A humanized antibody against HER2 antigen and its application

Abstract

The present invention belongs to the technical field of monoclonal antibody, and specifically relates to a humanized antibody against HER2 antigen and its application. A humanized antibody against HER2 antigen, wherein the amino acid sequences of complementarity determining region 1, complementarity determining region 2 and complementarity determining region 3 of the heavy chain are GYTFTSY, NTNTGN, CARRWLGYFDYW respectively; and the amino acid sequences of complementarity determining region 1, complementarity determining region 2 and complementarity determining region 3 of the light chain are QSISSYLN, AASSLQS, CQQSYSTPLTF respectively. The antibody sequence of this solution is different from other HER2 antibodies in the prior art, and the effect is consistent with that of trastuzumab. This technical solution can solve the technical problem of fewer types of antibodies against HER2 antigen in the prior art. The new antibody can overcome the problem of tumor drug resistance of a single drug, and is used for antibody combination therapy for cancer, having an ideal application and promotion prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of monoclonal antibody, and particularly relates to a humanized antibody against HER2 antigen and its application. Background Art

[0002] HER2 (human epidermal growth factor receptor 2, also known as Neu, ErbB-2, CD340 or p185) is a tyrosine kinase receptor membrane glycoprotein encoded by the ERBB2 proto-oncogene, and HER2 is a member of the epidermal growth factor receptor (EGFR / ErbB) family. It is known that amplification of the HER2 proto-oncogene or overexpression of the protein are found in various human tumors, including breast cancer, gastric cancer, ovarian cancer, lung cancer, prostate cancer, etc., which is clinically called HER2-positive tumors. Overexpression of the HER2 protein leads to an increase in the formation of HER2 homodimers and heterodimers, thereby causing effects such as cell proliferation, anti-apoptosis, invasion and angiogenesis by activating signaling pathways such as MAPK and P13K. HER2-positive tumors are characterized by high malignancy, strong invasion and metastasis ability, insensitivity to conventional chemotherapeutic drugs, and poor treatment prognosis.

[0003] Studies have shown that monoclonal antibody drugs targeting HER2 can play an anti-tumor role through direct and indirect mechanisms. The direct mechanism is mainly that the monoclonal antibody binds to the overexpressed HER2 on the surface of tumor cells, inhibits the formation of HER2 homodimers and heterodimers, and then blocks the activation of MAPK and P13K signaling pathways, ultimately playing an inhibitory role in tumor cell proliferation and invasion. The indirect mechanism is mainly that after the antibody binds to the overexpressed HER2 on the surface of tumor cells, the Fc end of the antibody tail can be recognized by the body's immune system, and the tumor cells are finally cleared through antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity.

[0004] Trastuzumab is the world's first humanized monoclonal antibody against HER2, which was approved by the FDA for marketing in 1998. Trastuzumab has greatly improved the prognosis of patients with HER2-positive breast cancer. However, approximately 50-60% of patients with HER2-positive breast cancer will develop resistance to the antibody, rendering it ineffective. Although there are some marketed monoclonal antibody drugs targeting HER2, after a period of treatment, drug resistance may occur, affecting the therapeutic effect of the drug. The applicant's prior patent CN118580359B (Anti-HER2 Monoclonal Antibody and Its Gene, Preparation Method and Application) immunized mice with a murine cell line overexpressing the HER2 protein antigen, obtained an antibody hybridoma cell line that produces an antibody with strong binding affinity to the HER2 protein through hybridoma fusion and positive clone screening, then performed sequence analysis to further obtain sequence information, and carried out humanization of the antibody. Finally, a monoclonal antibody against the HER2 protein antigen was obtained. This monoclonal antibody can effectively bind to the HER2 antigen and can achieve the killing of cancer cells expressing the HER2 antigen on the surface.

[0005] However, there are still a large number of other antigen sites on the cell surface expressing HER2. If monoclonal antibodies against different sites of a murine cell line overexpressing the HER2 protein antigen can be developed, the treatment efficiency can be effectively improved, and there will be other candidate antibodies after drug resistance occurs. Antibody cocktail therapy is a method of using a combination of multiple antibodies to treat diseases. This method attacks different parts of tumor-associated antigens by simultaneously using several different antibodies, thereby improving the therapeutic effect and reducing the possibility of cancer cell escape mutations. Although antibody cocktail therapy has shown potential in the treatment of certain viral infections and cancers, the antibody therapy for diseases related to HER2 overexpression is still in the research and development stage. If multiple antibodies against the HER2 antigen protein can be developed, these newly developed antibodies can be used as candidate antibodies for cocktail therapy, increasing the number of candidate antibodies for cocktail therapy. If a suitable variety of related antibodies for cocktail therapy can be developed, the treatment efficiency can be greatly improved, thereby achieving multi-target attack on cancer cells, reducing the risk of drug resistance, and enhancing the immune response, etc. Summary of the Invention

[0006] The present invention aims to provide a humanized antibody against the HER2 antigen to solve the technical problem of the small number of antibody types against the HER2 antigen in the prior art. Due to the small number of antibody types against the HER2 antigen, it is difficult to find candidate drugs to overcome after the occurrence of anti-cancer drug resistance, or there is a lack of effective candidate antibodies when using antibody combination therapy.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] A humanized antibody against the HER2 antigen, wherein the amino acid sequences of complementarity-determining region 1, complementarity-determining region 2, and complementarity-determining region 3 of the heavy chain are GYTFTSY, NTNTGN, and CARRWLGYFDYW respectively; and the amino acid sequences of complementarity-determining region 1, complementarity-determining region 2, and complementarity-determining region 3 of the light chain are QSISSYLN, AASSLQS, and CQQSYSTPLTF respectively.

[0009] Furthermore, the heavy chain framework region 1, heavy chain framework region 2, heavy chain framework region 3, and heavy chain framework region 4 are respectively:

[0010] QVQLVQSGSELKKPGASVKVSCKAS, AMNWVRQAPGQGLEWMGWI, PTYAQGFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYY, GQGTLVTVSS.

[0011] Furthermore, the light chain framework region 1, light chain framework region 2, light chain framework region 3, and light chain framework region 4 are respectively:

[0012] DIQMTQSPSSLSASVGDRVTITCRAS, WYQQKPGKAPKLLIY, GVPSRFSGSGSGTDFTLTISSLQPEDFATYY, GQGTKVEIK.

[0013] Furthermore, the amino acid sequence of its heavy chain is as shown in SEQ ID NO.5, and the amino acid sequence of its light chain is as shown in SEQ ID NO.7.

[0014] Furthermore, the nucleotide sequence of its heavy chain is as shown in SEQ ID NO.6; the nucleotide sequence of its light chain is as shown in SEQ ID NO.8.

[0015] This technical solution also provides an application of a humanized antibody against the HER2 antigen in the preparation of a drug for treating cancers in which the human epidermal growth factor receptor 2 protein is expressed on the cell surface, and the cancer is breast cancer. The monoclonal antibody obtained by this solution can be used to inhibit the growth of cancer cells expressing HER2. For example, the experiments in Example 4 verified the above effects.

[0016] This technical solution also provides an application of a humanized antibody against the HER2 antigen in the preparation of a reagent for detecting the human epidermal growth factor receptor 2 protein. The monoclonal antibody obtained by this solution can specifically bind to HER2. Therefore, it can be used for quantitative or qualitative detection of the HER2 protein. For example, detecting cancer cells expressing HER2. For example, the experiments in Example 4 verified the above effects.

[0017] The present technical solution also provides an application of a humanized antibody against HER2 antigen in the preparation of chimeric antigen receptor T cells, and the nucleotide sequence of the chimeric antigen receptor of the chimeric antigen receptor T cells is as shown in SEQ ID NO.9. On the basis of the sequence of the chimeric antigen receptor CAR in the prior art, the heavy chain variable region and the light chain variable region of the humanized antibody obtained in this solution are added, so that the constructed CAR-T cells have the ability to target HER2.

[0018] Furthermore, the chimeric antigen receptor T cells are used for treating breast cancer and / or cervical cancer. The experimental data in Examples 5 and 6 prove that the chimeric antigen receptor T cells containing the heavy chain variable region and the light chain variable region of the humanized antibody can effectively kill breast cancer and / or cervical cancer cells.

[0019] The present technical solution also provides a chimeric antigen receptor protein, and its nucleotide sequence is as shown in SEQ ID NO.9. The chimeric antigen receptor protein of this solution is a novel fusion protein. By conventional means in the prior art, expressing this new fusion protein in T cells can achieve CAR-T therapy targeting cells expressing HER2 protein on the surface.

[0020] In summary, the principle and beneficial effects of the present technical solution are as follows:

[0021] The present technical solution first constructs a murine cell line that stably overexpresses HER2 protein (antigen). Then, this cell line is used to immunize mice. Through hybridoma fusion and positive clone screening, a hybridoma cell line with strong binding ability to HER2 is obtained, and this cell line can produce an antibody with strong binding ability to HER2 protein. Sequence analysis is performed on the anti-HER2 hybridoma cell clones to further obtain sequence information, and humanization of the antibody is carried out. On this basis, artificial synthesis or expression of the antibody can be carried out to form a commercially available antibody against HER2 protein. In addition, the heavy chain and light chain sequences obtained in the present technical solution can also be used to prepare the chimeric antigen receptor of CAR-T cells to achieve the killing of cancer cells expressing HER2 antigen on the surface.

[0022] The novel humanized antibody obtained by this technical solution has a similar effect to existing trastuzumab, etc. It further solves the technical problem that it is difficult to overcome the tumor drug resistance problem caused by the lack of antibodies that recognize different HER2 epitopes in the prior art. The antibody (especially the complementarity-determining region) sequence obtained by this technical solution is different from any anti-HER2 antibody in the prior art and is also different from the antibody obtained by the applicant's prior patent CN118580359B. It can be used as an antibody for treating the same type of cancer and simultaneously overcome the tumor drug resistance problem brought by a single drug. And due to the increase in the number of candidate monoclonal antibodies against HER2, it makes it possible to further study the antibody cocktail therapy for related cancers, thereby effectively improving the treatment efficiency, and then achieving multi-target attack on cancer cells, reducing the risk of drug resistance, enhancing the immune response, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is the flow cytometry screening result of Example 1.

[0024] Figure 2 It is the immunofluorescence detection of the optimal hybridoma cell line in Example 1 (A: Fluorescence image of HER2 labeled with mCherry; B: Fluorescence image of the hybridoma cell line labeled with iFluor 488; C: Merged image of A and B).

[0025] Figure 3 It is the structural comparison diagram of the anti-HER2 humanized antibody and the murine antibody in Example 3.

[0026] Figure 4 It is the sequence comparison situation between the heavy chain of the humanized antibody of this solution and the heavy chain of the antibody in the prior patent in Example 3.

[0027] Figure 5 It is the sequence comparison situation between the light chain of the humanized antibody of this solution and the light chain of the antibody in the prior patent in Example 3.

[0028] Figure 6 It is the experimental result diagram of the humanized antibody detecting the HER2 protein on the surface of tumor cells in Example 4.

[0029] Figure 7 It is the experimental result diagram of the effect of the humanized antibody killing breast cancer cells in Example 4.

[0030] Figure 8 It is the experimental result of detecting the in vitro tumor killing ability of CAR-T cells in Example 6. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0031] The present invention will be further described in detail below in conjunction with embodiments, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art; the experimental methods used are all conventional methods and can be carried out according to the described recombinant techniques (see Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York). Moreover, the materials, reagents, etc. used can all be obtained through commercial channels.

[0032] Example 1:

[0033] Referring to the applicant's prior patent CN118580359B (Anti-HER2 monoclonal antibody and its gene, as well as preparation method and application), a murine cell line stably expressing HER2 protein was prepared, and then the prepared cell line was used as an antigen for immunizing mice. Then, through conventional methods in the prior art, several hybridoma cell lines to be screened were obtained.

[0034] By flow cytometry, hybridoma cells that could selectively bind to HER2 were selected from several hybridoma cell lines. The hybridoma cell lines were screened for subclones by the limiting dilution method. Flow cytometry was used to further detect their binding ability to HER2 protein. Finally, an optimal hybridoma cell line was obtained (see Figure 1 , the red frame part shows the hybridoma cell line with the best binding ability). Figure 1 The experimental data of

[0035] showed that the hybridoma cell line indicated by the red frame had the strongest HER2 binding ability. The binding of the antibodies secreted by other hybridoma cell lines to be screened to HER2 protein was not ideal. Through immunofluorescence experiments, we verified that the antibody of this hybridoma cell line could specifically bind to HER2. The specific steps were as follows: 24 hours in advance, the cell line 3T3-HER2 overexpressing HER2 and green fluorescent protein mCherry was plated in a 6-well plate. After the cells adhered and grew, an appropriate amount of the above-mentioned hybridoma supernatant with the best binding ability was added, and incubated at room temperature for 30 minutes. After washing off the excess supernatant, 1:500 diluted Figure 2 Goat anti-human IgG (H+L) antibody labeled with Alexa Fluor 488 was added, and images were collected under a microscope after incubation at room temperature for 30 minutes.

[0036] Example 2: Sequence analysis of anti-HER2 hybridoma cell clones

[0037] The ELISA method was used to determine that the Isotype of the antibody secreted by the hybridoma cell line screened in Example 1 was murine IgG2b. Suzhou Genewiz Biotechnology Co., Ltd. was commissioned to sequence the antibody variable region gene of this hybridoma cell.

[0038] Murine antibodies against the HER2 antigen include heavy and light chains. The amino acid sequence of the variable region of the murine antibody heavy chain (SEQ ID NO.1, double underlines indicate the framework regions in the variable region, and wavy lines indicate the complementarity-determining regions in the variable region):

[0039]

[0040] Among them, the variable region of the murine antibody heavy chain consists of complementarity-determining regions (CDRs) and framework regions (FRs), which are, in sequence:

[0041] Heavy chain framework region 1 (VH_FR1): QIQLVQSGPELKKPGETVKISCKAS;

[0042] Heavy chain complementarity-determining region 1 (VH_CDR1): GYTFTTY;

[0043] Heavy chain framework region 2 (VH_FR2): GMSWVKQAPGKGLKWMGWI;

[0044] Heavy chain complementarity-determining region 2 (VH_CDR2): NTYSGV;

[0045] Heavy chain framework region 3 (VH_FR3): PTYADDFKGRFAFSLETSASTAYLQNNNLKNEDTATYF;

[0046] Heavy chain complementarity-determining region 3 (VH_CDR3): CARRWLGYFDYW;

[0047] Heavy chain framework region 4 (VH_FR4): GQGTTLTVSS;

[0048] The connection order of the complementarity-determining regions and framework regions is: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0049] The nucleotide sequence of the variable region of the murine antibody heavy chain (SEQ ID NO.2, double underlines indicate the framework regions in the variable region, and wavy lines indicate the complementarity-determining regions in the variable region):

[0050]

[0051] The amino acid sequence of the variable region of the murine antibody light chain (SEQ ID NO.3, double underlines indicate the framework regions in the variable region, and wavy lines indicate the complementarity-determining regions in the variable region):

[0052]

[0053] Among them, the variable region of the murine antibody light chain consists of complementarity-determining regions (CDRs) and framework regions (FRs), which are, in sequence:

[0054] Light chain framework region 1 (VL_FR1): DIVMTQSHKFMSTSVGDRVSITCKAS;

[0055] Light chain complementarity-determining region 1 (VL_CDR1): QDVSTAVA;

[0056] Light chain framework region 2 (VL_FR2): WYQQKPGQSPKPLIY;

[0057] Light chain complementarity-determining region 2 (VL_CDR2): SASYRFT;

[0058] Light chain framework region 3 (VL_FR3): GVPDRFTGSGSGTDFTFTISSVQAEDLAVYY;

[0059] Light chain complementarity-determining region 3 (VL_CDR3): CQQHYSTTWTF;

[0060] Light chain framework region 4 (VL_FR4): GGGTKLEIK;

[0061] The connection order of the complementarity-determining regions and framework regions is: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0062] Nucleotide sequence of the variable region of the murine antibody light chain (SEQ ID NO.4, with the framework regions in the variable region indicated by double underlines and the complementarity-determining regions in the variable region indicated by wavy lines):

[0063]

[0064] Sequence comparison and analysis showed that the murine antibody is significantly different from known HER2 antibodies (and the HER2 antibody in the applicant's prior patent CN118580359B), indicating that it is a novel HER2 antibody.

[0065] Example 3: Humanization of the anti-HER2 murine monoclonal antibody

[0066] By analyzing large-scale bioinformatics data, we utilized artificial intelligence algorithms to humanize the murine antibody obtained in Example 2, reducing the immunogenicity of the antibody and thus minimizing the risk of immune responses in vivo. Additionally, intelligent design and optimization of the antibody framework and sequence were carried out, including enhancing antibody stability, improving the structure, reducing unnecessary modification sites, and optimizing the expression system to ensure optimal biological activity in the human body.

[0067] Humanized heavy chain amino acid sequence of the antibody (SEQ ID NO.5, the double underlines indicate the framework regions in the variable region, and the wavy lines indicate the complementarity-determining regions in the variable region):

[0068]

[0069] Among them, the variable region of the humanized heavy chain of the antibody consists of complementarity-determining regions (CDR) and framework regions (FR), which are in sequence:

[0070] Heavy chain framework region 1 (VH_FR1): QVQLVQSGSELKKPGASVKVSCKAS;

[0071] Heavy chain complementarity-determining region 1 (VH_CDR1): GYTFTSY;

[0072] Heavy chain framework region 2 (VH_FR2): AMNWVRQAPGQGLEWMGWI;

[0073] Heavy chain complementarity-determining region 2 (VH_CDR2): NTNTGN;

[0074] Heavy chain framework region 3 (VH_FR3): PTYAQGFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYY;

[0075] Heavy chain complementarity-determining region 3 (VH_CDR3): CARRWLGYFDYW;

[0076] Heavy chain framework region 4 (VH_FR4): GQGTLVTVSS;

[0077] The connection order of the complementarity-determining regions and the framework regions is: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0078] Humanized heavy chain nucleotide sequence of the antibody (SEQ ID NO.6, the double underlines indicate the framework regions in the variable region, and the wavy lines indicate the complementarity-determining regions in the variable region):

[0079]

[0080] GGAAGTACACACAGCTCAGACACAAACCCATAGAGAGGATTACAACAGTACTATCCG

[0081] GGTGGTCAGTGCCCTCCCCATCCAGCACCAGGACTGGATGAGTGGCAAGGAGTTCAA

[0082] ATGCAAGGTCAACAACAAAGACCTCCCATCACCCATCGAGAGAACCATCTCAAAAAT

[0083] TAAAGGGCTAGTCAGAGCTCCACAAGTATACATCTTGCCGCCACCAGCAGAGCAGTT

[0084] GTCCAGGAAAGATGTCAGTCTCACTTGCCTGGTCGTGGGCTTCAACCCTGGAGACATC

[0085] AGTGTGGAGTGGACCAGCAATGGGCATACAGAGGAGAACTACAAGGACACCGCACC

[0086] AGTCCTGGACTCTGACGGTTCTTACTTCATATACAGCAAGCTCGATATAAAAACAAGC

[0087] AAGTGGGAGAAAACAGATTCCTTCTCATGCAACGTGAGACACGAGGGTCTGAAAAATTACTACCTGAAGAAGACCATCTCCCGGTCTCCGGGTAAAtga。

[0088] Amino acid sequence of the humanized antibody light chain (SEQ ID NO.7, the framework regions in the variable region are indicated by double underlines, and the complementarity-determining regions in the variable region are indicated by wavy lines):

[0089] MMSSAQFLGLLLLCFQGTRC- DIQMTQSPSSLSASVGDRVTITCRAS QSISSYLN WYQ QKPGKAPKL LIY AASSLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYY CQQSYSTPLTF GQ GTKVEIK-RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLN SWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC。

[0090] The variable region of the humanized antibody light chain consists of complementarity-determining regions (CDRs) and framework regions (FRs), which are, in sequence:

[0091] Light chain framework region 1 (VL_FR1): DIQMTQSPSSLSASVGDRVTITCRAS;

[0092] Light chain complementarity-determining region 1 (VL_CDR1): QSISSYLN;

[0093] Light chain framework region 2 (VL_FR2): WYQQKPGKAPKLLIY;

[0094] Light chain complementarity-determining region 2 (VL_CDR2): AASSLQS;

[0095] Light chain framework region 3 (VL_FR3): GVPSRFSGSGSGTDFTLTISSLQPEDFATYY;

[0096] Light chain complementarity-determining region 3 (VL_CDR3): CQQSYSTPLTF;

[0097] Light chain framework region 4 (VL_FR4): GQGTKVEIK;

[0098] The connection order of the complementarity-determining regions and the framework regions is: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0099] The nucleotide sequence of the human antibody light chain (SEQ ID NO.8, with the framework regions in the variable region indicated by double underlines and the complementarity-determining regions in the variable region indicated by wavy lines):

[0100]

[0101] Sequence comparison and analysis showed that the humanized antibody has a high degree of similarity in spatial structure ( Figure 3), and the humanized antibody is highly similar to the human IgG2b gene sequence and is significantly different from known HER2 antibodies (e.g., trastuzumab, pertuzumab, the HER2 antibody in the applicant's prior patent CN118580359B). The comparison of the heavy and light chains of the humanized antibody of this solution with the HER2 antibody in the prior patent CN118580359B can be seen in detail in Figure 4 and Figure 5 . It can be seen that there are significant sequence differences between the heavy-chain CDRs and between the light-chain CDRs. The novel antibody with a sequence structure different from that of the prior patent was screened out in this technical solution, and the ideal antigen-binding efficiency and therapeutic effect can also be obtained (see the efficacy experimental data later). The humanized antibody obtained in this solution enriches the diversity of anti-HER2 antibodies and can be used as an effective means to cope with drug resistance. When cancer cells develop resistance to some monoclonal antibodies, antibodies with different CDR sequences can be used for continuous treatment. In addition, for antibody cocktail therapy, a novel humanized antibody provided by this patent can be used as a candidate for antibody cocktail therapy. The inventor will further study the antibody combination effect on mouse cell lines targeting the expression of HER2 protein antigen in the future.

[0102] Example 4: Expression, purification and functional verification of anti-HER2 humanized antibody

[0103] The construction, expression and purification of the anti-HER2 humanized antibody expression vector were carried out according to the method of the prior patent, which is a conventional method in the prior art and will not be elaborated here. According to the biological principle, after the heavy and light chains are synthesized in vivo, they will be assembled under the regulation of their respective signal peptides to form a complete antibody molecule. The complete antibody molecule will be used for subsequent experimental verification.

[0104] (1) Detection of HER2 protein on the surface of tumor cells by humanized antibody

[0105] By flow cytometry, different antibodies with the same concentration (10 μg / mL) (the humanized antibody of this solution, trastuzumab, control human IgG (Invitrogen, catalog number 02-7102)) were incubated with the human breast cancer cell line SK-BR-3 with high expression of HER2 protein at 4°C for 30 min, and then 1:500 diluted 647 goat anti-human IgG (H+L) antibody was added to measure the mean fluorescence intensity MFI of each antibody binding to the HER2 molecule on the cell surface. The detection results are shown in Figure 6 (sample size n = 5). There was no significant difference in the mean fluorescence intensity between the humanized group and the trastuzumab group, and both were significantly stronger than the control group. The results showed that the humanized antibody could specifically bind to the HER2 protein on the surface of tumor cells.

[0106] (2) Humanized antibody-mediated killing of breast cancer cells by immune cells

[0107] To prove that humanized antibodies can effectively mediate the killing of tumor cells by immune cells, the human breast cancer cell lines AU565 and BT-474 with high expression of HER2 protein were selected for in vitro antibody-dependent cell-mediated cytotoxicity (ADCC) experiments. The luciferase method was used to detect cell viability in the in vitro test. NK cells were co-cultured with the above AU565 and BT-474 cells at a ratio of 1:1. After incubating the co-cultured cells with humanized antibody, trastuzumab, and control IgG (0.5 μg / sample) for 48 h respectively, the cell viability was detected by an enzyme-linked immunosorbent assay (ELISA) reader, and the cell killing rate was statistically analyzed. The test results are as Figure 7 (Sample size n = 5), indicating that humanized antibodies can effectively mediate the killing of tumor cells by immune cells, and have a similar effect to trastuzumab.

[0108] Example 5: Construction of CAR-T cells based on the anti-HER2 humanized antibody sequence

[0109] CAR-T cells are conventional T cells modified by genetic engineering in the prior art. Mainly through transgenic technology, an expression plasmid containing the CAR gene is transferred into T cells, enabling T cells to express the CAR protein and enhancing the immunotherapeutic effect of T cells.

[0110] (1) Construction of a lentiviral vector plasmid expressing CAR and lentivirus packaging

[0111] The humanized antibody sequence of this solution is integrated into the chimeric antigen receptor CAR (HER2-CAR) targeting HER2 of this solution. The HER2-CAR of this solution includes the following parts, in order from extracellular to intracellular: signal peptide, light chain variable region (Iglv), hinge region (Hinge chain), heavy chain variable region (Ighv), CD28 extracellular region (CD28 Extra), CD28 transmembrane region (CD28 TM), CD28 intracellular region (CD28 Cyto), CD3zeta intracellular region (CD3zeta Cyto). The original lentiviral expression vector (empty vector) used is pLVX-IRES-mCherry (Biolight Biotechnology Co., Ltd., product number: 631237). This technical solution constructs anti-HER2-CAR using the light chain variable region and heavy chain variable region of the aforementioned humanized antibody, and expresses HER2-CAR in T cells to form CAR-T cells (HER2-CAR-T) for T cell therapy targeting HER2. HER2-CAR-T can recognize and bind to cells (cancer cells) expressing HER2, and T cells further achieve a killing effect on cells expressing HER2. The nucleotide sequence of HER2-CAR is shown in SEQ ID NO.9 (in the following sequence, the light chain variable region is closer to the 3' end, and the heavy chain variable region is closer to the 5' end; double underlines indicate the framework regions in the variable regions, wavy lines indicate the complementary determining regions in the variable regions, the light chain variable region is in the front, and the heavy chain variable region is in the back):

[0112]

[0113] GGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCC

[0114] GAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGG

[0115] CGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCAC

[0116] GATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGgccctgccccctcgc。

[0117] The constructed vector was subjected to conventional lentivirus packaging to obtain lentivirus for subsequent experiments. The construction process can be referred to the applicant's prior patent CN118580359B.

[0118] (2) Isolation and culture of primary mouse T lymphocytes

[0119] Isolation of mouse spleen T lymphocytes: Take the spleen of C57BL / 6 mice. After grinding through a filter screen, a single-cell suspension of the spleen is obtained. Treat this cell suspension with erythrocyte lysate, incubate on ice for 5 - 10 min, add 3 - 5 volumes of PBS for dilution, centrifuge the cells at 1500 rpm for 5 min, wash the cells once with PBS, incubate with anti-CD3 magnetic beads for 20 min, and separate CD3 + T cells by using a magnetic sorting column, count the cells, and wash the cells 1 - 2 times with PBS. CD3 + T lymphocytes represent total T lymphocytes, including helper / inducer T lymphocytes (CD3 + CD4 + ), suppressor / cytotoxic T lymphocytes (CD3 + CD8 + ), etc., all types of T lymphocytes.

[0120] In vitro stimulation and culture of T cells: Prepare a T lymphocyte stimulation solution, and the composition and ratio are shown in Table 5. Resuspend the cells with the prepared stimulation solution and culture them in a 12-well plate at 2 mL / well. Observe the cell density under the microscope, change the stimulation solution every day, stimulate continuously for three days, and then change the culture medium to a medium containing only IL-2 after the third day. The composition of the T lymphocyte stimulation solution is as follows: 90% lymphocyte medium, 10% fetal bovine serum (FBS), 100 U / mL penicillin / streptomycin double antibody, 10 ng / mL IL-2 (mouse), 0.6 μg / mL Anti-CD3 (mouse), 0.3 μg / mL Anti-CD28 (mouse).

[0121] (3) Viral infection of T cells

[0122] On the third day, seed the stimulated T cells in a six-well plate at a cell density of 2×10 6 cells / well, and prepare a viral infection solution, with the reagents shown in Table 6. Centrifuge the six-well plate (1000 g, 37 °C, 90 min), then place it in a cell incubator for 8 - 10 h, and then change to a normal medium (the medium contains IL-2). Fluorescence detection of the cells is performed 48 h after infecting the T cells to detect whether the transfection is successful. Thus, the construction of CAR-T cells is completed. For a single sample, the composition of the viral infection solution is: 2 mL of virus solution, 2 mL of cell solution, 8 μL of Polybrene (1 mg / ml), 40 μL of HEPES (1 M; Gibco).

[0123] In the above process, the processes of constructing lentiviral expression vectors, lentiviral packaging, obtaining T lymphocytes, and infecting T cells with viruses are all carried out in the conventional manner of the existing technology. The difference between this embodiment and the existing technology lies in that: on the basis of the sequence of the chimeric antigen receptor CAR in the existing technology, the heavy-chain variable region and the light-chain variable region of the humanized antibody obtained in this scheme are added, so that the constructed CAR-T cells have the ability to target HER2.

[0124] Example 6: Detection of the in vitro tumor-killing ability of CAR-T cells

[0125] To prove the in vitro tumor-killing ability of the CAR-T cells designed and constructed based on the humanized antibody sequence of this scheme, this example uses the co-culture experiment of in vitro tumor cells and CAR-T cells (experimental group) to detect the in vitro killing ability of CAR-T cells on tumor cells. And ordinary T cells are used as the control group. On day 0, the prepared CAR-T was inoculated into a 24-well plate at 1.0×10 6 cells / well. The breast cancer SK-BR-3 and cervical cancer SK-OV-3 tumor cell lines expressing the HER2 antigen and the thyroid cancer cell line BHT-101 not expressing HER2 were co-cultured with CAR-T cells at an effector-to-target ratio of 1:1. After 48 hours, the number of tumor cells was detected, and the tumor lysis rate of CAR-T cells was calculated. The experimental results are shown in Figure 8 (sample size n = 5), indicating that the CAR-T cells designed and constructed based on the humanized antibody sequence of this scheme have the ability to specifically kill tumor cells in vitro.

[0126] The above are only embodiments of the present invention. Specific technical solutions and / or common knowledge such as characteristics well known in the art are not described in detail herein. It should be pointed out that for those skilled in the art, without departing from the technical solution of the present invention, several modifications and improvements can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the implementation effect of the present invention and the practicality of the patent. The protection scope required by this application should be based on the content of its claims, and the specific implementation manners described in the specification can be used to interpret the content of the claims.

Claims

1. A humanized antibody against HER2 antigen, characterized in that, The amino acid sequences of complementarity-determining region 1, complementarity-determining region 2, and complementarity-determining region 3 of its heavy chain are GYTFTSY, NTNTGN, and CARRWLGYFDYW respectively; the amino acid sequences of complementarity-determining region 1, complementarity-determining region 2, and complementarity-determining region 3 of its light chain are QSISSYLN, AASSLQS, and CQQSYSTPLTF respectively.

2. The humanized antibody against HER2 antigen according to claim 1, wherein Its framework region 1 of the heavy chain, framework region 2 of the heavy chain, framework region 3 of the heavy chain, and framework region 4 of the heavy chain are: QVQLVQSGSELKKPGASVKVSCKAS, AMNWVRQAPGQGLEWMGWI, PTYAQGFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYY, GQGTLVTVSS.

3. A humanized antibody against HER2 antigen according to claim 1, characterized in that, Its framework region 1 of the light chain, framework region 2 of the light chain, framework region 3 of the light chain, and framework region 4 of the light chain are: DIQMTQSPSSLSASVGDRVTITCRAS, WYQQKPGKAPKLLIY, GVPSRFSGSGSGTDFTLTISSLQPEDFATYY, GQGTKVEIK.

4. A humanized antibody against HER2 antigen according to claim 1, characterized in that, The amino acid sequence of its heavy chain is as shown in SEQ ID NO.5, and the amino acid sequence of its light chain is as shown in SEQ ID NO.

7.

5. Use of a humanized antibody against HER2 antigen according to any one of claims 1-4 in the preparation of a medicament for treating cancer with human epidermal growth factor receptor 2 protein expressed on the cell surface, characterized in that, The cancer is breast cancer.

6. Use of a humanized antibody against HER2 antigen according to any one of claims 1-4 in the preparation of a reagent for detecting human epidermal growth factor receptor 2 protein.

7. Use of a humanized antibody against HER2 antigen according to any one of claims 1-4 in the preparation of chimeric antigen receptor T cells, characterized in that, The nucleotide sequence of the chimeric antigen receptor of the chimeric antigen receptor T cell is as shown in SEQ ID NO.

9.

8. A chimeric antigen receptor protein, characterized in that, Its nucleotide sequence is as shown in SEQ ID NO.9.

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