Humanized single-chain antibody against mic, immunoadsorbent and use thereof

Abstract

The application provides an anti-MIC humanized single-chain antibody, a coding gene, an expression vector, a host, a combination or conjugate, an adsorbent and uses thereof, the humanized single-chain antibody provided by the application can specifically combine with NKG2D ligands in human blood or plasma, and tumor immunosuppression caused by abnormal increase of soluble MIC protein is released. Compared with traditional antibodies, the humanized single-chain antibody provided by the application has the advantages of small molecular weight, strong stability, low immunogenicity, low cost, easy expression, high expression amount and suitability for large-scale production. The humanized single-chain antibody is combined with a solid-phase carrier in the application, has high adsorption efficiency, stable adsorption performance, low non-specific adsorption amount of other proteins in plasma, good specificity, good safety performance in clinical treatment and high economic benefits. Moreover, the humanized single-chain antibody provided by the application reduces side effects caused by shedding of adsorption ligands, and improves safety.

Description

Technical Field

[0001] This invention relates to the field of antibody technology, specifically to a humanized single-chain antibody against MIC, an immunosorbent, and its uses. Background Technology

[0002] NKG2D is a homodimeric type II transmembrane class C lectin receptor expressed in all NK cells, most CD8αβT cells, NKT cells, and γδT cells, as well as a small number of regulatory CD4αβT cells. It is one of the most important activating receptors in tumor cell recognition (Chitadze et al. 2013). The interaction between the NKG2D receptor and its ligands can activate immune cell-mediated killing. NKG2D ligands are usually not expressed or expressed at low levels in healthy human tissues, but are expressed at high levels on the cell membrane surface of infected cells and cancer cells. In humans, NKG2D ligands include MICA and MICB (class I MHC chain-associated proteins A and B), both encoded by MHC genes, with 84% sequence similarity (Lietal. 1999). MICA and MICB can be released from the surface of tumor cells, forming soluble MICA and MICB (sMIC for short). sMIC inhibits the recognition and killing of tumor cells by NK cells and T cells by downregulating NKG2D expression, thereby leading to tumor immune escape. Both MICA and MICB are highly polymorphic. Currently, 192 protein variants of MICA have been identified. In China, more than 80% of the population has the genotype MICA*002, 008, 009, 010, and 012. There are 42 protein variants of MICB, and their homology is higher than that of MICA in populations around the world, with MICB*005 being the most frequent.

[0003] The presence of soluble MICs in the blood of cancer patients and their inhibitory effect on effector cells make them a potential anti-tumor target. Cullinan Pharmaceuticals disclosed in CN112566659A / CN113056484A an anti-MICA / B antibody targeting the α3 domain of the MIC protein. This antibody can bind to membrane-bound MICA*004 and recombinant free MICA*001, 002, 004, 008, 009, and MICB, and can inhibit the shedding of MICA from the surface of PLC / PRF / 5 hepatocellular carcinoma cells. Jennifer Wu et al. developed an anti-MIC antibody using MICB as an antigen. This antibody, used in a tumor model constructed from transgenic mice capable of generating soluble MICB, enhanced the immune activity of the tumor microenvironment, reduced tumor quality, and inhibited metastasis without causing significant systemic toxicity. CN110088137A, WO2014140884A2, and US20200165343A1 disclose antibodies capable of binding to MICA / B, primarily binding to the α3 domain of the MICA / B protein. These antibodies effectively bind to MICA*002, 008, 009, 018, and MICB*005, and further enhance toxicity by binding to CD16 on immune cells. However, these antibodies do not enhance the toxicity of NK cells against tumor cells releasing MIC in exosome form. CN108779178A discloses an anti-MICA antibody that binds to the α2 domain on the lateral side of MICA near the NKG2D binding surface, specifically binding to MICA*001, 004, 007, and 008. Monoclonal antibody drugs require relatively large doses, and large-dose infusion of antibody drugs also poses certain safety risks.

[0004] Blood purification refers to the process of drawing a patient's blood out of the body and passing it through a purification device to remove certain pathogenic substances, thereby purifying the blood and treating diseases. Adsorption is one of the important principles of blood purification in removing pathogenic substances. Immunoadsorption combines highly specific antigens, antibodies, or substances with specific physicochemical affinity (ligands) with adsorption materials to form adsorption columns, allowing them to selectively or specifically adsorb corresponding pathogenic factors in the body. In combination with drug therapy, immunoadsorption can enhance the body's sensitivity to drug treatment, increase drug efficacy, reduce side effects, shorten the course of treatment, and reduce the recurrence rate. In recent years, immunoadsorption has been successfully used in the treatment of autoimmune diseases, cytokine storms, sepsis, etc., and also shows promise for the treatment of malignant tumors. US9707333 uses snow lotus lectin, anti-Fas antibodies, or aptamers as adsorption ligands, which are coupled to agarose microspheres, polyacrylate microspheres, or hollow fibers. Combined with membrane separation and immunoadsorption technology, it removes tumor-derived exosomes and restores the body's immune surveillance of tumor cells. Literature has documented the preparation of immunoadsorbents using protein A immunospheres to adsorb anti-MICA and anti-MICB antibodies. These immunoadsorbents effectively removed free MICA and MICB from plasma samples of patients with head and neck squamous cell carcinoma, and the antitumor activity of NK cells incubated with plasma treated with the adsorbent was restored. This research group also conjugated anti-MICA antibodies to microspheres to prepare immunoadsorbents. After three complete plasma volume exchanges, these immunoadsorbents effectively removed recombinant soluble MICA*004 injected into rhesus monkey plasma (Sandra Weil et al. 2017). The mouse monoclonal antibody developed by CN112574311A and the single-chain antibody designed based on its variable region sequence showed binding activity for MICA*002, 008, 009, 010, and MICB*005. Immunoadsorbents prepared using the mouse monoclonal antibody and single-chain antibody as adsorbent ligands exhibited adsorption properties for all five MIC proteins. However, both murine monoclonal antibodies and single-chain antibodies have murine-derived sequences, and the shedding of murine ligands into the human body can cause adverse immune responses. Summary of the Invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide a humanized single-chain antibody against MIC, an immunoadsorbent and its uses. The humanized single-chain antibody provided by the present invention has good adsorption performance on NKG2D ligand, reduces the side effects caused by the shedding of adsorbed ligand, and improves safety.

[0006] This invention provides a humanized single-chain antibody against MIC, comprising a light chain (VL) and a heavy chain (VH), wherein the amino acid sequences of the light chain and the heavy chain include:

[0007] (I) The amino acid sequence of the light chain is shown in SEQ ID No. 1 to 3; the amino acid sequence of the heavy chain is shown in any one of SEQ ID No. 4 to 6;

[0008] or

[0009] (II) Amino acid sequences obtained by substituting, deleting, or adding one or more amino acids to the amino acid sequence described in (I), and which have the same function as the amino acid sequence described in (I);

[0010] or

[0011] (III) An amino acid sequence that has more than 80% homology with the amino acid sequence described in (I) or (II).

[0012] In some specific embodiments, the humanized single-chain antibody further includes a linker sequence.

[0013] In some specific embodiments, the linker sequence consists of amino acids G / S.

[0014] In some specific implementations, the connecting subsequence is (GS)n, (GGS)n, (GGGS)n or (GGGGS)n, where n = 1 to 5.

[0015] In some specific implementations, the connector sequence is (GS)5, (GGS)2, (GGS)4, (GGGS)4, (GGGS)3, (GGGGS)1, (GGGGS)3, or (GGGGS)4.

[0016] In some specific implementations, the molecular structure of the antibody is VH-linker-VL or VL-linker-VH.

[0017] In some specific implementations, the amino acid sequence of the light chain is as shown in SEQ ID No. 1, and the amino acid sequence of the heavy chain is as shown in any one of SEQ ID No. 4 to 6;

[0018] Alternatively, the amino acid sequence of the light chain is as shown in SEQ ID No. 2, and the amino acid sequence of the heavy chain is as shown in any one of SEQ ID Nos. 4 to 6;

[0019] Alternatively, the amino acid sequence of the light chain may be as shown in SEQ ID NO.:3, and the amino acid sequence of the heavy chain may be as shown in any one of SEQ ID No.4 to 6.

[0020] Specifically, in some embodiments, the amino acid sequence of the light chain is as shown in SEQ ID No. 1, the amino acid sequence of the heavy chain is as shown in SEQ ID No. 4, and the linker sequence is (GGGGS)1;

[0021] Alternatively, the amino acid sequence of the light chain is as shown in SEQ ID No. 1, the amino acid sequence of the heavy chain is as shown in SEQ ID No. 5, and the linker sequence is (GGGS)4.

[0022] The amino acid sequence of the light chain is shown in SEQ ID No. 1, the amino acid sequence of the heavy chain is shown in SEQ ID No. 6, and the linker sequence is (GGGS)3;

[0023] The amino acid sequence of the light chain is shown in SEQ ID No. 2, the amino acid sequence of the heavy chain is shown in SEQ ID No. 4, and the linker sequence is (GGS)4;

[0024] The amino acid sequence of the light chain is shown in SEQ ID No. 2, the amino acid sequence of the heavy chain is shown in SEQ ID No. 5, and the linker sequence is (GGGGS)2;

[0025] The amino acid sequence of the light chain is shown in SEQ ID No. 2, the amino acid sequence of the heavy chain is shown in SEQ ID No. 6, and the linker sequence is (GGGGS)3;

[0026] The amino acid sequence of the light chain is shown in SEQ ID NO.:3, the amino acid sequence of the heavy chain is shown in SEQ ID No.4, and the linker sequence is (GGGS)4;

[0027] The amino acid sequence of the light chain is shown in SEQ ID No. 3, the amino acid sequence of the heavy chain is shown in SEQ ID No. 5, and the linker sequence is (GGS)2;

[0028] The amino acid sequence of the light chain is shown in SEQ ID No. 3, the amino acid sequence of the heavy chain is shown in SEQ ID No. 6, and the linker sequence is (GS)5.

[0029] In some specific embodiments, the amino acid sequence of the light chain is shown in SEQ ID No. 1, the amino acid sequence of the heavy chain is shown in SEQ ID No. 4, the linker sequence is (GGGGS)1, and the antibody structure is VH-linker-VL;

[0030] Alternatively, the amino acid sequence of the light chain is as shown in SEQ ID No. 1, the amino acid sequence of the heavy chain is as shown in SEQ ID No. 5, the linker sequence is (GGGS)4, and the antibody structure is VL-linker-VH;

[0031] The amino acid sequence of the light chain is shown in SEQ ID No. 1, the amino acid sequence of the heavy chain is shown in SEQ ID No. 6, the linker sequence is (GGGS)3, and the antibody structure is VH-linker-VL;

[0032] The amino acid sequence of the light chain is shown in SEQ ID No. 2, the amino acid sequence of the heavy chain is shown in SEQ ID No. 4, the linker sequence is (GGS)4, and the antibody structure is VL-linker-VH;

[0033] The amino acid sequence of the light chain is shown in SEQ ID No. 2, the amino acid sequence of the heavy chain is shown in SEQ ID No. 5, the linker sequence is (GGGGS)2, and the antibody structure is VH-linker-VL;

[0034] The amino acid sequence of the light chain is shown in SEQ ID No. 2, the amino acid sequence of the heavy chain is shown in SEQ ID No. 6, the linker sequence is (GGGGS)3, and the antibody structure is VL-linker-VH;

[0035] The amino acid sequence of the light chain is shown in SEQ ID NO.:3, the amino acid sequence of the heavy chain is shown in SEQ ID No.4, the linker sequence is (GGGS)4, and the antibody structure is VH-linker-VL;

[0036] The amino acid sequence of the light chain is shown in SEQ ID No. 3, the amino acid sequence of the heavy chain is shown in SEQ ID No. 5, the linker sequence is (GGS)2, and the antibody structure is VH-linker-VL;

[0037] The amino acid sequence of the light chain is shown in SEQ ID No. 3, the amino acid sequence of the heavy chain is shown in SEQ ID No. 6, the linker sequence is (GS)5, and the antibody structure is VL-linker-VH.

[0038] The present invention also provides a nucleic acid molecule encoding a humanized single-chain antibody against MIC as described in the above-described technical solution.

[0039] The present invention also provides an expression vector comprising nucleic acid molecules as described in the above technical solutions.

[0040] In one embodiment, the expression vector is a plasmid.

[0041] The present invention also provides a host for transforming or transfecting the expression vector described in the above technical solutions.

[0042] In one embodiment, the host is a bacterium, fungus, or animal cell.

[0043] In one embodiment, the bacteria are selected from Escherichia coli; the fungus is selected from Pichia pastoris; and the animal cells are selected from Sf9 cells, High Five cells, CHO cells, or 293 cells.

[0044] The present invention also provides a conjugate or conjugate comprising the humanized single-chain antibody against MIC as described in the above technical solution, and acceptable excipients or carriers.

[0045] The present invention also provides an immunoadsorbent comprising the humanized single-chain antibody against MIC and the carrier described in the above technical solution.

[0046] In some specific embodiments, the carrier is at least one of chitosan microspheres, agarose microspheres, dextran gel, resin, and cellulose microspheres.

[0047] The present invention also provides a chimeric antigen receptor, comprising the humanized single-chain antibody described in the above technical solution.

[0048] In some specific embodiments, the chimeric antigen receptor further includes an extracellular hinge region, a T-cell receptor transmembrane domain, and an intracellular T-cell receptor signal transduction domain.

[0049] This invention also provides the use of the humanized single-chain antibodies, conjugates or conjugates, immunoadsorbents, or chimeric antigen receptors against MIC described in the above-mentioned technical solutions in the preparation of drugs or formulations for the prevention and / or treatment of tumors and immune diseases, the uses including but not limited to:

[0050] (1) Enhance, strengthen or stimulate immune response or function;

[0051] (2) It has binding activity to soluble MICA*002, MICA*004, MICA*007, MICA*008, MICA*009, MICA*012, MICA*019, MICA*045 and MICB*002, MICB*005, MICB*008 and MICB*014 proteins.

[0052] (3) Inhibit tumor growth or kill tumor cells.

[0053] The present invention also provides a blood purification device, comprising the conjugate or coupling compound described in the above technical solution, or comprising the immunosorbent described in the above technical solution.

[0054] This invention provides a drug or drug combination that directly or indirectly includes the humanized single-chain antibody against MIC, a nucleic acid molecule, an expression vector, a host, a conjugate or conjugate, an immunoadsorbent or chimeric antigen receptor, and acceptable excipients as described in the above technical solutions.

[0055] The present invention also provides a detection reagent or detection kit, which directly or indirectly includes the anti-MIC humanized single-chain antibody, nucleic acid molecule, expression vector, host, conjugate or conjugate, immunoadsorbent or chimeric antigen receptor, and acceptable excipients and adjuvants as described in the above technical solutions.

[0056] The humanized single-chain antibody against MIC provided by this invention can specifically bind to NKG2D ligands in human blood or plasma, including soluble MICA / B molecules such as MICA*002, MICA*004, MICA*007, MICA*008, MICA*009, MICA*012, MICA*019, MICA*045 and MICB*002, MICB*005, MICB*008, MICB*014, or exosome-type MICA / B molecules and immune complexes composed of MICA / B and anti-MIC antibody drugs, thereby relieving the tumor immunosuppressive effect caused by abnormally elevated soluble MIC proteins. Compared with traditional antibodies, the humanized single-chain antibody provided by this invention has the advantages of small molecular weight, high stability, low immunogenicity, low cost, easy expression and high expression level, making it suitable for large-scale production. This invention combines humanized single-chain antibodies with a solid-phase carrier, enabling specific binding to soluble or exosome-type human MIC proteins. It exhibits high adsorption efficiency and stable adsorption performance, with low non-specific adsorption of other proteins in plasma, demonstrating good specificity and resulting in good safety and cost-effectiveness in clinical treatment. Furthermore, the humanized single-chain antibody provided by this invention reduces side effects caused by ligand shedding, thus improving safety. Attached Figure Description

[0057] Figure 1 The immunogenicity statistics of the antibodies provided in the examples. Detailed Implementation

[0058] The following non-limiting embodiments are intended to enable those skilled in the art to more fully understand the present invention, but do not limit the invention in any way. All technologies implemented based on the above description of the present invention should fall within the scope of protection claimed in this application.

[0059] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; the biological materials used in the reagent kits are commercially available unless otherwise specified.

[0060] Example 1: Preparation of free MIC protein

[0061] (1) Using pcDNA3 as the expression vector, the cDNA sequences encoding the extracellular regions of MICA*002, MICA*004, MICA*007, MICA*008, MICA*009, MICA*012, MICA*019, MICA*045 and MICB*002, MICB*005, MICB*008, and MICB*014 with His×6 tags at the C-terminus were ligated into the vector (synthesized by Guangzhou Aiji Biotechnology Co., Ltd.).

[0062] (2) After obtaining the transformant bacteria containing plasmids, ferment them with LB liquid medium, treat the bacterial solution with QIAGEN EndoFree Plasmid Kit and extract plasmids;

[0063] (3) Take 293F cells in the logarithmic growth phase with a survival rate ≥90% and dilute the cells to 4×10⁻⁶ using serum-free medium. 6 Cells / mL, placed in an Erlenmeyer flask for later use;

[0064] (4) Using physiological saline as a medium, add 120 μL / mL of Polyjet (SignaGen) to prepare solution A, and add 40 μg / mL of plasmid DNA to prepare solution B. Let stand at 20-30℃ for 5 min, mix solutions A and B in equal proportions and let stand for 10 min to obtain transfection complex. Then add the transfection complex to the prepared 293F cell suspension at a ratio of 1:20 and place it in a carbon dioxide shaking incubator for suspension culture at 130 rpm for 72 hours.

[0065] (5) Collect the expression supernatant by centrifugation at 2000 rpm for 10 min and filter it through a 0.45 μm filter membrane;

[0066] (6) Using PBS as a buffer system, the protein in the expression supernatant was purified by nickel ion affinity chromatography. The protein was eluted with PBS solution containing 500 mM imidazole. The imidazole in the protein solution was then removed by dialysis and concentrated to obtain soluble recombinant MICA*002, MICA*004, MICA*007, MICA*008, MICA*009, MICA*012, MICA*019, MICA*045 and MICB*002, MICB*005, MICB*008 and MICB*014 proteins, respectively.

[0067] Example 2: Preparation of exosomal MIC protein

[0068] Adherent HeLa cells (MICA*008 / 008) were cultured in DMEM medium containing 10% fetal bovine serum until 70% confluence. The culture supernatant was discarded, and the adherent cells were washed 1-2 times with 1X PBS, followed by 24 hours of culture in serum-free DMEM medium. The culture supernatant was collected and centrifuged at 2000xg for 30 minutes to remove cells and debris. The supernatant was then concentrated by ultrafiltration using an ultrafiltration tube with a molecular weight cutoff of 500kD to remove contaminating proteins, achieving a concentration factor of 20-fold. 0.5 volumes of total exosome separation reagent (ThermoFisher, 4478359) were added, mixed, and incubated overnight at 4°C. Subsequently, the cells were centrifuged at 10,000xg for 1 hour at 4°C. The supernatant was discarded, and the precipitate was resuspended in 1X PBS to obtain exosomes. Quantification and purity identification were performed using nanoparticle tracking analysis, and the exosomes were stored at 4°C for later use.

[0069] Example 3: Humanization of Anti-MIC Single-Chain Antibodies

[0070] This document provides three monoclonal cell lines, 3F5, 9C9, and 10A1, disclosed in Chinese Patent 202011464426.4, and the antibodies they produce. The light chain and heavy chain variable regions of the antibodies produced by each cell line are shown in Table 1. Table 1 contains the mouse antibody sequences.

[0071] Table 1. Murine antibody sequences

[0072] cell lines VL amino acid sequence VH amino acid sequence 3F5 SEQ ID NO: 7 SEQ ID NO: 8 9C9 SEQ ID NO: 9 SEQ ID NO: 10 10A1 SEQ ID NO: 11 SEQ ID NO: 12

[0073] SEQ ID No. 7:

[0074] DVLMTQIPLSLPVSLGDQASISCRSSQSIVHNNGVTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPRT FGGGTKLEIK

[0075] SEQ ID No. 8:

[0076] QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGVGWIRQPSGKGLEWLAHIWWDDDKRYNPALKSRLTISKDTSNNQVFLKIASVDTTDTATYYCVRMEDLTPYVMDY WGQGTSVTVSS

[0077] SEQ ID No. 9:

[0078] DVLSLPVSLGDQASISCRSSQSIVHNNGVTFLEWYLQKPGQSPKLLIYKASNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLYYCFQGSKVPRTFGGGTKLEIK

[0079] SEQ ID No.10:

[0080] QVTLKESGPGILQPSQTLSVTCSFSGFSLSTSGMGAGWIRQPSGKGLEWLAHIWWDEDKRYNPALKSRLTISKDTSNNQVFLKIASVDTTDTITYYCVRMEDLSPYVMDY WGQGTSVTVSS

[0081] SEQ ID No.11:

[0082] EVLMTQIPLSLPVSLGEQASISCRSSQSIVWNNGVTYLEWYLQKPGQSPKLLIYKVSNHFSGVPDRHSGSGSGTDHTLKISRVEAEDLGIYYCHQGSHVPRT FGGGTRLEIK

[0083] SEQ ID No.12:

[0084] QVTAKESGPGILQPSQTLSLTCSFSGFSLSTSGMGVHWIRQPSGKGIEWLAHIWWDDDKRYNPAVKSRLTISKDTTNNQVFLKIASVETTDTATYYCVRMEDLTPYVMDH WGQGTSVTVSS

[0085] The humanization process is as follows:

[0086] By comparing and analyzing the IGBLAST database, the human antibody sequences with the highest homology to the light chain variable region and heavy chain variable region of the 3F5 monoclonal antibody were identified as IGKV2-30*02 and IGHV2-5*02 germline genes, respectively. The CDR region of the murine antibody was combined with the FR region of the human antibody sequence, and individual amino acids were mutated at specific sites based on the differences between the human and murine antibody sequences. The linker was designed as (GGGS)3. The nucleotide sequences encoding the VL and VH of the humanized anti-MIC single-chain antibody were directly synthesized into VH-(GGGS)3-VL-his or VL-(GGGS)3-VH-his sequences, and inserted into the PET28a plasmid. The plasmid was transformed into E. coli BL21(DE3), cultured in kanamycin-resistant LB medium, and induced with 0.5 mM IPTG at 37°C for 6 hours. The bacterial cells were collected by centrifugation, resuspended in 1X PBS, and the supernatant was purified by cobalt affinity chromatography. Sequences with good performance in each indicator, such as solubility, expression level, activity, and stability, were screened. The same method was used to humanize the variable regions of the heavy and light chains of 9C9 and 10A1, resulting in three humanized light chain and three humanized heavy chain sequences. Nine single-chain antibodies were obtained by pairing the light chain and heavy chain sequences one by one, as shown in Table 2. Table 2 shows the humanized single-chain antibody sequences provided in the embodiments of the present invention.

[0087] Table 2. Humanized single-chain antibody sequences provided in the embodiments of the present invention.

[0088]

[0089] The sequence is as follows:

[0090] SEQ ID No. 1:

[0091] DVVMTQTPLSLPVTLGQPASISCRSSQSIVHNNGVTYLNWFQQRPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPRTFGGGTKLEIK

[0092] SEQ ID No. 2:

[0093] LSLPVTPGEPASISCRSSQSIVHNNGVTFLEWYLQKPGQSPQLLIYKASNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLYYCFQGSKVPRTFGQGTKVEIK

[0094] SEQ ID No. 3:

[0095] EVLMTQSPLSLPVSLGEPASISCRSSQSIVWNNGVTYLEWYQQKPGQSPRRLIYKVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDLGIYYCHQGSHVPRTFGGGTKLEIK

[0096] SEQ ID No. 4:

[0097] QVTLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVGWIRQPSGKALEWLAHIWWDDDKRYNPALKSRLTITKDTSNNQVFLTMTNMDTVDTATYYCVRMEDLTPYVMDYWGQGTLVTVSS

[0098] SEQ ID No. 5:

[0099] QVTLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGAGWIRQPSGKALEWLAHIWWDEDKRYNPALKSRLTITKDTSKNQVFLTMTNMDTVDTITYYCVRMEDLSPYVMDYWGQGTLVTVSS

[0100] SEQ ID No.6:

[0101] QVTLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVHWIRQPPGKALEWLAHIWWDDDKRYNPAVKSRLTITKDTSNNQVVLTMTNMEPTDTATYYCVRMEDLTPYVMDHWGQGTLVTVSS

[0102] Example 4 Synthesis of Immunosorbent

[0103] 1) The protein was expressed, purified, and collected according to Example 3;

[0104] 2) Take 5 mL of dextran microspheres Sephacryl S-1000SF, add 15 mL of 0.01 M sodium hydroxide, add 3.0 mL of epichlorohydrin solution, react at 37℃ and 120 rpm for 2 h, then wash the packing material and dry it.

[0105] 3) Add 5 mL of PBS solution and 3.0 mL of ethylenediamine solution. React at 37°C and 120 rpm for 3 hours. Then wash the packing material and dry it under vacuum.

[0106] 4) Add 5 mL of PBS solution and 7.5 mL of glutaraldehyde solution. React at 37°C and 120 rpm for 3 hours. Then wash the packing material and dry it under vacuum.

[0107] 5) Take 1 mL of the activated packing material into a disposable chromatography column, add 0.5 mL of a protein solution with a buffer system of 0.1 M sodium carbonate, 0.5 M sodium chloride, and pH = 8.0, and add 10 mg of each humanized single-chain antibody. Place the column on a decolorizing shaker and mix at 100 rpm at room temperature for 2 h. Then wash the packing material with 10 mL of pH = 8.0, 0.1 M Tris-HCl solution, add 5 mL of pH = 8.0, 0.1 M Tris-HCl solution to block at room temperature for 3 h, and finally wash the packing material with a large amount of purified water and store it.

[0108] Example 5: Evaluation of the adsorption performance of the immunosorbent for free MIC

[0109] 0.5 mL of the immunosorbent synthesized in Example 4 and 1 mL of free recombinant MICA*002, MICA*004, MICA*007, MICA*008, MICA*009, MICA*012, MICA*019, MICA*045 and MICB*002, MICB*005, MICB*008, MICB*014 protein solutions prepared in Example 1 were loaded into 10 mL EP tubes, with a protein concentration of 100 μg / mL. The mixture was stirred at 100 rpm at room temperature for 1 h. The packing material was then placed in a disposable affinity chromatography column, and the flow-through was collected. The packing material was washed with 0.5 mL of PBS, collected, and combined with the flow-through. A blank control of Sephacryl S-1000SF dextran microspheres was used. The change in total protein content in the solution before and after adsorption was detected using the Micro BCAProtein Assay kit (thermo), and the adsorption rate was calculated. The results are shown in Table 3. Table 3 shows the adsorption performance test results of the humanized single-chain antibody synthesis adsorbent provided in the embodiments of the present invention.

[0110] Table 3. Determination of Adsorption Performance of Adsorbents for Humanized Single-Chain Antibody Synthesis

[0111]

[0112] Example 6: Evaluation of the adsorption performance of immunosorbents on exosomal MICs

[0113] 0.5 mL of the immunosorbent synthesized in Example 4 and 1 mL of the solution containing MICA*008 exosomes prepared in Example 2 were respectively added to 10 mL EP tubes. The exosome concentration was 1 × 10⁻⁶. 10 / mL, after mixing at 100rpm at room temperature for 1-2h, the packing material was placed in a disposable affinity chromatography column, the flow-through was collected, and the packing material was washed with 0.5mL PBS, collected, and combined with the flow-through. The change in exosome concentration in the solution before and after adsorption was detected by nanoparticle tracer analysis, and the adsorption rate was calculated. The blank dextran microspheres Sephacryl S-1000SF were used as a control. The results are shown in Table 4. Table 4 shows the comparison of the scavenging rates of different adsorbents for exosomes containing MICA*008.

[0114] Table 4 Comparison of removal rates of MICA*008-containing exosomes by different adsorbents

[0115]

[0116]

[0117] Example 7: Stability Evaluation of Immunosorbents for Humanized Single-Chain Antibody Synthesis

[0118] The humanized single-chain antibody immunosorbent synthesized in Example 4 was stored in PBS containing 0.1% NaN3 and placed in a 4°C refrigerator for 7, 14, 30, and 60 days, respectively. The adsorption performance of the packing material for free MICA*008 and MICB*005 proteins, as well as MICA*008-containing exosomes, was then repeatedly measured according to the methods in Examples 5 and 6. The results are shown in Table 5. Within the detection error range of the kit, the adsorption performance of the packing material for MIC proteins did not decrease, and the adsorption performance of the adsorbent did not decrease within two months of storage at 4°C.

[0119] Table 5. Stability determination of adsorption of adsorbent for humanized single-chain antibody synthesis

[0120]

[0121]

[0122] Example 8: Cytotoxicity test of humanized single-chain antibody synthesis immunosorbent

[0123] 1) Culture L-929 cells in MEM complete medium containing 10% serum and 1% penicillin and streptomycin mixture until the logarithmic growth phase. Digest with trypsin and prepare cells to 1×10⁶ cells / year using MEM complete medium. 5 Cell suspension of 100 μL / well was seeded into a 96-well plate and incubated in a CO2 incubator for 24 hours. The plate was prepared with blank control, negative control, positive control and test sample group, with at least 3 wells in each group.

[0124] 2) Preparation of test solution: Take the sterile humanized single-chain antibody immunosorbent prepared in Example 4 and the blank dextran microspheres, add them to the complete culture medium at a ratio of 0.2 g / mL, mix thoroughly, and place in a carbon dioxide incubator for 24 hours.

[0125] 3) Preparation of negative control solution: Take high-density polyethylene material, wash it with ultrapure water and dry it. After irradiation with ultraviolet light, add it to the complete culture medium at a ratio of 0.2 g / mL, mix it thoroughly, and place it in a carbon dioxide incubator for 24 hours.

[0126] 4) Preparation of positive control solution: Take polyurethane material containing zinc diethyldithiocarbamate and diethyldithiocarbamate, wash it with ultrapure water and dry it. After irradiation with ultraviolet light, add it to the complete culture medium at a ratio of 0.2 g / mL, mix it thoroughly, and place it in a carbon dioxide incubator for 24 hours.

[0127] 5) Discard the original cell culture medium, add cell culture medium to the blank control group, add negative control solution to the negative control group, add positive control solution to the positive control group, and add test solution to the test sample group. Add 100 μL to each well and continue to incubate in a carbon dioxide incubator for 24 hours.

[0128] 6) After the culture is completed, remove the culture medium of the cells, add 50 μL of MTT solution with a concentration of 1 mg / mL to each well, continue to culture for 2 hours, discard the liquid in the well, add 100 μL of isopropanol, place on a shaker and shake for 10 minutes, measure the absorbance at a wavelength of 570 nm using a microplate reader, and calculate the survival rate.

[0129] The test results are shown in Table 6. The cell viability of the test sample group was not less than 70% of that of the blank, which meets the national standard.

[0130] Table 6. Cytotoxicity assay of adsorbents used in the synthesis of humanized single-chain antibodies.

[0131] OD570 average value Survival rate (%) Blank control 0.98 negative control 0.96 98.36 Positive control 0.03 2.67 1-4 0.86 88.25 1-5 0.88 90.09 1-6 0.89 91.14 2-4 0.90 91.45 2-5 0.87 88.63 2-6 0.85 86.91 3-4 0.85 89.82 3-5 0.85 87.03 3-6 0.90 91.49 Blank dextran microspheres 0.94 95.44

[0132] Example 9 Immunogenicity Test

[0133] (1) Select 20 healthy people, collect blood from healthy donors and separate PBMCs, and freeze a portion of them.

[0134] (2) Mononuclear cells were isolated from fresh PBMCs using a mononuclear cell isolation kit and an LS sorting column (Mitteni). The cells were resuspended in DC cell culture medium containing IL-4 (500 U / mL) and GM-CSF (1000 U / mL) and cultured.

[0135] (3) Change half the medium every 2 days. On the 4th day of culture, dilute the humanized single-chain antibody and mouse single-chain antibodies 3F5, 9C9 and 10A1 prepared in Example 3 with DC cell culture medium to a final concentration of 0.4 μM. At the same time, use keyhole hemocyanin of equal concentration as a positive control and DC cell culture medium as a negative control.

[0136] (4) After 24 hours, the cells were washed twice with PBS and then induced to mature with a culture medium containing TNFα (10 ng / mL), IL-1β (10 ng / mL) and IL-6 (1000 U / mL).

[0137] (5) Harvest mature DC cells, stain them with trypan blue, count the cells, calculate the cell viability, and then treat them with 30 Gy of γ rays.

[0138] (6) Resuscitate frozen PBMC cells, isolate CD4+ T cells using CD4+ T cell isolation kit (Mitten), stain with trypan blue, count the cells, and calculate cell viability.

[0139] (7) 2×10 5 / mL of CD4+ T cells and 2×10 4 DC cells of 200 μL / mL were added to a 96-well round-bottom microtiter plate and cultured for 2 days.

[0140] (8) Collect a small amount of culture supernatant and use a human IL-2 ELISA kit (BD Biosciences) to detect the IL-2 content in the culture supernatant.

[0141] (9) Add 50 μL of solution containing 1 μCi to each well. 3 Incubate cells in a medium containing H-thymidine for 6 hours. Collect cells into a glass fiber filter and measure [ ] using a β-scintillation counter. 3 The amount of H-thymidine inserted.

[0142] (10) Calculate the stimulation index (SI) for IL-2 and T cell proliferation, respectively. A positive result is defined as SI > 2. Calculate the percentage of all positive donors; see [link to results]. Figure 1 , Figure 1 The immunogenicity statistics of the antibodies provided in the examples are as follows: Figure 1 As shown, the immunogenicity of humanized single-chain antibodies is significantly reduced compared to murine single-chain antibodies 3F5, 9C9, and 10A1.

[0143] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. An anti-MIC humanized single chain antibody, characterized in that, comprises a light chain and a heavy chain, the amino acid sequence of the light chain is shown in SEQ ID No. 3; the amino acid sequence of the heavy chain is shown in any one of SEQ ID No. 4-6.

2. The humanized single chain antibody of claim 1, wherein, further comprising a linker sequence.

3. The humanized single chain antibody of claim 2, wherein, the linker sequence consists of amino acids G and S.

4. The humanized single chain antibody of claim 3, wherein, the linker sequence is (GS)n, (GGS)n, (GGGS)n or (GGGGS)n, n = 1-5.

5. The humanized single chain antibody of claim 4, wherein, the linker sequence is (GS)5, (GGS)2, (GGS)4, (GGGS)4, (GGGS)3, (GGGGS)1, (GGGGS)3 or (GGGGS)4.

6. The humanized single chain antibody according to any one of claims 1 to 5, wherein: the molecular structure of the antibody is VH-linker-VL or VL-linker-VH.

7. The humanized single-chain antibody of claim 1, wherein, the amino acid sequence of the light chain is shown in SEQ ID No. 3; the amino acid sequence of the heavy chain is shown in SEQ ID No. 4; and the linker sequence is (GGGS)4; the amino acid sequence of the light chain is shown in SEQ ID No. 3; the amino acid sequence of the heavy chain is shown in SEQ ID No. 5; and the linker sequence is (GGS)2; the amino acid sequence of the light chain is shown in SEQ ID No. 3; the amino acid sequence of the heavy chain is shown in SEQ ID No. 6; and the linker sequence is (GS)5.

8. The humanized single-chain antibody of claim 7, wherein, the amino acid sequence of the light chain is shown in SEQ ID No. 3; the amino acid sequence of the heavy chain is shown in SEQ ID No. 4; the linker sequence is (GGGS)4; and the antibody structure is VH-linker-VL; the amino acid sequence of the light chain is shown in SEQ ID No. 3; the amino acid sequence of the heavy chain is shown in SEQ ID No. 5; the linker sequence is (GGS)2; and the antibody structure is VH-linker-VL; the amino acid sequence of the light chain is shown in SEQ ID No. 3; the amino acid sequence of the heavy chain is shown in SEQ ID No. 6; the linker sequence is (GS)5; and the antibody structure is VL-linker-VH.

9. A nucleic acid molecule encoding the anti-MIC humanized single-chain antibody of any one of claims 1-8.

10. An expression vector comprising the nucleic acid molecule of claim 9.

11. The expression vector of claim 10, wherein, the vector is a plasmid.

12. A host transformed or transfected with the expression vector of claim 10 or 11.

13. The host of claim 12, wherein, the host is a bacterial, fungal or animal cell.

14. The host of claim 13, wherein, the bacterial is selected from E. coli; the fungal is selected from Pichia pastoris; and the animal cell is selected from Sf9 cell, High Five cell, CHO cell or 293 cell.

15. An immunoadsorbent, characterized in that, the anti-MIC humanized single-chain antibody of any one of claims 1-8 and the vector.

16. The immunoadsorbent of claim 15, wherein, the vector is at least one of chitosan microspheres, agarose microspheres, dextran gel, resin, cellulose microspheres.

17. A chimeric antigen receptor, characterized in that, The humanized single-chain antibody according to any one of claims 1 to 8.

18. The chimeric antigen receptor of claim 17, wherein, Further comprising an extracellular hinge region, a T cell receptor transmembrane domain, and an intracellular T cell receptor signaling domain.

19. Use of the anti-MIC humanized single-chain antibody according to any one of claims 1 to 8, the immunoabsorbent according to claim 15 or 16, or the chimeric antigen receptor according to claim 17 or 18 for the manufacture of an immunoabsorbent having adsorbing activity for soluble MICA*002, MICA*004, MICA*007, MICA*008, MICA*009, MICA*012, MICA*019, MICA*045 and MICB*002, MICB*005, MICB*008, MICB*014 proteins.

20. A blood purification apparatus characterized by comprising: The immunoabsorbent according to claim 15 or 16.

21. A medicament, a pharmaceutical combination, a detection reagent or a detection kit, characterized in that, The anti-MIC humanized single-chain antibody according to any one of claims 1 to 8, the nucleic acid molecule according to claim 9, the expression vector according to claim 10 or 11, the host according to claims 12 to 14, the immunoabsorbent according to claims 15 to 16, or the chimeric antigen receptor according to claims 17 to 18, and an acceptable excipient.

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