An anti-CRTAM monoclonal antibody and its preparation method and application

Abstract

The present invention discloses an anti-CRTAM monoclonal antibody and its preparation method and application, relating to the field of biomedical technology. The anti-CRTAM monoclonal antibody comprises CDR-H1, CDR-H2 and CDR-H3 with amino acid sequences such as SEQ ID NO.3-5, CDR-L1 with amino acid sequence such as SEQ ID NO.6, CDR-L2 with amino acid sequence RMS and CDR-L3 with amino acid sequence such as SEQ ID NO.7. The anti-CRTAM monoclonal antibody provided by the present invention can target and bind to mouse CRTAM protein, can significantly eliminate mouse CRTAM-positive T cells in preclinical models, has the effect of significantly alleviating immunotherapy-related adverse reactions, can be used for detection, blocking and / or elimination of mouse CRTAM protein, and is of great significance in research scenarios such as immunotherapy-related adverse reactions.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to an anti-CRTAM monoclonal antibody and a preparation method and application thereof. Background Art

[0002] Class I-restricted T cell-associated molecule (CRTAM) is a transmembrane protein. CRTAM belongs to the Ig-SF protein family, which contains multiple immunoregulatory and cell adhesion molecules and plays an important role in the formation, response and cell interaction of the immune system. CRTAM is specifically expressed in activated CD8 + and CD4 + T cells, whose ligand cell adhesion molecule 1 (CADM1) is highly expressed in non-tumor tissues.

[0003] Studies have shown that CRTAM is a key molecule in the development of immune-related adverse events (irAEs) associated with immune checkpoint blockade (ICB). T cells with high CRTAM expression are more likely to adhere to normal tissues by binding to CADM1, activating the three types of immunity centered around IL-17 and IL-23, causing inflammatory damage to normal tissues without affecting anti-tumor immunity.

[0004] Therefore, the development of monoclonal antibodies targeting CRTAM can provide an innovative drug testing and intervention basis for preclinical research on irAEs, help reveal the potential molecular mechanisms of irAEs, and have important clinical significance. Summary of the Invention

[0005] The present invention aims to provide an anti-CRTAM monoclonal antibody, its preparation method, and its application to address the problems of the prior art. This anti-CRTAM monoclonal antibody can target and bind to the CRTAM protein, significantly eliminate CRTAM-positive T cells in preclinical models, and significantly alleviate adverse reactions associated with immunotherapy. It can be used to detect, block, and / or eliminate CRTAM protein, and is of great significance in research scenarios such as adverse reactions associated with immunotherapy.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides an anti-CRTAM monoclonal antibody, which comprises a CDR-H1 with an amino acid sequence as shown in SEQ ID NO.3, a CDR-H2 with an amino acid sequence as shown in SEQ ID NO.4, a CDR-H3 with an amino acid sequence as shown in SEQ ID NO.5, a CDR-L1 with an amino acid sequence as shown in SEQ ID NO.6, a CDR-L2 with an amino acid sequence of RMS, and a CDR-L3 with an amino acid sequence as shown in SEQ ID NO.7.

[0008] Furthermore, the amino acid sequence of the heavy chain variable region of the anti-CRTAM monoclonal antibody is shown in SEQ ID NO.13, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.15.

[0009] Another aspect of the present invention provides a chimeric antigen receptor (CAR) fusion protein comprising the anti-CRTAM monoclonal antibody or antigen-binding fragment thereof of the present invention.

[0010] The present invention also provides a gene encoding the above-mentioned anti-CRTAM monoclonal antibody.

[0011] Furthermore, the anti-CRTAM monoclonal antibody encoding gene includes a heavy chain variable region encoding gene with a nucleotide sequence as shown in SEQ ID NO.14 and a light chain variable region encoding gene with a nucleotide sequence as shown in SEQ ID NO.16.

[0012] The present invention also provides a biomaterial, wherein the biomaterial is a substance shown in any one of (1) to (3):

[0013] (1) a gene expression cassette comprising a gene encoding the anti-CRTAM monoclonal antibody according to claim 3 or 4;

[0014] (2) a recombinant vector comprising the gene expression cassette;

[0015] (3) A recombinant host cell comprising the recombinant vector.

[0016] The host cell of the present invention is selected from prokaryotic cells and eukaryotic cells. In some embodiments, the host cell is a bacterium, preferably Escherichia coli. In another preferred embodiment, the host cell is a mammalian cell.

[0017] The present invention also provides a method for preparing the above-mentioned anti-CRTAM monoclonal antibody, comprising the steps of expressing the antibody using a recombinant host cell and isolating and obtaining the anti-CRTAM monoclonal antibody.

[0018] The present invention also provides the use of the above-mentioned encoding gene or biological material in preparing the above-mentioned anti-CRTAM monoclonal antibody.

[0019] The present invention also provides the use of the above-mentioned anti-CRTAM monoclonal antibody in the preparation of the product described in any one of the following (a)-(c):

[0020] (a) CRTAM protein detection kit;

[0021] (b) inhibitors of CRTAM protein;

[0022] (c) Drugs that suppress adverse reactions to immunotherapy.

[0023] The present invention also provides a CRTAM protein detection kit, comprising the above-mentioned anti-CRTAM monoclonal antibody.

[0024] The present invention also provides an inhibitor of CRTAM protein, the active ingredient of which includes the above-mentioned anti-CRTAM monoclonal antibody.

[0025] The present invention also provides a drug for suppressing adverse reactions of immunotherapy, wherein the active ingredient includes the above-mentioned anti-CRTAM monoclonal antibody. In some embodiments, the drug also contains other active ingredients, such as other antibodies, targeted drugs, etc. In some embodiments, the drug also includes a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient is selected from antioxidants, polypeptides, proteins, hydrophilic polymers, amino acids, sugars, chelating agents, sugar alcohols, ions and surfactants.

[0026] The present invention discloses the following technical effects:

[0027] The anti-CRTAM monoclonal antibody provided by the present invention can target and bind to CRTAM protein, can significantly eliminate CRTAM-positive T cells in preclinical models, has the effect of significantly alleviating immunotherapy-related adverse reactions, can be used for the detection, blocking and / or elimination of CRTAM protein, and is of great significance in research scenarios such as immunotherapy-related adverse reactions. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0029] Figure 1 Figure 1 is the ELISA test result of the purified anti-CRTAM monoclonal antibody 4F9;

[0030] Figure 2 Schematic diagram of the method for constructing a DSS-induced immunotherapy-related colitis mouse model;

[0031] Figure 3 The figures show the results of ultrasound measurement of colon wall thickness in mice with immunotherapy-related colitis after treatment with IgG or anti-CRTAM monoclonal antibody 4F9; A is the ultrasound detection image; B is the statistical graph of ultrasound intestinal wall thickness;

[0032] Figure 4 These are the results of flow cytometry detection of CRTAM-positive T cells in mice with immunotherapy-related colitis after treatment with IgG or anti-CRTAM monoclonal antibody 4F9; A is a flow cytometry detection graph; B is a statistical graph of A. DETAILED DESCRIPTION

[0033] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0034] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0035] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0036] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0037] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0038] Terminology Notes:

[0039] In an antibody molecule, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are arranged in a three-dimensional space relative to each other to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of the bound antigen, and the three hypervariable regions of each heavy chain and light chain are referred to as "complementarity determining regions" or "CDRs". The allocation of amino acids to each domain is according to Kabat << Sequences of proteins of interest to immunology >> (National Institutes of Health, Bethesda, Maryland (1987 and 1991)>) or Chothia and Lesk, J.Mol.Biol.196:901-917 (1987), Chothia et al., Nature 342:878-883 (1989) definition.

[0040] The "antibody" of the present invention refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. An antibody can be a complete antibody or any antigen-binding fragment or single chain thereof. The "antibody" of the present invention includes any protein or peptide that has at least a portion of an Ig molecule that has the biological activity of binding to an antigen. Examples of "antibodies" of the present invention include, but are not limited to, CDRs of a heavy or light chain or a ligand-binding portion thereof, a heavy or light chain variable region, a heavy or light chain constant region, a framework region, or any portion thereof.

[0041] The "antigen-binding fragment" of the present invention refers to Fab fragments, Fab' fragments, F(ab')2 fragments with antigen-binding activity, and Fv fragments and scFv fragments that bind to human CRTAM. The Fv fragment contains the variable regions of the heavy and light chains of an antibody, but lacks the constant region, and is the smallest antibody fragment with all antigen-binding sites. Generally, an Fv antibody also contains a polypeptide linker between the VH and VL domains, and is capable of forming the structure required for antigen binding. The two antibody variable regions can also be connected into a single polypeptide chain using various connectors, which is called a single-chain antibody or single-chain Fv (scFv). The anti-CRTAM monoclonal antibody of the present invention can be a single-chain variable region fragment (scFv), which is derived from a single-chain polypeptide of an antibody and retains the ability to bind to the antigen. Examples of scFv include antibody polypeptides formed by recombinant DNA technology, in which the Fv regions of immunoglobulin heavy chain (H chain) and light chain (L chain) fragments are linked via a spacer sequence. Various methods for preparing scFv are well known to those skilled in the art.

[0042] A monoclonal antibody (mAb) is an antibody derived from a single clonal cell line, which may be a eukaryotic, prokaryotic, or phage clone. Monoclonal antibodies or antigen-binding fragments can be recombinantly produced using techniques such as hybridoma technology, recombinant technology, phage display technology, synthetic techniques such as CDR grafting, or other existing technologies.

[0043] The "chimeric antibodies" described in this invention are antibodies created by fusing the variable region of a mouse antibody with the constant region of a human antibody. They can mitigate the immune response induced by mouse antibodies. To create chimeric antibodies, one must first establish a hybridoma that secretes mouse-specific monoclonal antibodies. The variable region genes are then cloned from the mouse hybridoma cells. Furthermore, the human constant region genes are cloned as needed. The mouse variable region genes and human constant region genes are then linked to form a chimeric gene, which is then inserted into a human vector. Finally, the chimeric antibody molecule is expressed in a eukaryotic or prokaryotic cell culture system.

[0044] Example 1 Preparation of anti-CRTAM monoclonal antibody cell line

[0045] 1. Selection of CRTAM recombinant immunogens

[0046] The mouse CRTAM protein amino acid sequence was queried in the Uniprot database, and the sequence encoded by the classic Q149L7-1 number was selected, as shown in SEQ ID NO. 1. According to the purpose of the present invention, the protein encoded by its extracellular segment amino acid sequence (Ala17-Gly289) was selected as the immunogen, and its amino acid sequence is shown in SEQ ID NO. 2. The corresponding recombinant protein for immunization (Cat. No. 94550ES25) was purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.

[0047] Full-length amino acid sequence of CRTAM protein (Q149L7-1):

[0048] MWWGALSLLFWVPVQAAFLKMETVTVEEGQTLTLTCVTSQTKNVSLQWLAPSGFTIFLNQHPALKSSKYQLLHHSATQLSISVSNVTLREEGVYTCLHYGSSVKTKQVRVTVLVTPFQPTVEALVLRRQNGEKSVVLKCSTERSKPPPQITWLLGEGLEIYGELNHEFEADGKICNTSSML IARAYGKNSTVHCIIQHEGLHGRKLVAPFQFEDLVADQETSDQETSDAPEQSSLSSQALQQPTSTVSMMENSSIPETDKEEKEHATQDPGLSTEASAQHTGLARRKSG ILLTLVSFLIFILFIIVQLFIMKLRKAHVVWKKESEISEQALESYRSRSNNEETSSQENSSQAPQSKRCMNYITRLYSGAKTKKSAQHWKLGGKHSRVPESIV(SEQ ID NO.1).

[0049] CRTAM protein extracellular segment amino acid sequence (Ala17-Gly289):

[0050] AFLKMETVTVEEGQTLTLTCVTSQTKNVSLQWLAPSGFTIFLNQHPALKSSKYQLLHHSATQLSISVSNVTLREEGVYTCLHYGSSVKTKQVRVTVLVTPFQPTVEALVLRRQNGEKSVVLKCSTERSKPPPQITWLL GEGLEIYGELNHEFEADGKICNTSSMLIARAYGKNSTVHCIIQHEGLHGRKLVAPFQFEDLVADQETSDQETSDAPEQSSLSSQALQQPTSTVSMMENSSIPETDKEEKEHATQDPGLSTEASAQHTGLARRKSG(SEQ ID NO.2).

[0051] 2. Immunization of BALB / c Mice

[0052] Healthy female BALB / c mice aged 6-8 weeks were selected, and the above-mentioned recombinant CRTAM protein (SEQ ID NO.2) was used as the immunogen. Multiple injections were performed subcutaneously on the back of the mice. The immunization schedule is shown in Table 1. The dosage of immunogen per mouse per injection was 100 μg. During the initial immunization, the immunogen was thoroughly mixed with an equal amount of Freund's complete adjuvant to form an emulsion, and the emulsion was injected subcutaneously at multiple points on the back. Every two weeks, an equal amount of immunogen and Freund's incomplete adjuvant were mixed into an emulsion and injected subcutaneously at multiple points on the back, for a total of three booster immunizations. On day 49, 10 μL of blood was collected from the caudal vein, and the antibody titer was determined using a conventional indirect enzyme-linked immunosorbent assay (ELISA). Mice with high serum titers were selected and sprint immunization was performed on day 56, with an intraperitoneal injection of the immunogen and an equal amount of normal saline.

[0053] Table 1 Mouse immunization scheme

[0054] One free (Day 1) Freund's complete adjuvant + CRTAM recombinant protein Second exemption (14th day) Freund's incomplete adjuvant + CRTAM recombinant protein Three exemptions (28th day) Freund's incomplete adjuvant + CRTAM recombinant protein Four free days (42nd day) Freund's incomplete adjuvant + CRTAM recombinant protein Five Free (Day 56) Normal saline + CRTAM recombinant protein

[0055] 3. Cell Fusion

[0056] On the fifth day after the five-immunostimulation shock, the spleen of the mouse was aseptically removed in an ultra-clean cell workbench, and the cell suspension was collected with RPMI-1640 medium after grinding on a 70 μm cell strainer. The cell suspension was centrifuged at 400 rcf for 5 min and washed three times with RPMI-1640 medium (centrifugation conditions were the same as described above). Finally, the cells were diluted to a certain volume and counted for later use.

[0057] Prepare 3×10 SP2 / 0 tumor cells in logarithmic growth phase 7The spleen cells were mixed with SP2 / 0 myeloma cells at a ratio of 5:1, centrifuged at 400 rcf for 5 minutes, and the supernatant discarded. Over 1 minute, 1 mL of PEG1450 at 37°C was slowly added to the cell pellet, followed by 2 minutes of stasis. The PEG1450 treatment was terminated by the addition of 30 mL of RPMI-1640 medium over 5 minutes. The fused cells were centrifuged at 350 rcf for 5 minutes, the supernatant discarded, and the cells were resuspended in RPMI-1640 selective culture medium supplemented with HAT (100 μM hypoxanthine, 0.4 μM aminopterin, 16 μM thymidine) and 20% fetal bovine serum. 200 μL was added to each well of a 96-well cell culture plate and incubated at 37°C in a 5% CO2 incubator.

[0058] 4. Subclone Screening

[0059] 14-16 days after fusion, the cell culture supernatant of the hybridoma mother clone was collected and the mother clones with strong binding ability were screened by ELISA. The positive mother clones were subcloned in the first round by limiting dilution. After culturing for 6-7 days, the above steps were repeated for the second round of subclone screening. After culturing for 5-6 days, the above steps were repeated for the third round of subclone screening.

[0060] 5. Cell Line Establishment

[0061] After the third round of subclone screening, positive cell lines were detected by ELISA, and a good monoclonal cell line was screened and labeled 4F9. The above cell lines were expanded and cultured until 90% of the culture dish was confluent. A small amount was transferred to a T25 flask, and the remaining cells were collected and frozen.

[0062] 6. Ascites Preparation

[0063] One week before cell injection, pre-stimulate mice with an intraperitoneal injection of mineral oil. Mix the hybridoma cells with sterile PBS to form a cell suspension. Use a sterile syringe to draw up the suspension and inject it intraperitoneally into the mouse. Observe the mice after hybridoma injection and monitor their general health and ascites production for at least every other day.

[0064] 7. Ascites Collection and Purification

[0065] Observe the mouse's abdominal cavity for 8-20 days. When the abdominal cavity becomes distended to the point of restricting movement, perform a paracentesis to collect ascites in a sterile 15mL conical-bottom centrifuge tube. Centrifuge the ascites at 1500rcf for 10 minutes to precipitate and remove cells. Transfer the ascites supernatant to a sterile 50mL centrifuge tube and keep frozen at -20°C during collection. If the mouse's health does not decline significantly, perform another paracentesis the next day. Euthanize the mouse 24-48 hours after the second aspiration and perform a third aspiration. All harvested ascites can be combined into the same centrifuge tube after centrifugation, centrifuged and filtered, diluted with PBS, and purified with Protein A / G to collect antibodies. The antibody concentration is adjusted to 0.5mg / mL.

[0066] Example 2 Analysis of Antibody Characteristics of Anti-CRTAM Monoclonal Antibody 4F9

[0067] The titer of the anti-CRTAM monoclonal antibody 4F9 was detected by ELISA as follows:

[0068] (1) Coating antigen

[0069] Mouse CRTAM recombinant protein (same as in Example 1) was diluted to 3 μg / mL with 0.05 mol / L carbonate solution (pH=9.6), 100 μL / well was plated, and the cells were incubated at 4°C overnight.

[0070] (2) Washing the plate

[0071] Each well was washed three times with 150 μL of PBST solution containing 0.05% Tween-20, for 3 minutes each time.

[0072] (3) Closed

[0073] Add 150 μL of PBST containing 5% skim milk powder to each well and incubate at 37°C for 2 hours for blocking.

[0074] (4) Washing the board

[0075] Each well was washed three times with 150 μL of PBST solution containing 0.05% Tween-20, for 3 minutes each time.

[0076] (5) Add primary antibody

[0077] The antibody collected from ascites was diluted 1:1000, then serially diluted to 1:512K, and incubated at 37°C for 1 hour.

[0078] (6) Washing the board

[0079] Each well was washed three times with 150 μL of PBST solution containing 0.05% Tween-20, for 3 minutes each time.

[0080] (7) Add secondary antibody

[0081] Horseradish enzyme-labeled goat anti-mouse IgG (H+L) at a dilution of 1:8000 was added to each well and incubated at 37°C for 45 minutes.

[0082] (8) Washing board

[0083] Each well was washed five times with 150 μL of PBST solution containing 0.05% Tween-20, 3 minutes each time.

[0084] (9) Color development

[0085] 100 μL of substrate solution (TMB) was added to each well, and the reaction was allowed to proceed for 5-10 minutes. Finally, 100 μL of 2 mol / L sulfuric acid was added to terminate the reaction.

[0086] (10) Measure OD value

[0087] The OD value was measured using a microplate reader at a wavelength of 450 nm.

[0088] like Figure 1 As shown in FIG, serum titer value ≥ 2.5 times of negative value was identified as positive. The experimental results showed that the anti-CRTAM monoclonal antibody 4F9 of the present invention has a good ability to bind to mouse CRTAM.

[0089] Example 3 Sequence determination of anti-CRTAM monoclonal antibody 4F9

[0090] Nanjing Detai Bioengineering Co., Ltd. was commissioned to sequence the antibody sequence secreted by the 4F9 hybridoma cell line. The specific method is as follows: hybridoma cells were lysed to extract total RNA, and the RNA was reverse transcribed into cDNA using rapid amplification of cDNA ends (RACE). The heavy and light chain variable region sequences were amplified by PCR. The target fragments were ligated to the vector using ligase, and the ligated products were transformed into competent E. coli cells. Single clones were then selected for sequencing analysis and annotation. The sequencing results are as follows:

[0091] The CDR sequences of the heavy chain variable region of the anti-CRTAM monoclonal antibody 4F9 are as follows:

[0092] CDR-H1: GFNIKDYF (SEQ ID NO.3);

[0093] CDR-H2: IDPENGDS (SEQ ID NO.4);

[0094] CDR-H3: DALMITMNYALDY (SEQ ID NO. 5).

[0095] The CDR sequence of the light chain variable region of the anti-CRTAM monoclonal antibody 4F9 is as follows:

[0096] CDR-L1: KSLLHSNGNTY (SEQ ID NO.6);

[0097] CDR-L2:RMS;

[0098] CDR-L3: MQHLEYPLT (SEQ ID NO. 7).

[0099] The nucleotide sequences encoding the heavy chain variable regions CDR-H1, CDR-H2, and CDR-H3 of the monoclonal antibody 4F9 are shown in SEQ ID NOs. 8-10:

[0100] SEQ ID NO.8: GGCTTCAACATTAAAGACTACTTT;

[0101] SEQ ID NO.9:ATTGATCCTGAAAATGGTGACTCT;

[0102] SEQ ID NO. 10: GATGCCCTTATGATTACGATGAACTATGCTTTGGACTAC.

[0103] The nucleotide sequences encoding the light chain variable regions CDR-L1 and CDR-L3 of the monoclonal antibody 4F9 are shown in SEQ ID NOs. 11-12: the nucleotide sequence encoding CDR-L2 is: CGGATGTCC;

[0104] SEQ ID NO.11: AAGAGTCTCCTGCATAGTAATGGCAACACTTAC;

[0105] SEQ ID NO. 12: ATGCAACATCTAGAATATCCGCTCACG.

[0106] The heavy chain subtype of the anti-CRTAM monoclonal antibody 4F9 is murine IgG2a, and the light chain subtype is murine-kappa.

[0107] The amino acid sequence of the heavy chain variable region of the anti-CRTAM monoclonal antibody 4F9 (SEQ ID NO.13):

[0108] EVQLLQSGAELVRSGASVKLSCTASGFNIKDYFIHWMKQRPEQGLEWIGWIDPENGDS KYAPNFKGKATMTADTSSNTAYLQLISLTSEDTAVYFCDALMITMNYALDYWGQGTSVTVS S。

[0109] The nucleotide sequence (SEQ ID NO.14) of the encoding gene for the heavy chain variable region:

[0110] GAGGTTCAGCTGCTGCAGTCTGGGGCTGAACTTGTGAGGTCAGGGGCCTCAGTCAAGTTGTCCTGCACAGCTTCTGGCTTCAACATTAAAGACTACTTTATCCACTGGATGAAGCAGAGGCCTGAACAGGGCCTGGAATGGATTGGATGGATTGATCCTGAAAATGGTGACTCTAAATATGCCCCGAATTTCAAGGGCAAGGCCACTATGACTGCAGACACATCATCCAACACAGCCTACCTGCAGCTCATCAGCCTGACATCTGAGGACACTGCCGTCTATTTCTGTGATGCCCTTATGATTACGATGAACTATGCTTTGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGAGGTTCAGCTGCTGCAGTCTGGGGCTGAACTTGTGAGGTCAGGGGCCTCAGTCAAGTTGTCCTGCACAGCTTCTGGCTTCAACATTAAAGACTACTTTATCCACTGGATGAAGCAGAGGCCTGAACAGGGCCTGGAATGGATTGGATGGATTGATCCTGAAAATGGTGACTCTAAATATGCCCCGAATTTCAAGGGCAAGGCCACTATGACTGCAGACACATCATCCAACACAGCCTACCTGCAGCTCATCAGCCTGACATCTGAGGACACTGCCGTCTATTTCTGTGATGCCCTTATGATTACGATGAACTATGCTTTGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA。

[0111] The amino acid sequence (SEQ ID NO.15) of the light chain variable region of the anti-CRTAM monoclonal antibody 4F9:

[0112] DIVMTQAAPSVPVTPGESVSISCRSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRMSNL ASGVPDRFSGSGSGTAFTLRISRVETEDVGIYYCMQHLEYPLTFGAGTKLELK.

[0113] The nucleotide sequence of the gene encoding the light chain variable region (SEQ ID NO.16):

[0114] .

[0115] Example 4 Application of anti-CRTAM monoclonal antibodies in preclinical models of adverse reactions to immunotherapy

[0116] 1. Experimental Materials

[0117] 1.1 Experimental Animals

[0118] C57BL / 6J mice, female, 6-8 weeks old, weighing 16-20 g.

[0119] 1.2 Reference substances and test substances

[0120] The control substance was IgG, which was used as a negative control. Before the experiment, the anti-CRTAM monoclonal antibody 4F9 of the present invention was prepared with sterile PBS to a concentration of 0.5 mg / mL and set aside.

[0121] 2 Experimental methods

[0122] Mice were intraperitoneally administered with 100 μg of anti-PD1 and 100 μg of anti-CTLA4 monoclonal antibodies on days -3, 0, and 3, and 100 μg of IgG or anti-CRTAM monoclonal antibody 4F9, respectively. They were then given a 3% DSS solution made of dextran sodium sulfate DSS from day 0 to day 5 to establish a preclinical model of DSS-induced immunotherapy-related colitis in mice ( Figure 2 The thickness of the mouse colon was measured by small animal ultrasound to assess the severity of colitis in the mouse ( Figure 3 ), the ability of anti-CRTAM monoclonal antibody 4F9 to eliminate mouse CRTAM-positive T cells was detected by flow cytometry ( Figure 4 The results showed that the anti-CRTAM monoclonal antibody 4F9 of the present invention can effectively inhibit the occurrence of immunotherapy-related colitis in mice.

[0123] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. An anti-CRTAM monoclonal antibody, characterized in that It comprises: CDR-H1 with an amino acid sequence as shown in SEQ ID NO.3, CDR-H2 with an amino acid sequence as shown in SEQ ID NO.4, CDR-H3 with an amino acid sequence as shown in SEQ ID NO.5, CDR-L1 with an amino acid sequence as shown in SEQ ID NO.6, CDR-L2 with an amino acid sequence of RMS, and CDR-L3 with an amino acid sequence as shown in SEQ ID NO.

7.

2. The anti-CRTAM monoclonal antibody according to claim 1, characterized in that The amino acid sequence of the heavy chain variable region of the anti-CRTAM monoclonal antibody is shown in SEQ ID NO.13, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

15.

3. A gene encoding an anti-CRTAM monoclonal antibody according to claim 2, characterized in that: The encoding genes include a heavy chain variable region encoding gene whose nucleotide sequence is shown as SEQ ID NO.14 and a light chain variable region encoding gene whose nucleotide sequence is shown as SEQ ID NO.

16.

4. A biomaterial, characterized in that The biological material is any one of (1) to (3): (1) A gene expression cassette comprising a gene encoding the anti-CRTAM monoclonal antibody according to claim 3; (2) a recombinant vector comprising the gene expression cassette; (3) A recombinant host cell containing the recombinant vector.

5. Use of the coding gene according to claim 3 or the biomaterial according to claim 4 in preparing the anti-CRTAM monoclonal antibody according to claim 2.

6. Use of the anti-CRTAM monoclonal antibody according to claim 1 or 2 in the preparation of the product according to any one of the following (a)-(b): (a) CRTAM protein detection kit; (b) A drug that suppresses an adverse reaction to immunotherapy; the adverse reaction to immunotherapy is colitis.

7. A detection kit for CRTAM protein, characterized in that: The invention comprises the anti-CRTAM monoclonal antibody according to claim 1 or 2.

8. An inhibitor of CRTAM protein, characterized in that The active ingredient comprises the anti-CRTAM monoclonal antibody according to claim 1 or 2.

9. A drug for suppressing adverse reactions of immunotherapy, characterized in that: The active ingredient comprises the anti-CRTAM monoclonal antibody according to claim 1 or 2.

10. A method for preparing an anti-CRTAM monoclonal antibody according to claim 1 or 2, characterized in that: The method comprises the steps of utilizing recombinant host cells to express the antibody and isolating and obtaining the anti-CRTAM monoclonal antibody.

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