CN112574311B - Antibody with double MIC binding activity and application thereof - Google Patents
Antibody with double MIC binding activity and application thereof Download PDFInfo
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Abstract
The invention discloses an antibody with double MIC binding activity and application thereof, wherein the antibody comprises at least one heavy chain variable region containing three CDRs and at least one light chain variable region containing three CDRs, and the CDR sequences of the light chain variable region and the heavy chain variable region are shown in SEQ ID NO. 1-30. The anti-MIC antibody has good affinity effect on a plurality of MICA and MICB proteins, particularly has binding activity on soluble MICA 008, MICA 010, MICA 002, MICA 009 and MICB 005, and can relieve the tumor immunosuppressive effect caused by abnormal rising of MIC proteins by adsorbing NKG2D ligand in human blood or blood plasma, including soluble MICA, MICB molecules and immune complexes formed by MICA/B and anti-MIC monoclonal antibody medicaments.
Description
Technical Field
The invention relates to the field of proteins, in particular to an antibody with double MIC binding activity and application thereof, and specifically relates to treatment of tumors related to abnormal expression of soluble MICA and MICB molecules. Antibodies for resisting MICA and MICB are expressed and coupled to a solid phase carrier, and free soluble MIC family protein in serum of a tumor patient is removed in an immunoadsorption mode, so that the immune monitoring effect of NK cells is enhanced.
Background
The prevention and treatment of cancer is a great problem in human health and life in this century, and immunotherapy provides a safe and effective tumor treatment scheme in recent years. Natural killer cells (NK cells) are an important member of the innate immune system, primarily against pathogens and cancer. NKG2D expressed in NK cells and CD8+T cells, also expressed in part of CD4+T cells, invariant natural killer T cells (iNKT), and γ δ T cells. Humans have at least 8 genes encoding ligands for NKG2D, including MICA, MICB, RAET1E, RAET1G, RAET1H, RAET1I, RAET1L, and RAET1N, some with extensive allelic polymorphism (Lanier, l.l., 2015). The MIC gene family includes 7 members of MICA, MICB, MICC, MICD, MICE, MICF and MICG, of which only MICA and MICB are functional genes. The NKG2D receptor and the ligand thereof interact with each other to activate the killing function mediated by immune cells, and the NKG2D can also be used as a co-stimulatory molecule to enhance the signal transduction of the T cell receptor so as to mediate the tumor killing function of T cells.
MIC molecules are classified into membrane-type MICs and soluble MICs (sMIC), with sMIC being present in the sera of many malignant patients. In vitro experiments showed that NKG 2D-dependent cytotoxicity of NK cells was significantly reduced in the presence of soluble MICA or RAET1E protein, or patient serum containing high levels of soluble MICA (WANG H, 2008; bang, 2010). Therefore, methods for inhibiting shedding of MIC molecules or neutralizing sMIC with antibody drugs are becoming new directions for treating tumors and are receiving more and more attention.
The treatment of malignant tumor at present mainly includes operation, radiotherapy and chemotherapy, targeted medicine, immunotherapy and other treatment means. Surgery is the most basic treatment, but the treatment requirements for surgery are relatively early and the patient's lesions are localized. For a part of patients in middle and advanced stages, chemoradiotherapy and targeted drug therapy are used in the treatment process. Radiation therapy is also required for a portion with local symptoms of pressure or local infiltration metastasis. In the aspect of drug therapy, the side effect of the traditional chemical drugs is large, and in recent years, antibody drugs are a new research hotspot for tumor drug therapy. CN110088137A/WO2014140884a2 and US20200165343a1 disclose an antibody capable of binding MICA/B, primarily to the α 3 domain of MICA/B protein. CN108779178A discloses an anti-MICA antibody, mainly binding MICA 001, MICA 004, MICA 007 and MICA 008. CN110267678A provides an antibody capable of binding MICA 008, MICA 002, MICA 004 and MICB 005. US20180085400a1 discloses a method of treating cancer using bispecific single chain antibodies BiTEs composed of an anti-MICA antibody and a molecule such as CD 3. CN105517572A discloses a monoclonal antibody for neutralizing soluble MIC for treating cancer, treating MIC positive tumor. However, the dosage required by the monoclonal antibody drug is relatively large, and the large-dosage antibody drug input also has some safety risks. In conclusion, there is currently no universally effective way of treating tumors.
The blood adsorption treatment is based on the principles of pore adsorption, and/or hydrophobic effect, and/or electrostatic adsorption, and/or biological affinity (complement combination, Fc segment combination and antigen-antibody combination), and can be used for selectively or specifically adsorbing pathogenic factors in blood through an extracorporeal circulation technology without influencing the normal functions of the organism, so as to achieve the purposes of purifying blood and relieving the disease condition. The blood purification technology commonly used in clinic includes blood perfusion, blood filtration, hemodialysis and the like, and at present, blood purification becomes a third treatment mode after surgery and medicines, and is successfully applied to treatment of autoimmune diseases and treatment of liver failure, renal failure and medicine poisoning. In recent years, blood purification has also been widely used for the treatment of inflammatory factors, sepsis, and severe pancreatitis. Blood purification can also be used for treating severe new coronary pneumonia.
In the aspect of blood purification and tumor treatment, US20180231569 fixes aglucon-snowdrop agglutinin which is a ligand of glycoprotein on the surface of tumor exosomes and specifically adsorbs the glycoprotein on a polyether sulfone or cellulose acetate membrane, eliminates the content of the tumor exosomes through a blood filtration system, relieves the inhibition of the exosomes on an immune system, and restores the immunity of an organism. There are documents that protein A filler is used for adsorbing anti-MICA monoclonal antibody, then sMICA in serum of a liver cancer patient is adsorbed, then a NKG2D receptor pathway is used for activation, which leads to Green Fluorescent Protein (GFP) expression of receptor cells to identify the activity of NKG2D (Luo Q, 2020), and the result shows that the number of GFP positive cells is greatly reduced after sMICA is added; after sMICA was adsorbed, GFP positive cell numbers were close to negative control healthy human serum numbers, further demonstrating that sMICA inhibited activation of NKG 2D. In the method used in this document, the protein A and the antibody IgG are mainly bound by affinity, and the antibody adsorbed on the surface of the filler is further adsorbed to MICA, which is weak and unstable in binding force. The mouse monoclonal antibody (fragment) resisting the MIC protein is directly connected to the surface of the microsphere in a covalent bond mode, the density of the ligand is greatly increased, the ligand is prevented from falling off, and the synthesized immunoadsorbent has stronger adsorption capacity on the MIC protein of the target protein. In addition, the immunoadsorbent also has an adsorption effect on an immune complex consisting of the target protein and the antibody drug, so the immunoadsorbent can be used for an auxiliary treatment mode of tumor drug treatment.
MICA and MICB have gene polymorphism, and more than 200 MICA/B alleles are found at present, so that the antibody binding activity of one antibody to all alleles is almost impossible to achieve by screening. Therefore, the invention mainly selects several MIC proteins with higher content in Chinese population to screen corresponding antibodies.
Disclosure of Invention
It is an object of the present invention to overcome at least one of the disadvantages of the prior art and to provide anti-MIC antibodies with dual MIC binding activity and uses thereof.
The technical scheme adopted by the invention is as follows:
in a first aspect of the present invention, there is provided:
an anti-MIC antibody comprising at least one heavy chain variable region comprising three CDRs and at least one light chain variable region comprising three CDRs, wherein:
the light chain CDR1, light chain CDR2, and light chain CDR3 of the light chain variable region have the amino acid sequences as set forth in SEQ ID No.: 1-3, or as set forth in SEQ ID No.: 4-6, or as set forth in SEQ ID No.: 7-9, or as set forth in SEQ ID No.: 10-12, or as set forth in SEQ ID No.: 13-15;
the heavy chain variable region heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 sequences are respectively as shown in SEQ ID No.: 16-18, or as set forth in SEQ ID No.: 19-21, or as set forth in SEQ ID No.: 22-24, or as set forth in SEQ ID No.: 25-27, or as set forth in SEQ ID No.: 28-30.
In some examples, the amino acid sequences of the light chain variable regions are set forth in SEQ ID No.: 31. SEQ ID No.: 33. SEQ ID No.: 35. SEQ ID No.: 37 and SEQ ID No.: shown at 39.
In some examples, the amino acid sequences of the heavy chain variable regions are set forth in SEQ ID No.: 32. SEQ ID No.: 34. SEQ ID No.: 36. SEQ ID No.: 38 and SEQ ID No.: shown at 40.
In some examples, the light chain CDR1, light chain CDR2, and light chain CDR3 of the light chain variable region have amino acid sequences as set forth in SEQ ID No.: 1-3, the heavy chain variable region heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 sequences are respectively as shown in SEQ ID No.: 16-18; or
The light chain CDR1, light chain CDR2, and light chain CDR3 of the light chain variable region have the amino acid sequences as set forth in SEQ ID No.: 4-6, the heavy chain variable region heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 sequences are respectively as shown in SEQ ID No.: 19-21; or
The light chain CDR1, light chain CDR2, and light chain CDR3 of the light chain variable region have the amino acid sequences as set forth in SEQ ID No.: 7-9, the heavy chain variable region heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 sequences are respectively as shown in SEQ ID No.: 22-24; or
The light chain CDR1, light chain CDR2, and light chain CDR3 of the light chain variable region have the amino acid sequences as set forth in SEQ ID No.: 10-12, the heavy chain variable region heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 sequences are respectively as shown in SEQ ID No.: 25-27; or
The amino acid sequences of the light chain CDR1, light chain CDR2, and light chain CDR3 of the light chain variable region are SEQ ID No.: 13-15, the heavy chain variable region heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 sequences are respectively as shown in SEQ ID No.: 28-30 parts of; or
The amino acid sequence of the light chain variable region is shown as SEQ ID No.: 31, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID No.: 32 is shown; or
The amino acid sequence of the light chain variable region is shown as SEQ ID No.: 33, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID No.: 34; or
The amino acid sequence of the light chain variable region is shown as SEQ ID No.: 35, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID No.: 36 is shown; or
The amino acid sequence of the light chain variable region is shown as SEQ ID No.: 37, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID No.: 38; or
The amino acid sequence of the light chain variable region is shown as SEQ ID No.: 39, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID No.: shown at 40.
Antibodies comprise a variable region (V region) and a constant region (H region), wherein the composition and arrangement of the amino acids in the V region determine the antigen-binding specificity of the antibody. There is a higher degree of variation in the amino acid composition and arrangement order of certain localized regions in VL (light chain variable region) and VH (heavy chain variable region), which are called hypervariable regions (HVRs). The amino acid composition and arrangement of non-HVR sites in the V region is relatively conserved, called the framework region (framework region). The hypervariable region is where the antibody antigen binds and is therefore referred to as the complementarity-determining region (CDR). HVR1, HVR2 and HVR3 for VL and VH may also be referred to as CDR1, CDR2 and CDR3, respectively, with generally higher degrees of CDR3 hypermutation. In the invention, the full-length sequences and the complementarity determining region sequences of the 5 antibodies with higher binding force are determined by screening and sequencing analysis of monoclonal cell strains.
In some examples, the anti-MIC antibody is expressed using a eukaryotic cell.
In some examples, the anti-MIC antibody is expressed using CHO cells or insect cells.
In some examples, the antibody is a single chain antibody having the molecular structure VH-X1-VL, and X1 is a linker sequence.
In some examples, the linker sequence consists of amino acids G/S.
In some examples, the linker sequence is (GS) n, (GGS) n, (GGGS) n, or (GGGGS) n, n =1 ~ 5.
In some examples, the linker sequence is GGGGSGGGGSGGGGS.
In some examples, the anti-MIC antibody is expressed using prokaryotic cells.
In some examples, the prokaryotic cell is e.
In a second aspect of the present invention, there is provided:
an immunoadsorbent comprising a solid support having coupled thereto an anti-MIC antibody according to the first aspect of the invention.
In some examples, the solid support is at least one of chitosan microspheres, agarose microspheres, sephadex, resin, cellulose microspheres.
In some examples, the immunoadsorbent has binding activity against soluble MICA 008, MICA 010, MICA 002, MICA 009, and MICB 005.
In some examples, the coupling is by covalent bond linkage.
In a third aspect of the present invention, there is provided:
use of an immunoadsorbent, as described in the second aspect of the invention, for the preparation of a blood purification agent.
In some examples, the main purpose of the immunoadsorbent is to adsorb NKG2D ligand in human blood or plasma, thereby relieving tumor immunosuppression caused by abnormal rise of MIC protein.
In some examples, the NKG2D ligand includes soluble MICA, MICB molecules, and immune complexes of MICA/B with anti-MIC monoclonal antibody drugs.
In some examples, the immunoadsorbent treats or ameliorates tumor suppression caused by abnormal elevation of soluble MIC proteins by blood purification.
In some examples, the tumor of interest includes, but is not limited to, melanoma, lung cancer, plasma cell cancer, leukemia, lymphoma, ovarian cancer, colon cancer, pancreatic or prostate cancer, brain cancer, liver cancer, stomach cancer, testicular cancer, cervical cancer, vaginal cancer, squamous cell cancer, malignant mesothelioma cancer, oral cancer, head and neck cancer, throat cancer, thymus cancer, gastrointestinal stromal tumor (GIST) cancer, nasopharyngeal cancer, esophageal cancer, colon cancer, anal cancer, breast cancer, prostate cancer, bladder cancer, pancreatic cancer, neuroblastoma, glioma, or renal cancer.
In a fourth aspect of the present invention, there is provided:
a nucleotide sequence expressing an anti-MIC antibody, which may encode a protein sequence of an anti-MIC antibody according to the first aspect of the invention.
In a fifth aspect of the present invention, there is provided:
an expression vector capable of expressing a corresponding anti-MIC antibody based on the nucleotide sequence of the fourth aspect of the invention.
In some examples, the expression body is a eukaryotic cell or a prokaryotic cell.
In some examples, the eukaryotic cell is selected from a CHO cell or an insect cell.
In some examples, the prokaryotic cell is e.
In a sixth aspect of the present invention, there is provided:
a kit for detecting MICA/B comprising a MIC antibody directed against according to the first aspect of the invention.
The invention has the beneficial effects that:
in some embodiments of the invention, anti-MIC antibodies have good affinity for a variety of MICA and MICB proteins, particularly binding activity against soluble MICA 008, 010, 002, 009, and 005.
Some examples of the invention, using solid phase carriers coupled with anti-MIC antibodies, to simulate blood purification, have shown that it is possible to effectively adsorb minute amounts of MICA and MICB proteins in plasma, in particular soluble MICA 008, 010, 002, 009, and 005.
The immunoadsorbent of the embodiments of the invention can adsorb NKG2D ligand in human blood or plasma, including soluble MICA and MICB molecules and an immune complex consisting of MICA/B and anti-MIC monoclonal antibody drugs, thereby relieving the tumor immune suppression effect caused by abnormal increase of MIC protein.
Drawings
FIG. 1 shows the preliminary screening results of monoclonal antibody cell lines;
FIG. 2 is an electrophoretogram of each monoclonal antibody after purification;
FIG. 3 shows the results of ELISA assay for binding activity of each monoclonal antibody to MIC protein;
FIG. 4 is a single chain antibody plasmid cleavage map;
FIG. 5 is an electrophoretogram of purified single-chain antibody.
Detailed Description
The technical scheme of the invention is further explained by combining experiments.
Example 1 preparation of MIC protein expressed in Membrane and immunization of animals
cDNA expressing full-length MICA 002, MICA 008, MICA 009, MICA 010 and MICB 005 (sequence details are shown in http:// hla. alloles. org/alloles/text _ index. html. MICA full-length sequence comprises signal peptide, amino acid sequence 1-366; extracellular region sequence comprises signal peptide, amino acid sequence 1-284. MICB full-length sequence comprises signal peptide, amino acid sequence 1-360; extracellular region sequence comprises signal peptide, amino acid sequence 1-286) is connected to lentiviral vector pCDH-CMV-EF 1-Puro, and then transferred into 293T cells together with helper plasmid, lentivirus is produced from 293T cells, and viral supernatant CHO cells are collected. Take 5 x 106Mixing transfected cells (5 cells with equal content) with Freund's complete adjuvant, shaking and mixing uniformly, and performing abdominal subcutaneous injection to immunize mice for the first time; three weeks later, 5X 106Mixing transfected cells with Freund's incomplete adjuvant, and performing intraperitoneal injection for second immunization; three weeks later, 5X 106The transfected cells were mixed with physiological saline and injected intraperitoneally for the 3 rd immunization. The cells were boosted prior to fusion in the same manner as 3 rd immunization. Mixing spleen and myeloma cells at a ratio of 5: 1, fusing with PEG, and culturing in methylcellulose semisolid culture medium to obtain single hybridTumor clones were formed.
Example 2 preparation of secreted MIC proteins
The cDNA (C-terminal designed his tag) of the extracellular regions of MICA 002, MICA 008, MICA 009, MICA 010 and MICB 005 was ligated to a lentiviral vector pCDH-CMV-MCS-EF1-Puro, and transferred to 293T cells together with a helper plasmid to produce viruses, and the viral supernatant was collected and infected into CHO cells. And collecting the CHO cell culture solution, and purifying by using metal ion chelating filler to obtain the target protein. The MICA protein has a molecular weight of about 55KD, and the MICB protein has a molecular weight of about 45 KD.
EXAMPLE 3 screening of monoclonal antibody cell lines
Screening 30 monoclonal cell strains, taking the monoclonal cells under microscope, inoculating to 96-well plate, adding 5% CO at 37 deg.C2The incubator was incubated for 3 days, and the supernatant was collected. ELISA plates were coated with native MICA 008, 100 ng/well, incubated overnight at 4 ℃, primary antibody was 100 μ L/well of cell culture supernatant diluted in gradient (2, 4, 8, 16, 32, 64, 128 fold diluted with PBS respectively), incubated at 37 ℃ for two hours, secondary antibody: goat anti-mouse antibody (HRP labeled) 1: diluting by 5000, incubating at 37 ℃ for 1h at 100 muL/hole, developing for 10min by using TMB developing solution, and detecting light absorption value at 450 nm. The 5 cell lines with high binding force with MICA 008 were selected to be 1E7, 3F5, 8B6, 9C9 and 10a1 cell lines, and the results are shown in fig. 1.
The anti-MIC antibodies produced by the 5 monoclonal cell lines were detected and sequenced, and the light chain variable region and the heavy chain variable region of the antibody produced by each cell line were as follows:
further analysis of the sequencing results showed that:
SEQ ID No.: 31 comprises SEQ ID No.: 1-3 light chain CDR1, light chain CDR2 and light chain CDR 3;
SEQ ID No.: 32 comprises SEQ ID No.: 16-18, and a heavy chain CDR1, 2, and 3;
SEQ ID No.: 33 comprises SEQ ID No.: 4-6, a light chain CDR1, a light chain CDR2, and a light chain CDR 3;
SEQ ID No.: 34 comprises SEQ ID No.: 19-21, a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR 3;
SEQ ID No.: 35 comprises SEQ ID No.: 7-9, a light chain CDR1, a light chain CDR2, and a light chain CDR 3;
SEQ ID No.: 36 comprises SEQ ID No.: 22-24, a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR 3;
SEQ ID No.: 37 comprises SEQ ID No.: 10-12 of a light chain CDR1, a light chain CDR2, and a light chain CDR 3;
SEQ ID No.: 38 comprises SEQ ID No.: 25-27 of a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR 3;
SEQ ID No.: 39 comprises SEQ ID No.: 13-15 light chain CDR1, light chain CDR2 and light chain CDR 3;
SEQ ID No.: 40 comprises SEQ ID No.: 28-30, and a heavy chain CDR1, 2, and 3.
EXAMPLE 4 Mass expression and purification of monoclonal antibodies
Selecting adult Balb/c mice, injecting 0.5ml liquid paraffin into abdominal cavity of each mouse, after ten days, centrifuging the 5 hybridoma cells cultured to logarithmic growth phase, resuspending with PBS, and counting by 1 × 106Per ml, each mouse was injected with 0.5ml of cell suspension intraperitoneally, three cells per strain, and ascites was collected ten days later. Purifying mouse anti-ascites with protein A chromatography filler, adding 5mL protein A filler into 1mL ascites, balancing with PBS, statically adsorbing, eluting with eluent with pH =3.0 and containing 100mM glycine-HCl, collecting elution peak to obtain purified mouse anti-ascites, ultrafiltering, concentrating, and replacing with PBS buffer solution. The purity of the antibody was checked with 12% separation gel. The results are shown in FIG. 2.
Example 5 binding assays of each monoclonal antibody to other MIC proteins
The microplate was coated with MICA 002, MICA 009, MICA 010, MICB 005 protein, 100 ng/well, washed, and then added with a primary antibody, mouse monoclonal antibody purified from 5 cell lines, at an initial concentration of 1ug/ml, and a secondary antibody, goat anti mouse-HRP labeled 1: and (5) 5000 dilution. Incubating for 1 hour at 37 ℃, developing for 10min by using TMB developing solution, and detecting the light absorption value at 450 nm. The results are shown in the figure below, and the adsorption effect on several proteins is comprehensively compared, and 3F5, 9C9 and 10A1 are excellent cell strains, and the generated monoclonal antibodies have higher binding force on 4 MICA/B. The results are shown in FIG. 3.
EXAMPLE 6 preparation of Single chain antibody
Recovering frozen 9C9 cell strain with RPMI-1640 culture medium, collecting 2-5 x 106Sequencing heavy chain and light chain genes of each/mL cell by entrusted Shanghai's work (extracting total RNA by using an RNA extraction kit, carrying out reverse transcription to obtain cDNA, using the cDNA as a template, designing degenerate primers according to a variable region and a framework region of a BALB/c murine monoclonal antibody, amplifying a VH gene and a VL gene by using the primers, recovering a PCR product by using the kit, connecting the PCR product to a T vector, sequencing and identifying4Ser)3Direct synthesis of VH- (Gly) based on VH and VL sequencing results4Ser)3VL-his sequence, PCR product (size about 800 bp) ligated into PET30a vector and sequence verified. Plasmid 10 mu L is taken and transferred into BL21 (DE)3To competent cells, kanamycin resistance was added and positive clones were selected on plates. And then selecting monoclonal bacteria from the plate, transferring the monoclonal bacteria into an LB culture medium for culture, collecting the bacteria after amplification, crushing the bacteria by using a high-pressure homogenizer, collecting supernatant, purifying by using metal chelating filler, collecting protein solution, and carrying out electrophoresis identification, wherein the molecular weight of the purified protein is about 27 KD. The plasmid map after digestion is shown in FIG. 4 (in the figure, 1 is DNA marker; 2 is digestion map), and the protein electrophoresis map is shown in FIG. 5 (in the figure, 1 is pre-stained marker; 2 is purified protein).
Example 7 Synthesis of immunoadsorbent and evaluation of adsorption Performance
1mL of cyanogen bromide activated agarose was placed in a disposable chromatographic column and the pad was washed with about 30mL of 1mmol HCl solution and drained. Then 0.5mL of 1mmol HCl solution and 0.5mL of buffer system of 0.1M Na were added2CO30.5M NaCl, protein solution with pH =8.0 (15 mg of protein is added by 9C9 monoclonal antibody, 2.8mg of single-chain antibody is added, the same mole number is ensured), placing on a decoloring shaker, 100rpm, and mixing uniformly at room temperature for 2 h; the packing was then washed with 10mL of 0.1M Tris-HCl, pH8.0, and then 1mL of 0.1M Tris-HCl, pH8.0, was added and blocked for 3h at room temperature. Finally, theWith 0.2M CH3And cleaning the filler with the COOH solution and storing.
1mL of the above-mentioned synthetic filler and 2mL of secretory MICA 002, MICA 008, MICA 009, MICA 010 and MICB 005 protein solutions (see example 2) were charged into a 10mL EP tube, and after contacting at 28 ℃ and 100rpm for 1 to 2 hours, the filler was placed in a disposable affinity chromatography column and the filler was drained. The change in MICA and MICB content in the supernatant before and after adsorption was measured with MICA ELISA kit (abcam) and MICB ELISA kit (abcam), respectively. Blank agarose GE Sepharose 6FF was used as a control. The results are shown in tables 1 and 2.
Experimental results show that the murine monoclonal antibody produced by the cell strain 9C9 and the single-chain antibody constructed based on the murine monoclonal antibody have good affinity for various MIC proteins, and the prepared adsorbent can effectively adsorb sMIC proteins in blood.
SEQ ID No.: 1-40, wherein the underlined sequences are CDR region amino acid sequences.
SEQ ID NO.:1:RTSQSIVHNNGVTYLE
SEQ ID NO.:2:KASNRFT
SEQ ID NO.:3:FQGSHVPRS
SEQ ID NO.:4:RSSQSIVHNNGVTYLE
SEQ ID NO.:5:KVSNRFS
SEQ ID NO.:6:FQGSHVPRT
SEQ ID NO.:7:RSSQSIVHNNGLTYLE
SEQ ID NO.:8:RVSNRFS
SEQ ID NO.:9:FQGSHVYRT
SEQ ID NO.:10:RSSQSIVHNNGVTFLE
SEQ ID NO.:11:KASNRFS
SEQ ID NO.:12:FQGSKVPRT
SEQ ID NO.:13:RSSQSIVWNNGVTYLE
SEQ ID NO.:14:KVSNHFS
SEQ ID NO.:15:HQGSHVPRT
SEQ ID NO.:16:SSGMGVG
SEQ ID NO.:17:HINWDDDKRYNPALKG
SEQ ID NO.:18:MEDATPYVMDY
SEQ ID NO.:19:TSGMGVG
SEQ ID NO.:20:HIWWDDDKRYNPALKS
SEQ ID NO.:21:MEDLTPYVMDY
SEQ ID NO.:22:TSGVGVG
SEQ ID NO.:23:HIWGDDDKRYNPALKG
SEQ ID NO.:24:MEDLTPYVMEY
SEQ ID NO.:25:TSGMGAG
SEQ ID NO.:26:HIWWDEDKRYNPALKS
SEQ ID NO.:27:MEDLSPYVMDY
SEQ ID NO.:28:TSGMGVH
SEQ ID NO.:29:HIWWDDDKRYNPAVKS
SEQ ID NO.:30:MEDLTPYVMDH
SEQ ID NO.:31:DVLMTQTTSYLPVSLGDQASISCRTSQSIVHNNGVTYLEWYLQKPGQSPKLLIYKASNRFTGVPDRFSGSGSGTDYSLKISRVEAEDLGVYYCFQGSHVPRSFGGGTTLEIK
SEQ ID NO.:32:QVTLKDSGPGILQPSQTLSLTCSFSGYSLSSSGMGVGWIRQPSGKGLEWLAHIN WDDDKRYNPALKGRLTISKDTSNNQVFLHIASVDTTDTATYYCVRMEDATPYVMDYWGQGTSVTVSS
SEQ ID NO.:33:DVLMTQIPLSLPVSLGDQASISCRSSQSIVHNNGVTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPRTFGGGTKLEIK
SEQ ID NO.:34:QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGVGWIRQPSGKGLEWLAHIW WDDDKRYNPALKSRLTISKDTSNNQVFLKIASVDTTDTATYYCVRMEDLTPYVMDYWGQGTSVTVSS
SEQ ID NO.:35:
DVLMTQIPLSLPVSLGDQASISCRSSQSIVHNNGLTYLEWYLQKPGQSPKLLIYRVSNRFSGVPDRFSGSGTDFTLKIAEDLGVYYCFQGSHVYRTFGGGTKLEIK
SEQ ID NO.:36:QVTLRESGPGILQPSQTLSLTCSFSGFSLSTSGVGVGWIRQPSGKGLEWLAHIW GDDDKRYNPALKGRLTISKDTSNNQVFLKIATVDTTDTATYYCVRMEDLTPYVMEYWGQGTSATVSS
SEQ ID NO.:37:DVLSLPVSLGDQASISCRSSQSIVHNNGVTFLEWYLQKPGQSPKLLIYKASNRF SGVPDRFSGSGSGTDFTLKISRVEAEDLYYCFQGSKVPRTFGGGTKLEIK
SEQ ID NO.:38:QVTLKESGPGILQPSQTLSVTCSFSGFSLSTSGMGAGWIRQPSGKGLEWLAHIW WDEDKRYNPALKSRLTISKDTSNNQVFLKIASVDTTDTITYYCVRMEDLSPYVMDYWGQGTSVTVSS
SEQ ID NO.:39:EVLMTQIPLSLPVSLGEQASISCRSSQSIVWNNGVTYLEWYLQKPGQSPKLLIYKVSNHFSGVPDRHSGSGSGTDHTLKISRVEAEDLGIYYCHQGSHVPRTFGGGTRLEIK
SEQ ID NO.:40:QVTAKESGPGILQPSQTLSLTCSFSGFSLSTSGMGVHWIRQPSGKGIEWLAHIW WDDDKRYNPAVKSRLTISKDTTNNQVFLKIASVETTDTATYYCVRMEDLTPYVMDHWGQGTSVTVSS。
SEQUENCE LISTING
<110> Guangzhou Kangsheng Biotechnology GmbH
<120> antibody with double MIC binding activity and application thereof
<130> MIC
<160> 40
<170> PatentIn version 3.5
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Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr
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Lys Ala Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu
65 70 75 80
Asp Leu Tyr Tyr Cys Phe Gln Gly Ser Lys Val Pro Arg Thr Phe Gly
85 90 95
Gly Gly Thr Lys Leu Glu Ile Lys
100
<210> 38
<211> 121
<212> PRT
<213> Artificial sequence
<400> 38
Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Gln Pro Ser Gln
1 5 10 15
Thr Leu Ser Val Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr Ser
20 25 30
Gly Met Gly Ala Gly Trp Ile Arg Gln Pro Ser Gly Lys Gly Leu Glu
35 40 45
Trp Leu Ala His Ile Trp Trp Asp Glu Asp Lys Arg Tyr Asn Pro Ala
50 55 60
Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Asn Asn Gln Val
65 70 75 80
Phe Leu Lys Ile Ala Ser Val Asp Thr Thr Asp Thr Ile Thr Tyr Tyr
85 90 95
Cys Val Arg Met Glu Asp Leu Ser Pro Tyr Val Met Asp Tyr Trp Gly
100 105 110
Gln Gly Thr Ser Val Thr Val Ser Ser
115 120
<210> 39
<211> 112
<212> PRT
<213> Artificial sequence
<400> 39
Glu Val Leu Met Thr Gln Ile Pro Leu Ser Leu Pro Val Ser Leu Gly
1 5 10 15
Glu Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val Trp Asn
20 25 30
Asn Gly Val Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn His Phe Ser Gly Val Pro
50 55 60
Asp Arg His Ser Gly Ser Gly Ser Gly Thr Asp His Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Leu Gly Ile Tyr Tyr Cys His Gln Gly
85 90 95
Ser His Val Pro Arg Thr Phe Gly Gly Gly Thr Arg Leu Glu Ile Lys
100 105 110
<210> 40
<211> 121
<212> PRT
<213> Artificial sequence
<400> 40
Gln Val Thr Ala Lys Glu Ser Gly Pro Gly Ile Leu Gln Pro Ser Gln
1 5 10 15
Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr Ser
20 25 30
Gly Met Gly Val His Trp Ile Arg Gln Pro Ser Gly Lys Gly Ile Glu
35 40 45
Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ala
50 55 60
Val Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Thr Asn Asn Gln Val
65 70 75 80
Phe Leu Lys Ile Ala Ser Val Glu Thr Thr Asp Thr Ala Thr Tyr Tyr
85 90 95
Cys Val Arg Met Glu Asp Leu Thr Pro Tyr Val Met Asp His Trp Gly
100 105 110
Gln Gly Thr Ser Val Thr Val Ser Ser
115 120
Claims (18)
1. An anti-MIC antibody comprising at least one heavy chain variable region comprising three CDRs and at least one light chain variable region comprising three CDRs, wherein:
the light chain CDR1, light chain CDR2, and light chain CDR3 of the light chain variable region have the amino acid sequences as set forth in SEQ ID No.: 1-3, the heavy chain variable region heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 sequences are respectively as shown in SEQ ID No.: 16-18; or
The light chain CDR1, light chain CDR2, and light chain CDR3 of the light chain variable region have the amino acid sequences as set forth in SEQ ID No.: 4-6, the heavy chain variable region heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 sequences are respectively as shown in SEQ ID No.: 19-21; or
The light chain CDR1, light chain CDR2, and light chain CDR3 of the light chain variable region have the amino acid sequences as set forth in SEQ ID No.: 7-9, the heavy chain variable region heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 sequences are respectively as shown in SEQ ID No.: 22-24; or
The light chain CDR1, light chain CDR2, and light chain CDR3 of the light chain variable region have the amino acid sequences as set forth in SEQ ID No.: 10-12, the heavy chain variable region heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 sequences are respectively as shown in SEQ ID No.: 25-27; or
The amino acid sequences of the light chain CDR1, light chain CDR2, and light chain CDR3 of the light chain variable region are SEQ ID No.: 13-15, the heavy chain variable region heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 sequences are respectively as shown in SEQ ID No.: 28-30 parts of; or
The amino acid sequence of the light chain variable region is shown as SEQ ID No.: 31, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID No.: 32 is shown; or
The amino acid sequence of the light chain variable region is shown as SEQ ID No.: 33, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID No.: 34; or
The amino acid sequence of the light chain variable region is shown as SEQ ID No.: 35, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID No.: 36 is shown; or
The amino acid sequence of the light chain variable region is shown as SEQ ID No.: 37, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID No.: 38; or
The amino acid sequence of the light chain variable region is shown as SEQ ID No.: 39, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID No.: shown at 40.
2. The anti-MIC antibody of claim 1, wherein: the anti-MIC antibodies were expressed using eukaryotic cells.
3. The anti-MIC antibody of claim 2, wherein: the anti-MIC antibodies were expressed using CHO cells, 293ft cells, or insect cells.
4. The anti-MIC antibody of claim 1, wherein: the antibody is a single-chain antibody with a molecular structure of VH-X1-VL, and X1 is a linker sequence.
5. The anti-MIC antibody of claim 4, wherein: the linker sequence consists of amino acids G/S.
6. The anti-MIC antibody of claim 5, wherein: the linker sequence is (GS) n, (GGS) n, (GGGS) n or (GGGGS) n, wherein n = 1-5.
7. The anti-MIC antibody of claim 6, wherein: the linker sequence is GGGGSGGGGSGGS.
8. An anti-MIC antibody according to any one of claims 4 to 7, characterised in that: the anti-MIC antibodies were expressed using prokaryotic cells.
9. The anti-MIC antibody of claim 8, wherein: the anti-MIC antibodies were expressed using e.
10. An anti-MIC antibody according to any one of claims 1 to 7, characterised in that: the anti-MIC antibodies have binding activity to MICA/B protein.
11. A nucleotide sequence expressing an anti-MIC antibody, characterized in that: the nucleotide sequence may encode a protein sequence of an anti-MIC antibody according to any one of claims 1 to 10.
12. A kit for detecting MICA/B, comprising: comprising an anti-MIC antibody according to any one of claims 1 to 10.
13. An immunoadsorbent comprising a solid support, wherein: an anti-MIC antibody according to any one of claims 1 to 10 coupled to a solid support.
14. The immunoadsorbent of claim 13, wherein: the solid phase carrier is at least one of chitosan microspheres, agarose microspheres, sephadex, resin and cellulose microspheres.
15. The immunoadsorbent of claim 13 or 14, wherein: it has binding activity to soluble MICA 008, 010, 002, 009 and 005.
16. The application of the immunoadsorbent in the preparation of the blood purifying agent is characterized in that: the immunoadsorbent of any one of claims 13 to 15.
17. Use according to claim 16, characterized in that: the immunoadsorbent is mainly used for adsorbing NKG2D ligand in human blood or blood plasma, so that the tumor immunosuppression effect caused by abnormal rising of MIC protein is relieved.
18. Use according to claim 17, characterized in that: the NKG2D ligand is selected from soluble MICA, MICB molecule and immune complex composed of MICA/B and anti-MIC monoclonal antibody drug.
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CN117843790A (en) * | 2021-12-30 | 2024-04-09 | 广州康盛生物科技股份有限公司 | MIC protein and ULBP protein bivalent nano antibody and application thereof |
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CN108779178A (en) * | 2016-03-15 | 2018-11-09 | 依奈特制药公司 | Anti- MICA antibody |
CN110088137A (en) * | 2016-10-19 | 2019-08-02 | 诺瓦罗技科斯生物科技有限公司 | For the antibody of MICA and MICB albumen |
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US8753640B2 (en) * | 2011-05-31 | 2014-06-17 | University Of Washington Through Its Center For Commercialization | MIC-binding antibodies and methods of use thereof |
AU2014229476B2 (en) * | 2013-03-15 | 2019-07-11 | Novelogics Biotechnology, Inc. | Antibodies to MICA and MICB proteins |
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CN108779178A (en) * | 2016-03-15 | 2018-11-09 | 依奈特制药公司 | Anti- MICA antibody |
CN110088137A (en) * | 2016-10-19 | 2019-08-02 | 诺瓦罗技科斯生物科技有限公司 | For the antibody of MICA and MICB albumen |
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