Anti-CRTAM monoclonal antibody as well as preparation method and application thereof

Abstract

The invention discloses an anti-CRTAM monoclonal antibody as well as a preparation method and application thereof, and relates to the technical field of biomedicine. The anti-CRTAM monoclonal antibody comprises a CDR-H1, a CDR-H2 and a CDR-H3 of which the amino acid sequences are shown as SEQ ID NO.3-5, a CDR-L1 of which the amino acid sequence is shown as SEQ ID NO.6, a CDR-L2 of which the amino acid sequence is RMS, and a CDR-L3 of which the amino acid sequence is shown as SEQ ID NO.7. The anti-CRTAM monoclonal antibody disclosed by the invention has the advantages that the anti-CRTAM monoclonal antibody can be used for preparing the anti-CRTAM monoclonal antibody; the anti-CRTAM monoclonal antibody provided by the invention can be combined with mouse CRTAM protein in a targeted manner, can remarkably remove mouse CRTAM positive T cells in a preclinical model, has the effect of remarkably relieving immunotherapy-related adverse reactions, can be used for detecting, blocking and / or removing the mouse CRTAM protein, and has important significance in research scenes such as the 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] Existing studies have shown that CRTAM is a key molecule in the occurrence of immune-related adverse events (irAEs) associated with immune checkpoint blockade (ICB). T cells with high expression of CRTAM tend to adhere to normal tissues by binding to CADM1, while activating the three types of immunity centered on 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 basis for drug testing and intervention in preclinical studies of irAEs, help reveal the potential molecular mechanisms of irAEs, and have important clinical significance. Summary of the invention

[0005] The purpose of the present invention is to provide an anti-CRTAM monoclonal antibody and a preparation method and application thereof to solve the problems existing in the above-mentioned prior art. The anti-CRTAM monoclonal antibody 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.

[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 coding gene of the anti-CRTAM monoclonal antibody includes a heavy chain variable region coding gene with a nucleotide sequence as shown in SEQ ID NO.14 and a light chain variable region coding 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, wherein the gene expression cassette comprises 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 encoding gene or biological material in preparing the above anti-CRTAM monoclonal antibody.

[0019] The present invention also provides the use of the above 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 detection kit for CRTAM protein, comprising the above-mentioned anti-CRTAM monoclonal antibody.

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

[0025] The present invention also provides a drug for inhibiting adverse reactions of immunotherapy, wherein the active ingredient includes the above-mentioned anti-CRTAM monoclonal antibody. In some embodiments, the drug also includes other active components, 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 drawings required for use in the embodiments will be briefly introduced below. 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 creative work.

[0029] Figure 1 This is a diagram showing the ELISA test results 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 results of ultrasonic measurement of colon wall thickness in mice with immunotherapy-related colitis after treatment with IgG or anti-CRTAM monoclonal antibody 4F9; A is an ultrasonic detection image; B is a statistical graph of ultrasonic 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; wherein A is a flow cytometry detection graph; and 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 should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

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

[0035] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation 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 a conflict with any incorporated document, the content 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 of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are 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 Note:

[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 relative position to each other in three-dimensional space 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 in 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. The antibody can be a complete antibody and any antigen-binding fragment or single chain thereof. The "antibody" of the present invention includes any protein or peptide that contains at least a portion of an Ig molecule having a biological activity of binding to an antigen. Examples of the "antibody" of the present invention include, but are not limited to, a CDR 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" described in the present invention refers to a Fab fragment, a Fab' fragment, a F(ab')2 fragment, and an Fv fragment and a scFv fragment that bind to human CRTAM that have antigen binding activity. The Fv fragment contains the variable region of the heavy chain and the variable region of the light chain of the antibody, but has no constant region, and is the smallest antibody fragment with all antigen binding sites. Generally, the Fv antibody also contains a polypeptide linker between the VH and VL domains, and is capable of forming the structure required for antigen binding. Two antibody variable regions can also be connected into a polypeptide chain using different 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 an 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 connected via spacer sequences. Various methods for preparing scFv are well known to those skilled in the art.

[0042] Monoclonal antibodies (mAbs) are antibodies obtained from a single cloned cell line, which is not limited to eukaryotic, prokaryotic or phage cloned cell lines. Monoclonal antibodies or antigen-binding fragments can be obtained by recombination using hybridoma technology, recombinant technology, phage display technology, and synthetic technology such as CDR grafting or other existing technologies.

[0043] The "chimeric antibody" described in the present invention is an antibody formed by fusing the variable region of a mouse antibody with the constant region of a human antibody, which can reduce the immune response induced by the mouse antibody. To establish a chimeric antibody, a hybridoma that secretes mouse-specific monoclonal antibodies must first be established, and then the variable region gene must be cloned from the mouse hybridoma cells, and then the constant region gene of the human antibody must be cloned as needed, and the mouse variable region gene and the human constant region gene must be connected into a chimeric gene and then inserted into a human vector, and finally the chimeric antibody molecule must be expressed in a eukaryotic or prokaryotic industrial system.

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

[0045] 1. CRTAM recombinant immunogen selection

[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 was shown in SEQ ID NO. 2. The corresponding recombinant protein for immunization (Cat. No.: 94550ES25) was purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.

[0047] The 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 an immunogen. Multiple injections were performed subcutaneously on the back of the mice. The immunization scheme is shown in Table 1. The amount of immunogen used for each injection of each mouse was 100 μg. During the initial immunization, the immunogen was fully mixed with an equal amount of Freund's complete adjuvant to form an emulsifier, and multiple injections were performed subcutaneously on the back; every 2 weeks, an equal amount of immunogen and Freund's incomplete adjuvant were mixed with an emulsion and injected subcutaneously on the back at multiple points, and a total of 3 booster immunizations were performed. On the 49th day, 10 μL of blood was collected from the tail vein, and the antibody titer was determined by conventional indirect enzyme-linked immunosorbent assay (ELISA). Mice with high serum titers were selected, and sprint immunization was performed on the 56th day, and the immunogen and an equal amount of saline were injected intraperitoneally.

[0053] Table 1 Mouse immunization scheme

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

[0055] 3. Cell Fusion

[0056] On the 5th day after the five-immune shock, the spleen of the mouse was removed aseptically in an ultra-clean cell workbench, and the cell suspension was collected with RPMI-1640 medium after grinding on a 70 μm cell filter. The cells were centrifuged at 400 rcf for 5 min, and the cells were washed 3 times with RPMI-1640 medium (the 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 min, and the supernatant was discarded. Within 1 min, 1 mL of PEG1450 at 37°C was slowly added to the cell pellet, then allowed to stand for 2 min, and then 30 mL of RMPI-1640 medium was added dropwise within 5 min to terminate the effect of PEG1450. The fused cells were centrifuged at 350 rcf for 5 min, the supernatant was discarded, and the cells were resuspended in RPMI-1640 screening culture system containing HAT (100 μM hypoxanthine, 0.4 μM aminopterin, 16 μM thymidine) and 20% newborn fetal bovine serum, and 200 μL was added to each well of a 96-well cell culture plate, and placed at 37°C, 5% CO 2 Culture in an incubator.

[0058] 4. Subclone Screening

[0059] 14-16 days after fusion, the cell culture supernatant of the hybridoma mother clone was taken, and the mother clones with stronger binding ability were screened by ELISA. The positive mother clones were subjected to the first round of subcloning by the limiting dilution method. 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 as 4F9. The above cell lines were expanded and cultured. After 90% of the culture dishes were filled, a small amount was taken and inoculated into a T25 bottle, and the remaining cells were collected and frozen.

[0062] 6. Ascites Preparation

[0063] One week before cell injection, mice were pre-stimulated by intraperitoneal injection of mineral oil. Hybridoma cells were mixed with sterile PBS to form a cell suspension, and the mixed cell suspension was drawn up with a sterile syringe and injected into the mouse peritoneum. The mice after hybridoma cell injection were observed, and their general health and ascites production were observed at least every other day thereafter.

[0064] 7. Ascites Collection and Purification

[0065] Observe the abdominal cavity of the mouse within 8-20 days. When the abdominal cavity is swollen to the point of limiting movement, perform a puncture and aspiration to collect ascites in a sterile 15mL conical centrifuge tube. Centrifuge the ascites at 1500rcf for 10min to precipitate and remove the cells in the ascites. Transfer the ascites supernatant to a sterile 50mL centrifuge tube and keep it frozen at -20℃ during the collection period. If the health of the mouse does not decline significantly, perform another puncture and aspiration every other day. Euthanize the mouse 24-48 hours after the second aspiration and perform a third puncture and aspiration. All harvested ascites can be combined into the same centrifuge tube after centrifugation, centrifuged and filtered, diluted with PBS, and purified by ProteinA / 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 anti-CRTAM monoclonal antibody 4F9 was detected by ELISA method as follows:

[0068] (1) Coating antigen

[0069] The 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 incubated at 4°C overnight.

[0070] (2) Washing plate

[0071] Each well was washed three times with 150 μL of PBST solution containing 0.05% Tween-20, 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 plate

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

[0076] (5) Add primary antibody

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

[0078] (6) Washing plate

[0079] Each well was washed three times with 150 μL of PBST solution containing 0.05% Tween-20, 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 rendering

[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) OD value measurement

[0087] The OD value was measured with an enzyme-labeled instrument at a wavelength of 450 nm.

[0088] like Figure 1 As shown, serum titer values ​​≥ 2.5 times the negative value were 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: the hybridoma cells were lysed to extract total RNA, and the RNA was reverse transcribed into cDNA using rapid amplification of cDNA ends (RACE), and the heavy chain and light chain variable region sequences were obtained by PCR amplification. The target fragments were connected to the vector using ligase, and the ligation products were transformed into Escherichia coli competent cells, and then single clones were selected for sequencing analysis and annotation. The sequencing results are as follows:

[0091] The CDR sequence of the heavy chain variable region of the anti-CRTAM monoclonal antibody 4F9 is 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 above 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 above 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 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 of the encoding gene of the heavy chain variable region (SEQ ID NO.14):

[0110] GAGGTTCAGCTGCTGCAGTCTGGGGCTGAACTTGTGAGGTCAGGGGCCTCAGTCAAGTTGTCCTGCACAGCTTCTGGCTTCAACATTAAAGACTACTTTATCCACTGGATGAAGCAGAGGCCTGAACAGGGCCTGGAATGGATTGGATGGATTGATCCTGAAAATGGTGACTCTAAATATGCCCCGAATTTCAAGGGCAAGGCCACTATGACTGCAGACACATCATCCAACACAGCCTACCTGCAGCTCATCAGCCTGACATCTGAGGACACTGCCGTCTATTTCTGTGATGCCCTTATGATTACGATGAACTATGCTTTGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGAGGTTCAGCTGCTGCAGTCTGGGGCTGAACTTGTGAGGTCAGGGGCCTCAGTCAAGTTGTCCTGCACAGCTTCTGGCTTCAACATTAAAGACTACTTTATCCACTGGATGAAGCAGAGGCCTGAACAGGGCCTGGAATGGATTGGATGGATTGATCCTGAAAATGGTGACTCTAAATATGCCCCGAATTTCAAGGGCAAGGCCACTATGACTGCAGACACATCATCCAACACAGCCTACCTGCAGCTCATCAGCCTGACATCTGAGGACACTGCCGTCTATTTCTGTGATGCCCTTATGATTACGATGAACTATGCTTTGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA。

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

[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 for later use.

[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, and then drank 3% DSS solution prepared with dextran sodium sulfate DSS from day 0 to day 5 to construct 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 mice ( 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 only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined 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 the anti-CRTAM monoclonal antibody as claimed in claim 2; 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 a substance shown in any one of (1) to (3): (1) a gene expression cassette, wherein the gene expression cassette comprises 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 comprising the recombinant vector.

5. Use of the coding gene according to claim 3 or the biological material 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 described in any one of the following (a)-(c): (a) CRTAM protein detection kit; (b) inhibitors of CRTAM protein; (c) Drugs that suppress adverse reactions to immunotherapy.

7. A detection kit for CRTAM protein, characterized in that: Comprising 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 inhibiting 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 antibodies and separating and obtaining the anti-CRTAM monoclonal antibody.

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