Humanized antibody aiming at HER2 antigen and application thereof

Abstract

The invention belongs to the technical field of monoclonal antibodies, and particularly relates to a humanized antibody aiming at an HER2 antigen and application of the humanized antibody. The invention relates to a humanized antibody aiming at an HER2 antigen. The amino acid sequences of a complementarity determining region 1, a complementarity determining region 2 and a complementarity determining region 3 of a heavy chain of the humanized antibody are respectively GYTFTSY, NTNTGN and CARRWLGYFDYW; the amino acid sequences of a complementarity determining region 1, a complementarity determining region 2 and a complementarity determining region 3 of a light chain are QSISSYLN, AASSLQS and CQQSYSTPLTF respectively. The antibody sequence in the scheme is different from that of other HER2 antibodies in the prior art, and the action effect is consistent with that of trastuzumab. According to the technical scheme, the technical problem that in the prior art, the number of antibody types aiming at the HER2 antigen is small can be solved, the problem of tumor drug resistance of a single drug can be solved through the new antibody, the new antibody can be used for antibody combination therapy of cancers, and the ideal application and popularization prospects are achieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of monoclonal antibodies, and in particular relates to a humanized antibody targeting HER2 antigen and an application thereof. Background Art

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

[0003] Studies have shown that monoclonal antibody drugs targeting HER2 can exert anti-tumor effects through direct and indirect mechanisms. The direct mechanism is mainly that the monoclonal antibody binds to HER2 overexpressed on the surface of tumor cells, inhibiting the formation of HER2 homodimers and heterodimers, thereby blocking the activation of MAPK and P13K signaling pathways, and ultimately inhibiting tumor cell proliferation and invasion. The indirect mechanism is that after the antibody binds to HER2 overexpressed 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 eliminated through antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity.

[0004] Trastuzumab is the world's first humanized monoclonal antibody targeting HER2, and was approved for marketing by the FDA in 1998. Trastuzumab has greatly improved the prognosis of patients with HER2-positive breast cancer. However, about 50-60% of HER2-positive breast cancer patients will develop resistance to the antibody, making it ineffective. Although there are some monoclonal antibody drugs targeting HER2 on the market, drug resistance may occur after a period of treatment, affecting the therapeutic effect of the drug. The applicant's prior patent CN118580359B (anti-HER2 monoclonal antibody and its gene, preparation method and application) uses a mouse cell line overexpressing HER2 protein antigen to immunize mice, obtains an antibody hybridoma cell line with strong binding force to HER2 protein through hybridoma fusion and positive clone screening, and then performs sequence analysis to further obtain sequence information, and performs humanization treatment of the antibody, and finally obtains a monoclonal antibody against HER2 protein antigen. The monoclonal antibody can effectively bind to HER2 antigen and can kill cancer cells expressing HER2 antigen on the surface.

[0005] However, the surface of cells expressing HER2 still has a large number of other antigenic sites. If multiple monoclonal antibodies that can target different sites of mouse cell lines that overexpress HER2 protein antigens can be developed, the treatment efficiency can be effectively improved, and other candidate antibodies will be available after drug resistance develops. Antibody cocktail therapy is a method of treating diseases using a combination of multiple antibodies. This method uses several different antibodies to attack different parts of tumor-associated antigens at the same time, thereby improving the treatment 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, antibody therapy for diseases related to HER2 overexpression is still in the research and development stage. If multiple antibodies can be developed against HER2 antigen proteins, these newly developed antibodies can be used as candidate antibodies for cocktail therapy, increasing the number of candidate antibodies for cocktail therapy. If a variety of appropriate related antibodies that can be used for cocktail therapy can be developed, the treatment efficiency can be greatly improved, thereby achieving multi-target attacks on cancer cells, reducing the risk of drug resistance, and enhancing immune response. Summary of the invention

[0006] The present invention is intended to provide a humanized antibody against HER2 antigen to solve the technical problem that there are few types of antibodies against HER2 antigen in the prior art. Due to the small number of antibody types against HER2 antigen, it is difficult to find candidate drugs to overcome the drug resistance of anticancer drugs, or it leads to a lack of effective candidate antibodies when using antibody combination therapy.

[0007] In order to achieve the above object, the present invention adopts the following technical scheme:

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

[0009] Further, its 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, its light chain framework region 1, light chain framework region 2, light chain framework region 3, and light chain framework region 4 are respectively:

[0012] DIQMTQSPSSSLSASVGDRVTITCRAS,WYQQKPGKAPKLLIY,GVPSRFSGSGSGTDFTLTISSLQPEDFATYY,GQGTKVEIK.

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

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

[0015] This technical solution also provides a use of a humanized antibody against HER2 antigen in the preparation of a drug for treating cancer expressing human epidermal growth factor receptor 2 protein on the cell surface, wherein the cancer is breast cancer. The monoclonal antibody obtained in this solution can be used to inhibit the growth of cancer cells expressing HER2, and the experiment in Example 4, for example, verifies the above effect.

[0016] The technical solution also provides a use of a humanized antibody against HER2 antigen in the preparation of a reagent for detecting human epidermal growth factor receptor 2 protein. The monoclonal antibody obtained in this solution can specifically bind to HER2, and therefore can be used for quantitative or qualitative detection of HER2 protein, for example: detecting cancer cells expressing HER2, for example, the experiment in Example 4 verifies the above effect.

[0017] This 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 cell is shown in SEQ ID NO. 9. The heavy chain variable region and light chain variable region of the humanized antibody obtained in this solution are added to the sequence of the chimeric antigen receptor CAR in the prior art, so that the constructed CAR-T cells have the ability to target HER2.

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

[0019] The present technical solution also provides a chimeric antigen receptor protein, whose nucleotide sequence is shown in SEQ ID NO. 9. The chimeric antigen receptor protein of the present solution is a new type of fusion protein. By expressing this new fusion protein in T cells through conventional means of the prior art, CAR-T therapy targeting surface-expressed HER2 protein can be achieved.

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

[0021] This technical solution first constructs a mouse cell line that stably overexpresses HER2 protein (antigen). The cell line is then used to immunize mice, and a hybridoma cell line with strong binding to HER2 is obtained through hybridoma fusion and positive clone screening. The cell line can produce antibodies with strong binding to HER2 protein. Sequence analysis is performed on anti-HER2 hybridoma cell clones to further obtain sequence information, and humanization of the antibodies is performed. On this basis, antibodies can be artificially synthesized or expressed to form commercially available anti-HER2 protein antibodies. In addition, the heavy chain and light chain sequences obtained by this technical solution can also be used to prepare chimeric antigen receptors for CAR-T cells to achieve the killing of cancer cells that express HER2 antigens on their surfaces.

[0022] The novel humanized antibody obtained by this technical solution has similar effects as the existing trastuzumab and the like. It further solves the difficult-to-overcome technical problem of tumor drug resistance caused by the lack of antibodies that recognize different HER2 antigen epitopes in the prior art. The antibody (especially the complementary 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 similar cancers and overcome the problem of tumor drug resistance caused by a single drug. And because the number of candidate monoclonal antibodies for HER2 has increased, it has become possible to further study antibody cocktail therapy for related cancers, thereby effectively improving the treatment efficiency, thereby achieving multi-target attack on cancer cells, reducing the risk of drug resistance, and enhancing immune response. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0024] Figure 2 This 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 hybridoma cell line labeled with iFluor 488; C: fusion image of A and B).

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

[0026] Figure 4 The figure shows the sequence alignment between the humanized antibody heavy chain of the present embodiment 3 and the antibody heavy chain of the prior patent.

[0027] Figure 5 The figure shows the sequence alignment between the humanized antibody light chain of the present embodiment 3 and the antibody light chain of the prior patent.

[0028] Figure 6 This is a diagram showing the experimental results of the humanized antibody in Example 4 detecting HER2 protein on the surface of tumor cells.

[0029] Figure 7 This is a graph showing the experimental results of the killing effect of the humanized antibody in Example 4 on breast cancer cells.

[0030] Figure 8 These are the experimental results for detecting the in vitro tumor killing ability of CAR-T cells in Example 6. DETAILED DESCRIPTION

[0031] The present invention is further described in detail below with reference to the examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following examples 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 technology (see Molecular Cloning, Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York). The materials, reagents, etc. used can all be obtained from commercial sources.

[0032] Embodiment 1:

[0033] Referring to the applicant's prior patent CN118580359B (anti-HER2 monoclonal antibodies and genes thereof, as well as preparation methods and applications), a mouse cell line stably expressing HER2 protein is prepared, and the prepared cell line is used as an antigen for immunizing mice, and then a number of hybridoma cell lines to be screened are obtained by conventional methods of the prior art.

[0034] By flow cytometry, hybridoma cells that can selectively bind to HER2 were selected from several hybridoma cell lines. Subcloned hybridoma cell lines were screened by limiting dilution method. Flow cytometry was used to further detect their ability to bind to HER2 protein. Finally, an optimal hybridoma cell line was screened (see Figure 1 , the red frame shows the hybridoma cell line with the best binding ability). Figure 1 The experimental data showed that the hybridoma cell line indicated by the red box has the strongest HER2 binding ability. However, the antibodies secreted by other hybridoma lines to be screened did not bind well to the HER2 protein.

[0035] Through immunofluorescence experiments, we verified that the antibody of the hybridoma cell line can specifically bind to HER2. The specific steps are 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 to the wall and grew, an appropriate amount of the hybridoma supernatant with the best binding ability was added, incubated at room temperature for 30 minutes, and after washing away the excess supernatant, a 1:500 dilution of 488 goat anti-human IgG (H+L) antibody, incubate at room temperature for 30 min, and collect images under a microscope. Figure 2 The immunofluorescence images show that the antibody from the hybridoma cell line with the best binding ability can specifically bind to HER2.

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

[0037] The antibody Isotype secreted by the hybridoma cell line screened in Example 1 was determined by ELISA to be mouse IgG2b. Suzhou Genewise Biotechnology Co., Ltd. was commissioned to sequence the antibody variable region gene of the hybridoma cell line.

[0038] The mouse antibody against HER2 antigen includes a heavy chain and a light chain. The amino acid sequence of the variable region of the heavy chain of the mouse antibody (SEQ ID NO.1, double underlines indicate the framework region in the variable region, and wavy lines indicate the complementary determining region in the variable region):

[0039]

[0040] Among them, the variable region of the mouse antibody heavy chain consists of the complementarity-determining region (CDR) and the framework region (FR), which are:

[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 the framework regions is: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0049] The nucleotide sequence of the variable region of the heavy chain of the mouse antibody (SEQ ID NO.2, the double underline indicates the framework region in the variable region, and the wavy line indicates the complementary determining region in the variable region):

[0050]

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

[0052]

[0053] Among them, the variable region of the mouse antibody light chain consists of the complementarity-determining region (CDR) and the framework region (FR), which are:

[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 the framework regions is: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0062] The nucleotide sequence of the light chain variable region of the mouse antibody (SEQ ID NO.4, double underlines indicate the framework region in the variable region, and wavy lines indicate the complementary determining region in the variable region):

[0063]

[0064] Sequence comparison analysis showed that the mouse antibody was significantly different from known HER2 antibodies (as well as the HER2 antibody of the applicant's prior patent CN118580359B), indicating that it was a completely new HER2 antibody.

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

[0066] By analyzing large-scale bioinformatics data, we used artificial intelligence algorithms to humanize the mouse antibody obtained in Example 2, reducing the immunogenicity of the antibody, thereby reducing the risk of immune response in the body. In addition, the antibody framework and sequence were intelligently designed and optimized, including improving antibody stability, improving structure, reducing unnecessary modification sites, and optimizing the expression system to ensure optimal biological activity in the human body.

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

[0068]

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

[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] Human antibody heavy chain nucleotide sequence (SEQ ID NO.6, double underlines indicate the framework region in the variable region, and wavy lines indicate the complementary determining region in the variable region):

[0079]

[0080] GGAAGTACACACAGCTCAGACACAAACCCATAGAGAGGATTACAACAGTACTATCCG

[0081] GGTGGTCAGTGCCCTCCCCATCCAGCACCAGGACTGGATGAGTGGCAAGGAGTTCAA

[0082] ATGCAAGGTCAACAACAAAGACCTCCCATCACCCATCGAGAGAACCATCTCAAAAAT

[0083] TAAAGGGCTAGTCAGAGCTCCACAAGTATACATCTTGCCGCCACCAGCAGAGCAGTT

[0084] GTCCAGGAAAGATGTCAGTCTCACTTGCCTGGTCGTGGGCTTCAACCCTGGAGACATC

[0085] AGTGTGGAGTGGACCAGCAATGGGCATACAGAGGAGAACTACAAGGACACCGCACC

[0086] AGTCCTGGACTCTGACGGTTCTTACTTCATATACAGCAAGCTCGATATAAAAACAAGC

[0087] AAGTGGGAGAAAACAGATTCCTTCTCATGCAACGTGAGACACGAGGGTCTGAAAAATTACTACCTGAAGAAGACCATCTCCCGGTCTCCGGGTAAAtga.

[0088] Humanized antibody light chain amino acid sequence (SEQ ID NO.7, double underlines indicate the framework region in the variable region, and wavy lines indicate the complementarity determining region in the variable region):

[0089] MMSSAQFLGLLLLCFQGTRC- DIQMTQSPSSLSASVGDRVTITCRAS QSISSYLN Wlq QKPGKAPKL LIY AASSLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYY CQQSYSTPLTF GQ GTKVEIK-RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLN SWTDQDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC.

[0090] The variable region of the humanized antibody light chain consists of the complementarity-determining region (CDR) and the framework region (FR), in order:

[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] Human antibody light chain nucleotide sequence (SEQ ID NO.8, double underlines indicate the framework region in the variable region, and wavy lines indicate the complementary determining region in the variable region):

[0100]

[0101] Sequence comparison analysis showed that the humanized antibody has a high degree of similarity with the 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, and the HER2 antibody of the applicant's prior patent CN118580359B). For details on the comparison of the heavy chain and light chain of the humanized antibody of this scheme and the HER2 antibody of the prior patent CN118580359B, see Figure 4 and Figure 5 , it can be seen that there are large sequence differences between the heavy chain CDRs and between the light chain CDRs. This technical solution screens out new antibodies with sequence structures different from those in the prior patents, and can also obtain ideal antigen binding efficiency and therapeutic effects (see the efficacy experimental data below for details). The humanized antibodies obtained in this solution enrich the diversity of anti-HER2 antibodies and can be used as an effective means to deal with drug resistance. When cancer cells become resistant to some monoclonal antibodies, other antibodies with different CDR sequences can be used to continue treatment. In addition, for antibody cocktail therapy, a new humanized antibody provided by this patent can be used as a candidate for antibody cocktail therapy. The inventor will further study the combination effect of antibodies targeting mouse cell lines expressing HER2 protein antigens.

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

[0103] The construction, expression and purification of the anti-HER2 humanized antibody expression vector were carried out in accordance with the prior patent method, which is a conventional method in the prior art and will not be described here. According to biological principles, after the heavy chain and light chain 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) Humanized antibody detection of HER2 protein on the surface of tumor cells

[0105] Using flow cytometry, different antibodies (humanized antibodies in this protocol, trastuzumab, and control human IgG (Invitrogen, Cat. No. 02-7102)) at the same concentration (10 μg / mL) were incubated with SK-BR-3, a human breast cancer cell line that highly expresses HER2 protein, at 4°C for 30 min, and then a 1:500 dilution of 647 goat anti-human IgG (H+L) antibody, measure the mean fluorescence intensity (MFI) of each antibody binding to the HER2 molecule on the cell surface. Figure 6 (Sample size n=5), there was no significant difference in the average 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 can specifically bind to the HER2 protein on the surface of tumor cells.

[0106] (2) Humanized antibodies mediate immune cell killing of breast cancer cells

[0107] In order to prove that humanized antibodies can effectively mediate the killing effect of immune cells on tumor cells, 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 in vitro experiment used the luciferase method to detect cell viability. NK cells were co-cultured with the above-mentioned AU565 and BT-474 cells at a 1:1 ratio. After incubation with humanized antibodies, trastuzumab, and control IgG (0.5μg / sample) for 48 hours, the cell viability was detected by an enzyme reader, and the cell killing rate was statistically analyzed. The experimental results are as follows Figure 7 (Sample size n=5), indicating that humanized antibodies can effectively mediate immune cells to kill tumor cells, and the effect is similar to that of trastuzumab.

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

[0109] CAR-T cells are conventional genetically modified T cells in existing technologies. They are mainly produced by transferring expression plasmids containing CAR genes into T cells through transgenic technology, so that T cells express CAR proteins and enhance the immunotherapy effect of T cells.

[0110] (1) Construction of CAR expression lentiviral vector plasmid and lentiviral packaging

[0111] The chimeric antigen receptor CAR (HER2-CAR) targeting HER2 of this scheme integrates the humanized antibody sequence of this scheme. The HER2-CAR of this scheme 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 (Baori Medical Biotechnology Co., Ltd., product number: 631237). This technical scheme uses the light chain variable region and heavy chain variable region of the aforementioned humanized antibody to construct anti-HER2-CAR, and express 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 kill 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; the double underline indicates the framework region in the variable region, the wavy line indicates the complementary determining region in the variable region, the light chain variable region is in 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 packaged with conventional lentivirus to obtain lentivirus for subsequent experiments. The construction process can be found in the applicant's prior patent CN118580359B.

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

[0119] Isolation of mouse spleen T lymphocytes: C57BL / 6 mouse spleen was taken and ground through a filter to obtain a spleen single cell suspension. The cell suspension was treated with red blood cell lysis buffer, incubated on ice for 5-10 minutes, diluted with 3-5 times the volume of PBS, centrifuged at 1500rpm for 5 minutes, washed with PBS once, incubated with anti-CD3 magnetic beads for 20 minutes, and separated by magnetic separation column CD3 + T cells, count the cells, and wash the cells 1-2 times with PBS. + T lymphocytes represent all T lymphocytes, including helper / inducer T lymphocytes (CD3 + CD4 + ), suppressor / cytotoxic T lymphocytes (CD3 + CD8 + ) and all types of T lymphocytes.

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

[0121] (3) T cell viral infection

[0122] On the third day, the stimulated T cells were plated in a six-well plate at a cell density of 2×10 6 / well, prepare the virus infection solution, the reagents are shown in Table 6. The six-well plate was centrifuged (1000g, 37°C, 90min), and then placed in a cell incubator for 8-10h, and then replaced with normal culture medium (containing IL-2). After 48h of T cell infection, the cells were fluorescently detected to detect whether the transfection was successful. At this point, the construction of CAR-T cells was completed. For a single sample, the composition of the virus infection solution was: 2mL of virus solution, 2mL of cell solution, 8μl of Polybrene (1mg / ml), and 40μl of HEPES (1M; Gibco).

[0123] In the above process, the processes of constructing lentiviral expression vector, lentiviral packaging, obtaining T lymphocytes, and T cell virus infection are all carried out in conventional manners of the prior art. The difference between this embodiment and the prior art is that the heavy chain variable region and light chain variable region of the humanized antibody obtained in this scheme are added to the sequence of the chimeric antigen receptor CAR of the prior art, so that the constructed CAR-T cells have the ability to target HER2.

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

[0125] In order to prove the in vitro tumor killing ability of CAR-T cells designed and constructed based on the humanized antibody sequence of this protocol, this example uses an in vitro tumor cell and CAR-T cell (experimental group) co-culture experiment 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 cells were injected with 1.0×10 6 100 cells / well were inoculated in a 24-well plate, and the breast cancer SK-BR-3 and cervical cancer SK-OV-3 tumor cell lines expressing HER2 antigen and the thyroid cancer cell line BHT-101 that does not express HER2 were co-cultured with CAR-T cells at an effector-target ratio of 1:1. The number of tumor cells was detected after 48 hours, 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 is only an embodiment of the present invention, and the common knowledge such as the known specific technical solutions and / or characteristics in the solution is not described in detail here. 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 effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records 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 the complementary determining region 1, complementary determining region 2 and complementary determining region 3 of its heavy chain are GYTFTSY, NTNTGN, CARRWLGYFDYW, respectively; the amino acid sequences of the complementary determining region 1, complementary determining region 2 and complementary determining region 3 of its light chain are QSISSYLN, AASSLQS, CQQSYSTPLTF, respectively.

2. A humanized antibody against HER2 antigen according to claim 1, characterized in that: Its heavy chain framework region 1, heavy chain framework region 2, heavy chain framework region 3, and heavy chain framework region 4 are: QVQLVQSGSELKKPGASVKVSCKAS, AMNWVRQAPGQGLEWMGWI, PTYAQGFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYY, and GQGTLVTVSS.

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

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

7.

5. A humanized antibody against HER2 antigen according to claim 4, characterized in that: The nucleotide sequence of its heavy chain is shown in SEQ ID NO.6; The nucleotide sequence of its light chain is shown in SEQ ID NO.

8.

6. Use of a humanized antibody against HER2 antigen according to any one of claims 1 to 5 in the preparation of a drug for treating cancers expressing human epidermal growth factor receptor 2 protein on the cell surface, characterized in that: The cancer is breast cancer.

7. Use of a humanized antibody against HER2 antigen according to any one of claims 1 to 5 in the preparation of a reagent for detecting human epidermal growth factor receptor 2 protein.

8. Use of a humanized antibody against HER2 antigen according to any one of claims 1 to 5 in preparing chimeric antigen receptor T cells, characterized in that: The nucleotide sequence of the chimeric antigen receptor of the chimeric antigen receptor T cell is shown in SEQ ID NO.

9.

9. The use of a humanized antibody against HER2 antigen in preparing chimeric antigen receptor T cells according to claim 8, characterized in that: The chimeric antigen receptor T cells are used to treat breast cancer and / or cervical cancer.

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

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