGCCGCAATTTTCGTTATTATCCTGTACGTTCTGTGTTGCAAGAAAAAGACTTCAGCCGCCGAAGGCCCCGAGCTCGTGAGTCTGCGCACCCTGGATCAGCACCCCGTTGGTACTGGAGATCACCGTGACGCAACC(SEQ ID NO:27)。

ATGGGTCTGGGAAGAGTATTGCTGTTTCTGGGTCGAGTCCTGCTGTTTCTCGCCTCCATTTTCTCTTTTACTCGACCTCGAGCCGCTGCTGAGCTGCACTCACTCAGATACAACGTGACCGTCTTGTCTAGAGATGGGTCAGTGCAGAGCGAGTTTTTGGCAGAGGGCCATTTGGATGGTCAGCTCTTTGTGAGGTACGATAGGGAAACACGACGTGCTAGACCTCAGGGACAGTGGGCAGAGGCAGTGTTGGGGGACCAGGAGACAGAGGACTTGACCGAGAACGCACAGGACCTGCGCAGAACACTGACCCACATTGAAGGCCAGAAGGGCGGCCTGCATAGTCTGCAGAAGATAAAAATCTGCGAAATTTATGAGGATGGCTCTACTGGCGGATTTTGGCATTTTTATTACGACGGGGAGCTGTTTCTGTCCCTTAATCTGGAGACACAGAAGTGGACAGTAGCTCAGTCATCCCGCGCTCAAACCCTGGCAATGAATTTCTGGAAGGAGGACACTATGAAAACTAATACACACTATAGAGCCATGAGGGCCGATTGCTTGAAAAAACTGTGGAGATACCAGAAGTCTCGGGTTGCTGTGAGAAGGACAGTTCCCCCTATGGTGAATGTCACTCATGGGGAGGCCTCTGAGGGCAACATAACAGTCACATGCAGGGCTTCAGGCTTTTACCCTGGAAACATCACTCTGACTTGGAGACAAGATGGCGTGTCCCTGAGTCACGACGCACAACAGTGGGGCGACGTCCTGCCTGATTGGAATGGCACCTATCAGACATGGGTCGCCACAAGAATCCGACAGGGAGAGGAACAGCGTTTTGCCTGTTACATGGAGCATAGCGGCAATCATTCCACACATCCTGTGCCTTCTGGCAAGCCACCAGTGCTCCAGTCCGAACGGTTGAATCTGCTGTATGTCCCCGTAGCAGTGGCTGTTGCTGTTGTCACTGCCTTTATCATTATCTGCGTTCACCGATGTAAAAAGAAAAAAACATCAGCTGCCGAGGGCCCAGAGTTGGTATCCCTCAGAACTCTTGACCAACACCCTGTCGGAACTGGGGATCATAGAGATGCTACCCAGCTGGGATTCCAGCCTCTCATGAGCGCTCCAGGGTCCACCGGATCAACTGAAGGGGCC(SEQ ID NO:28)。

MGLGRVLLFLAVAFPFAPPAAAAEPHSLRYNLMVLSQDESVQSGFLAEGHLDGQPFLRYDRQKRRAKPQGQWAEDVLGAKTWDTETEDLTENGQDLRRTLTHIKDQKGGLHSLQEIRVCEIHEDSSTRGSRHFYYDGELFLSQNLETQESTVPQSSRAQTLAMNVTNFWKEDAMKTKTHYRAMQADCLQKLQRYLKSGVAIRRTVPPMVNVTCSEVSEGNITVTCRASSFYPRNITLTWRQDGVSLSHNTQQWGDVLPDGNGTYQTWVATRIRQGEEQRFTCYMEHSGNHGTHPVPSGKVLVLQSQRTDFPYVSAAMPCFVIIIILCVPCCKKKTSAAEGPELVSLQVLDQHPVGTGDHRDAAQLGFQPLMSATGSTGSTEGA(SEQ ID NO:29)。

MGPGRVLLFLTSIFPFARPGAPTELHSLRYNVTVLSRDGSVQSDFLAEGHLDSQLFVRYDRETRRARPQGQWAEAVLGAKTWDTETGDLTENGKDLRRTLTHIEGQKGGLHSLQDIQNCEIYEDGSTGGSRHFYYDGERFLSLNLATQEWTVAQSSRAQTLAMNFWKEDTMKTNTHYHAMQADCLKKLRRYQKSRVAVRRTAPPMVNVTHSEASEGNITVTCRASGFYPRNIALTWRQDGVSLNHDAQQWGGILPDQNGTYQTWVATRIRQGEEQRFACYMEHSGNHSTHPVPSGKVLVFQSQWLDIPYVLGVAAAAVAAAAAIFVIILYVLCCKKKTSAAEGPELVSLRTLDQHPVGTGDHRDAT(SEQ ID NO:30)。

MGLGRVLLFLGRVLLFLASIFSFTRPRAAAELHSLRYNVTVLSRDGSVQSEFLAEGHLDGQLFVRYDRETRRARPQGQWAEAVLGDQETEDLTENAQDLRRTLTHIEGQKGGLHSLQKIKICEIYEDGSTGGFWHFYYDGELFLSLNLETQKWTVAQSSRAQTLAMNFWKEDTMKTNTHYRAMRADCLKKLWRYQKSRVAVRRTVPPMVNVTHGEASEGNITVTCRASGFYPGNITLTWRQDGVSLSHDAQQWGDVLPDWNGTYQTWVATRIRQGEEQRFACYMEHSGNHSTHPVPSGKPPVLQSERLNLLYVPVAVAVAVVTAFIIICVHRCKKKKTSAAEGPELVSLRTLDQHPVGTGDHRDATQLGFQPLMSAPGSTGSTEGA(SEQ ID NO:31)。

Example 5 MICA antibody flow cytometry binding assay

This example was conducted to examine the binding properties of MICA antibodies by flow cytometry, and the binding properties of the antibodies to MICA, MICB, Rhesuus MIC1, Rhesuus MICB were judged by adding the MICA antibodies to the CHO-K1-MICA, CHO-K1-MICB, CHO-K1-Rhesuus MIC1, and CHO-K1-Rhesuus MICB cells obtained in example 4 and by determining the intensity of the signals after the addition of the antibodies.

CHO-K1-MICA, CHO-K1-MICB, CHO-K1-Rhesuus MIC1, CHO-K1-Rhesuus MICB cells were diluted to 2X 10 with PBS⁶PermL, added to a 1.5mL EP tube in a volume of 100. mu.L/tube, to which 10. mu.L/tube goat serum was added, and blocked at 4 ℃ for 30 min. Adding different concentrations (10)^-4、10^-3、10^-2、10^-1、10⁰、10¹、10²μ G/mL) of MICA antibody (No. 11G3) or mouse IgG1(Biolegend, MG1-45) were incubated at 4 ℃ for 30 min. 1mL of PBS was added to the EP tube, centrifuged at 3500rpm at 4 ℃ for 5min, and the supernatant was discarded and washed with PBS. After centrifugation, the supernatant was discarded, the cells were resuspended in 100. mu.L/tube of PBS, and 0.1. mu.L/tube of Alexa-647-labeled goat anti-mouse antibody secondary antibody (Biolegend, 405322) was added thereto, and the mixture was incubated at 4 ℃ for 30min in the absence of light. Washed twice with PBS, centrifuged and the supernatant discarded. Resuspend cells in 200. mu.L/tube PBS, and detect by flow cytometry, the results are shown in FIGS. 3, 4, 5, and 6, further showing that the antibody of the invention can bind MICA, MICB, Rhesuus MIC1, Rhesuus MICB protein.

Example 6 MICA antibody flow cytometry in combination with tumor cell assays

The flow cytometry experiment is used for detecting the binding property of the MICA antibody to tumor cells, the MICA antibody is added into lung cancer NCI-H1299 cells, and the binding property of the antibody and the tumor cells is judged based on the strength of signals after the antibody is added.

NCI-H1299 cells were diluted 2X 10 with PBS⁶PermL, added to a 1.5mL EP tube in a volume of 100. mu.L/tube, to which 10. mu.L/tube goat serum was added, and blocked at 4 ℃ for 30 min. Adding different concentrations (10)^-4、10^-3、10^-2、10^-1、10⁰、10¹、10²μ g/mL) of MICA antibody, incubated at 4 ℃ for 30 min. 1mL of PBS was added to the EP tube, centrifuged at 4 ℃ and 3500rpm for 5min, the supernatant was discarded, and the precipitate was washed with PBS. Centrifuging, discarding the supernatant, resuspending the cells with 100. mu.L/tube PBS, adding 0.1. mu.L/tube Alexa-647 labeled goat anti-mouse antibody secondary antibody (Biolegend, 405322), incubating at 4 deg.C in the dark for 30min, washing twice with PBS after incubation, centrifuging, and discarding the supernatant; the cell pellet was resuspended in 200. mu.L/tube PBS and assayed by flow cytometry, as shown in FIG. 7, which further shows that the antibody of the present invention is capable of binding to lung cancer NCI-H1299 cells.

Example 7 determination of epitope Properties of MICA antibodies

Western blot experiments were used to determine epitope properties of MICA antibodies.

CHO-K1-MICA and CHO-K1 cells were harvested and washed twice with PBS. Then adding RIPA lysate, lysing on ice for 15min, centrifuging at 4 deg.C and 15000g for 5min, and collecting supernatant. Protein quantification was performed on the lysed supernatants using the BCA kit. Samples were then prepared using an SDS-PAGE loading buffer at 50 ug/well. After electrophoresis, the membrane is transferred to a nitrocellulose membrane and sealed for 1h at room temperature by using 5 percent skim milk powder. Then, 1:2000(w/v) dilution of MICA antibody was added and incubated overnight at 4 ℃. The membrane was washed 5min x 6 times with 1% o PBST, then HRP-goat anti-mouse IgG diluted 1:5000(v/v) was added and incubated for 1h at room temperature. The membrane was washed 5min x 6 times with 1% PBST. Chemiluminescence was detected by development with ECL substrate. The results are shown in figure 8, further showing that the antibodies of the invention are capable of binding linear epitopes of MICA protein.

Example 8 screening of antibodies to facilitate the ability of MICA to bind NKG2D

The MICA antibody promotes the signaling pathway between MICA and its receptor NKG2D by binding to the extracellular domain of MICA, and the present example examines the effect of MICA antibody on the binding of MICA to its receptor NKG2D by flow cytometry.

CHO-K1-MICA cells (supra) were diluted to 2X 10 with PBS⁶To 1.5mL of EP tube was added 100. mu.L/tube, 10. mu.L/tube of mouse serum was added, and the mixture was blocked at 4 ℃ for 30 min. MICA antibody was added and incubated at 4 ℃ for 30 min. 2 μ g/tube of NKG2D extracellular domain Fc fusion protein (NKG2D-Fc, SEQ ID NO:32, obtained by cell expression by the inventors) was added and incubated at 4 ℃ for 30 min. 1mL of PBS was added to the EP tube, centrifuged at 3500 rpm. times.5 min at 4 ℃ and the supernatant was discarded and washed with PBS. After centrifugation, the supernatant was discarded, the cells were resuspended in 100. mu.L/tube of PBS, and 1. mu.L/tube of 647-labeled mouse anti-human antibody secondary antibody (Biolegend, HP6017) was added thereto, and incubated at 4 ℃ for 30min in the absence of light. Washed twice with PBS, centrifuged and the supernatant discarded. The cells were resuspended in 200. mu.L/tube PBS and assayed by flow cytometry, the results are shown in FIG. 9, and it can be seen that the antibodies of the invention promote the binding of MICA to the receptor NKG 2D.

IWSAVFLNSLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTVPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:32)。

Example 9 MICA antibody promotes NK92 cell anti-tumor

This example is an in vitro killing experiment to examine the effect of MICA on the killing of tumor cells by NK92 cells (ATCC accession number CRL-2407). A total of 2 groups were established, in which melanoma A375 cells (ATCC accession number CRL-1619) were used as tumor cells, and the experimental procedures were as follows:

(1) the A375 cells were washed twice with serum-free DMEM medium, centrifuged at 200g at 4 ℃ for 5min, and the supernatant was discarded. Resuspend with 1mL serum-free DMEM medium, add CTV to a final concentration of 5. mu.M, incubate for 30min at 37 ℃.

(2) After the incubation was completed, 200. mu.L of precooled fetal calf serum stop marker was added, and the mixture was centrifuged at 4 ℃ and 200g for 5min, and the supernatant was discarded. The cell pellet was washed twice with complete RPMI-1640 medium containing 10% fetal bovine serum and the supernatant was discarded. Resuspend with complete RPMI-1640 medium and count. Diluting with complete RPMI-1640 medium to 1.25 × 10⁵one/mL.

(3) NK92 cells were diluted to a range of concentrations (2X 10) in complete RPMI-1640 medium containing 10% fetal bovine serum⁵4X 10 units/mL⁵8X 10 pieces/mL⁵1.6X 10 units/mL⁶one/mL).

(4) Diluted PBMC were added to a 96-well round bottom plate at a volume of 100. mu.L/well.

(5) MICA antibody or control mouse IgG was diluted to 50. mu.g/mL with complete RPMI-1640 medium and the diluted MICA antibody or mouse IgG was added to a 96-well round bottom plate at a volume of 20. mu.L/well.

(6) The diluted tumor cells were added to a 96-well round bottom plate in a volume of 80. mu.L/well.

(7) The 95-hole round bottom plate is centrifuged for 1min at 1500rpm and 4 ℃, and the centrifuged 96-hole plate is incubated for 4h at 37 ℃.

(8) 7-AAD (BD, 559925) was added to a 96-well round bottom plate in a volume of 1. mu.L/well and mixed well. All the liquid in the hole is transferred into a flow tube, and a flow cytometer is used for detection, and the specific experimental result is shown in fig. 10, so that the antibody disclosed by the invention can promote NK92 cells to kill tumors.

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.

Claims

1. An antibody or antigen-binding fragment comprising a CDR sequence selected from at least one of:

heavy chain variable region CDR sequences: 1-3 of SEQ ID NO;

light chain variable region CDR sequences: 4-6 of SEQ ID NO.

2. The antibody or antigen-binding fragment of claim 1, comprising:

a heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO. 1 or a conservatively modified form thereof,

a heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO. 2 or a conservatively modified form thereof,

a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO. 3 or a conservatively modified form thereof,

light chain CDR1 having the amino acid sequence set forth in SEQ ID NO. 4 or a conservatively modified form thereof,

light chain CDR2 having the amino acid sequence set forth in SEQ ID NO. 5 or a conservatively modified form thereof,

light chain CDR3 having the amino acid sequence set forth in SEQ ID NO. 6 or a conservatively modified form thereof.

3. The antibody or antigen-binding fragment of claim 1 or 2, comprising: a heavy chain variable region and a light chain variable region, wherein:

(i) the heavy chain variable region comprises an amino acid sequence with at least 80 percent of homology of at least one of the amino acid sequence shown in SEQ ID NO. 7 and the amino acid sequence of a conservative modification form thereof; and/or, (ii) the light chain variable region comprises an amino acid sequence that is at least 80% homologous to at least one of the amino acid sequence set forth in SEQ ID NO. 8 and conservatively modified versions thereof.

4. The antibody or antigen-binding fragment of any of claims 1-3, wherein the heavy chain variable region comprises an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% homologous to a heavy chain variable region selected from (i); (iii) the light chain variable region comprises an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% homologous to the light chain variable region selected from (ii).

5. The antibody or antigen-binding fragment of claim 3 or 4, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 7 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 8.

6. The antibody or antigen-binding fragment of any one of claims 1 to 5, comprising: a heavy chain comprising the amino acid sequence shown in SEQ ID NO. 9 and a light chain comprising the amino acid sequence shown in SEQ ID NO. 10.

7. The antibody or antigen-binding fragment thereof of any one of claims 1-6, wherein the antibody is a monoclonal antibody, a murine antibody, a chimeric antibody, a humanized antibody, a human antibody, an Fv, an scFv, an Fab '-SH, or an F (ab') 2.

8. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, wherein the antibody or antigen-binding fragment thereof is capable of binding to at least a portion of the amino acid sequence set forth in SEQ ID NO. 11.

9. An immunoconjugate comprising a therapeutic agent and the antibody or antigen-binding fragment of any one of claims 1-8, wherein the antibody or antigen-binding fragment is coupled to the therapeutic agent.

10. A composition comprising the antibody or antigen-binding fragment of any one of claims 1 to 8 and/or the immunoconjugate of claim 9.

11. A kit for detecting MICA comprising the antibody or antigen-binding fragment of any one of claims 1 to 8.

12. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-8 in the preparation of a kit for detecting MICA.

13. A medicament, comprising: the antibody or antigen-binding fragment of any one of claims 1-8 and/or the immunoconjugate of claim 9 and/or the composition of claim 10.

14. Use of the antibody or antigen-binding fragment of any one of claims 1-8 and/or the immunoconjugate of claim 9 and/or the composition of claim 10 in the manufacture of a medicament for the prevention and/or treatment of a MICA-mediated disease;

optionally, the MICA-mediated disease is cancer or transplant rejection, an autoimmune disease, an infectious disease;

optionally, the cancer is at least one of lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, stomach cancer, esophageal cancer, oral squamous cell carcinoma, and head and neck cancer.

15. A nucleic acid comprising a sequence encoding the antibody or antigen-binding fragment of any one of claims 1-8; optionally, the nucleic acid has the nucleotide sequence shown in SEQ ID NO 12 and 13.

16. A recombinant vector or transformant containing the nucleic acid of claim 15.

17. A recombinant cell carrying the nucleic acid of claim 15, the expression vector or transformant of claim 16, or the antibody or antigen-binding fragment of any one of claims 1 to 8.