CN119735686B - A humanized antibody targeting HER2 antigen and its application - Google Patents

Abstract

The invention belongs to the technical field of monoclonal antibodies, and particularly relates to a humanized antibody aiming at HER2 antigen and application thereof. A humanized antibody against HER2 antigen has the amino acid sequences of complementarity determining region 1, complementarity determining region 2 and complementarity determining region 3 of the heavy chain of GYTFTSY, NTNTGN, CARRWLGYFDYW, and the amino acid sequences of complementarity determining region 1, complementarity determining region 2 and complementarity determining region 3 of the light chain of QSISSYLN, AASSLQS, CQQSYSTPLTF, respectively. The antibody sequences of this protocol are different from other HER2 antibodies of the prior art and the effect is consistent with trastuzumab. The technical scheme can solve the technical problem of less antibody types aiming at HER2 antigen in the prior art, the novel antibody can overcome the problem of tumor drug resistance of single medicine, and the antibody combination therapy for cancers has ideal application and popularization prospect.

Description

Humanized antibody aiming at HER2 antigen and application thereof

Technical Field

The invention belongs to the technical field of monoclonal antibodies, and particularly relates to a humanized antibody aiming at HER2 antigen and application thereof.

Background

HER2 (human epidermal growth factor receptor 2,human epidermal growth factor receptor 2, abbreviated as HER2, also known as Neu, erbB-2, CD340, or p 185) is a tyrosine kinase receptor membrane glycoprotein encoded by ErbB2 proto-oncogene, HER2 being a member of the epidermal growth factor receptor (EGFR/ErbB) family. It is known that amplification of the HER2 protooncogene or overexpression of a protein is found in various human tumors including breast cancer, stomach cancer, ovarian cancer, lung cancer, prostate cancer, etc., and is clinically referred to as HER2 positive tumor. Overexpression of the HER2 protein results in increased HER2 homodimer formation, which leads to effects such as cell proliferation, anti-apoptosis, invasion and angiogenesis by activating MAPK and P13K signaling pathways. HER2 positive tumors are clinically shown to be high in malignancy, strong in infiltration and metastatic capacity, insensitive to conventional chemotherapeutic drugs and poor in treatment prognosis.

Studies have shown that monoclonal antibody drugs targeting HER2 can exert antitumor effects through direct and indirect mechanisms. The direct mechanism is mainly that the monoclonal antibody is combined with HER2 which is overexpressed on the surface of tumor cells, so that the formation of HER2 homodimers and heterodimers is inhibited, the activation of MAPK and P13K signal paths is further blocked, and the effect of inhibiting proliferation and invasion of the tumor cells is finally exerted. The indirect mechanism is mainly that after the antibody binds to HER2 over-expressed on the surface of tumor cells, the Fc end of the antibody tail can be recognized by the immune system of the organism, and the clearance of the tumor cells is finally realized through the cell-mediated cytotoxicity effect and the complement-dependent cytotoxicity effect of the antibody.

Trastuzumab (Trastuzumab) is the first humanized mab against HER2 worldwide and was marketed by the FDA in 1998. Trastuzumab greatly improves prognosis in HER 2-positive breast cancer patients, however, approximately 50-60% of HER 2-positive breast cancer patients develop resistance to antibodies, rendering them ineffective. Although there are some monoclonal antibody drugs that target HER2 on the market, resistance may develop after a period of treatment, affecting the therapeutic effect of the drug. The applicant's prior patent CN118580359B (anti-HER 2 monoclonal antibody and gene thereof, and preparation method and application thereof) uses a murine cell line that overexpresses HER2 protein antigen to immunize mice, obtains antibody hybridoma cell lines that produce antibodies with stronger binding force with HER2 protein through hybridoma fusion and positive clone screening, then performs sequence analysis, further obtains sequence information, and performs humanized treatment of the antibodies, finally obtains monoclonal antibodies against HER2 protein antigen. The monoclonal antibody can effectively bind to HER2 antigen, and can kill cancer cells aiming at surface expression of HER2 antigen.

However, the cell surface expressing HER2 still has a large number of other antigenic sites, and if a variety of monoclonal antibodies can be developed against different sites of murine cell lines that overexpress HER2 protein antigens, the therapeutic efficiency can be effectively improved and other candidate antibodies can be generated after drug resistance. Antibody cocktail therapy is a method of treating a disease using a combination of multiple antibodies. This approach increases the therapeutic effect and reduces the likelihood of cancer cell escape mutations by using several different antibodies simultaneously to attack different sites of tumor-associated antigen. Although antibody cocktail therapy has shown potential in certain viral infections and cancer treatments, antibody therapies directed against diseases associated with HER2 overexpression are still currently under investigation and development. If multiple antibodies can be developed against the HER2 antigen protein, these newly developed antibodies can be used as candidates for cocktail therapy, increasing the number of antibodies that are candidates for cocktail therapy. If a proper variety of related antibodies for cocktail therapy can be developed, the therapeutic efficiency can be greatly improved, thereby realizing multi-target attack on cancer cells, reducing drug resistance risk, enhancing immune response and the like.

Disclosure of Invention

The invention aims to provide a humanized antibody aiming at a HER2 antigen so as to solve the technical problem of less antibody types aiming at the HER2 antigen in the prior art. Because of the low number of antibody types against HER2 antigen, it is difficult to find candidate drugs to overcome after development of resistance to anticancer drugs, or it results in a lack of effective candidate antibodies when antibody combination therapies are used.

In order to achieve the above purpose, the invention adopts the following technical scheme:

A humanized antibody against HER2 antigen has the amino acid sequences of complementarity determining region 1, complementarity determining region 2 and complementarity determining region 3 of the heavy chain of GYTFTSY, NTNTGN, CARRWLGYFDYW, and the amino acid sequences of complementarity determining region 1, complementarity determining region 2 and complementarity determining region 3 of the light chain of QSISSYLN, AASSLQS, CQQSYSTPLTF, respectively.

Further, the heavy chain framework region 1, the heavy chain framework region 2, the heavy chain framework region 3 and the heavy chain framework region 4 are respectively:

QVQLVQSGSELKKPGASVKVSCKAS、AMNWVRQAPGQGLEWMGWI、PTYAQGFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYY、GQGTLVTVSS。

further, the light chain framework region 1, the light chain framework region 2, the light chain framework region 3 and the light chain framework region 4 are respectively:

DIQMTQSPSSLSASVGDRVTITCRAS、WYQQKPGKAPKLLIY、GVPSRFSGSGSGTDFTLTISSLQPEDFATYY、GQGTKVEIK。

Further, the amino acid sequence of the heavy chain is shown as SEQ ID NO.5, and the amino acid sequence of the light chain is shown as SEQ ID NO. 7.

Further, the nucleotide sequence of the heavy chain is shown as SEQ ID NO.6, and the nucleotide sequence of the light chain is shown as SEQ ID NO. 8.

The technical scheme also provides application of the humanized antibody aiming at the HER2 antigen in preparing medicines for treating cancers expressing the HER2 protein on the cell surface, wherein the cancers are breast cancers. The monoclonal antibodies obtained in this protocol can be used to inhibit the growth of HER2 expressing cancer cells, as demonstrated by experiments such as in example 4.

The technical scheme also provides application of the humanized antibody aiming at the HER2 antigen in preparing a reagent for detecting the HER2 protein. The monoclonal antibodies obtained in this protocol can specifically bind to HER2 and thus can be used for quantitative or qualitative detection of proteins directed against HER2, e.g. for detection of HER2 expressing cancer cells, e.g. experiments in example 4 confirm the above effect.

The technical scheme also provides application of the humanized antibody aiming at the HER2 antigen in preparation of chimeric antigen receptor T cells, wherein the nucleotide sequence of the chimeric antigen receptor T cells is shown as SEQ ID NO. 9. The heavy chain variable region and the light chain variable region of the humanized antibody obtained by the scheme are added on the basis of the sequence of the chimeric antigen receptor CAR in the prior art, so that the constructed CAR-T cell has the capability of targeting HER 2.

Further, the chimeric antigen receptor T cells are useful for treating breast cancer and/or cervical cancer. Experimental data in examples 5 and 6 demonstrate that chimeric antigen receptor T cells containing the heavy and light chain variable regions of humanized antibodies can effectively kill breast and/or cervical cancer cells.

The technical scheme also provides a chimeric antigen receptor protein, and the nucleotide sequence of the chimeric antigen receptor protein is shown as SEQ ID NO. 9. The chimeric antigen receptor protein of the scheme is a novel fusion protein, and the novel fusion protein is expressed in T cells by conventional means in the prior art, so that CAR-T therapy aiming at surface expression of HER2 protein can be realized.

To sum up, the principle of the technical scheme and the beneficial effects are that:

The technical scheme firstly constructs a murine cell line for stably and overexpressing HER2 protein (antigen). Then the cell line is used for immunizing mice, hybridoma cell lines with stronger binding force with HER2 are obtained through hybridoma fusion and positive clone screening, and the cell lines can generate antibodies with stronger binding force with HER2 protein. Sequence analysis was performed on anti-HER 2 hybridoma clones, further sequence information was obtained, and humanized treatment of the antibodies was performed. On this basis, the antibody can be artificially synthesized or expressed to form a commercialized antibody against HER2 protein. In addition, the heavy chain and light chain sequences obtained by the technical scheme can be used for preparing chimeric antigen receptors of CAR-T cells, so that killing of cancer cells aiming at surface expression of HER2 antigen can be realized.

The novel humanized antibody obtained by the technical scheme has similar action and effects as the existing trastuzumab and the like. Further solves the technical problem that the tumor drug resistance problem caused by the lack of antibodies for recognizing different HER2 epitopes in the prior art is difficult to overcome. The antibody (particularly the complementary determining region) sequence obtained by the technical scheme is different from any anti-HER 2 antibody in the prior art and is also different from the antibody obtained by the prior patent CN118580359B of the applicant, and can be used as an antibody for treating similar cancers and simultaneously solve the problem of tumor drug resistance caused by single drug. And because the number of the candidate monoclonal antibodies aiming at HER2 is increased, the antibody cocktail therapy for further researching related cancers becomes possible, so that the treatment efficiency is effectively improved, and the multi-target attack on cancer cells, the drug resistance risk reduction, the immune response enhancement and the like are realized.

Drawings

FIG. 1 shows the results of the flow cytometry screening of example 1.

FIG. 2 is an immunofluorescence assay for the optimal hybridoma cell line of example 1 (A: fluorescent image using mCherry marker HER 2; B: fluorescent image using iFluor 488-labeled hybridoma cell line; C: fusion image of A and B).

Fig. 3 is a structural alignment diagram of the anti-HER 2 humanized antibody and the murine antibody of example 3.

FIG. 4 is a sequence alignment of the humanized antibody heavy chain of the present embodiment of example 3 and the antibody heavy chain of the prior patent.

FIG. 5 is a sequence alignment of the humanized antibody light chain of the present embodiment of example 3 and the antibody light chain of the prior patent.

FIG. 6 is a graph showing the experimental results of the humanized antibody of example 4 for detecting HER2 protein on the surface of tumor cells.

FIG. 7 is a graph showing the experimental results of the effect of the humanized antibody of example 4 on killing breast cancer cells.

Fig. 8 is the results of an in vitro tumor killing assay for CAR-T cells of example 6.

Detailed Description

The present invention will be described in further detail with reference to examples, but embodiments of the present invention are not limited thereto. Unless otherwise indicated, the techniques used in the examples below are conventional and are well known to those skilled in the art, and the experimental procedures used are conventional and may be carried out according to the recombinant techniques described (see molecular cloning, laboratory Manual, 2 nd edition, cold spring harbor laboratory Press, cold spring harbor, N.Y.). And the materials, reagents and the like used are all commercially available.

Example 1:

referring to the prior patent CN118580359B (anti-HER 2 monoclonal antibody, gene, preparation method and application thereof), a murine cell line for stably expressing HER2 protein is prepared, the prepared cell line is used as an antigen for mouse immunization, and a plurality of hybridoma cell strains to be screened are obtained by screening in a conventional mode in the prior art.

From several hybridoma cell lines, hybridoma cells that selectively bind HER2 are preferred by flow cytometry. Subcloned hybridoma cell lines were screened by limiting dilution. The ability to bind HER2 protein was further examined using flow cytometry. Finally, an optimal hybridoma cell strain is obtained by screening (see FIG. 1, the red frame part shows the hybridoma cell strain with optimal binding capacity). The experimental data of fig. 1 demonstrate that the red-framed hybridoma cell line has the strongest HER2 binding capacity. While the binding of antibodies secreted by other hybridoma strains to be screened to HER2 protein is not ideal.

Through immunofluorescence experiments, we verified that antibodies of the hybridoma cell line can specifically bind to HER 2. The specific steps are as follows, spreading cell strain 3T3-HER2 over-expressing HER2 and green fluorescent protein mCherry in a 6-well plate 24h in advance, adding a proper amount of hybridoma supernatant with optimal binding force after cell wall-adhering growth, incubating for 30min at room temperature, washing off redundant supernatant, and adding 1:500 dilution488 Goat anti-human IgG (h+l) antibody, images were collected under a microscope after incubation for 30min at room temperature. The immunofluorescence image of fig. 2 shows that antibodies of the hybridoma cell line that have optimal binding capacity can specifically bind to HER 2.

Example 2 sequence analysis of anti-HER 2 hybridoma cell clones

The antibody Isotype secreted by the hybridoma cell line screened in example 1 was determined to be murine IgG2b by ELISA. The hybridoma cells were subjected to antibody variable region gene sequencing by the company Soujin Zhi Biotech Co.

Murine antibodies to HER2 antigen include heavy and light chains. The murine antibody heavy chain variable region amino acid sequence (SEQ ID NO.1, double underlined indicates the framework regions in the variable region, wavy lines indicate complementarity determining regions in the variable region):

Wherein the variable region of the murine heavy chain consists of complementarity-DETERMINING REGION (CDR) and Framework Regions (FR) in the order:

Heavy chain framework region 1 (VH_FR1): QIQLVQSGPELKKPGETVKISCKAS;

heavy chain complementarity determining region 1 (VH_CDR1): GYTFTTY;

heavy chain framework region 2 (VH_FR2): GMSWVKQAPGKGLKWMGWI;

Heavy chain complementarity determining region 2 (VH_CDR2): NTYSGV;

Heavy chain framework region 3 (VH_FR3): PTYADDFKGRFAFSLETSASTAYLQNNNLKNEDTATYF;

Heavy chain complementarity determining region 3 (VH_CDR3): CARRWLGYFDYW;

heavy chain framework region 4 (VH_FR4): GQGTTLTVSS;

The sequence of the complementarity determining region and the framework region is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

Murine antibody heavy chain variable region nucleotide sequence (SEQ ID NO.2, double underlined indicates the framework regions in the variable region, wavy lines indicate complementarity determining regions in the variable region):

the murine antibody light chain variable region amino acid sequence (SEQ ID NO.3, double underlined indicates the framework regions in the variable region, wavy lines indicate the complementarity determining regions in the variable region):

wherein the variable region of the murine antibody light chain consists of complementarity-DETERMINING REGION (CDR) and Framework Regions (FR) in the order:

light chain framework region 1 (VL_FR1): DIVMTQSHKFMSTSVGDRVSITCKAS;

light chain complementarity determining region 1 (VL_CDR1): QDVSTAVA;

Light chain framework region 2 (VL_FR2): WYQQKPGQSPKPLIY;

light chain complementarity determining region 2 (VL_CDR2): SASYRFT;

light chain framework region 3 (VL_FR3): GVPDRFTGSGSGTDFTFTISSVQAEDLAVYY;

light chain complementarity determining region 3 (VL_CDR3): CQQHYSTTWTF;

Light chain framework region 4 (VL_FR4): GGGTKLEIK;

The sequence of the complementarity determining region and the framework region is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

Murine antibody light chain variable region nucleotide sequence (SEQ ID NO.4, double underlined indicates the framework regions in the variable region, wavy lines indicate complementarity determining regions in the variable region):

Sequence comparison analysis showed that murine antibody is significantly different from the known HER2 antibody (and from HER2 antibody of applicant's prior patent CN 118580359B), indicating that it is a completely new HER2 antibody.

Example 3 humanization of anti-HER 2 murine monoclonal antibody

By analyzing large-scale bioinformatic data, we performed humanization of the murine antibody obtained in example 2 using an artificial intelligence algorithm, reducing the immunogenicity of the antibody and thus reducing the risk of immune responses in vivo. In addition, intelligent design and optimization of antibody frameworks and sequences is performed, including improved antibody stability, improved structure, reduced unnecessary modification sites, and expression system optimization, to ensure optimal biological activity in humans.

Humanized antibody heavy chain amino acid sequence (SEQ ID NO.5, double underlined indicates the framework regions in the variable region, wavy lines indicate complementarity determining regions in the variable region):

Wherein the variable region of the heavy chain of the human antibody consists of complementarity-DETERMINING REGION (CDR) and Framework Regions (FR) in this order:

Heavy chain framework region 1 (VH_FR1): QVQLVQSGSELKKPGASVKVSCKAS;

heavy chain complementarity determining region 1 (VH_CDR1): GYTFTSY;

Heavy chain framework region 2 (VH_FR2): AMNWVRQAPGQGLEWMGWI;

Heavy chain complementarity determining region 2 (VH_CDR2): NTNTGN;

heavy chain framework region 3 (VH_FR3): PTYAQGFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYY;

Heavy chain complementarity determining region 3 (VH_CDR3): CARRWLGYFDYW;

heavy chain framework region 4 (VH_FR4): GQGTLVTVSS;

The sequence of the complementarity determining region and the framework region is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

Human antibody heavy chain nucleotide sequence (SEQ ID NO.6, double underlined indicates the framework regions in the variable region, wavy lines indicate complementarity determining regions in the variable region):

GGAAGTACACACAGCTCAGACACAAACCCATAGAGAGGATTACAACAGTACTATCCG

GGTGGTCAGTGCCCTCCCCATCCAGCACCAGGACTGGATGAGTGGCAAGGAGTTCAA

ATGCAAGGTCAACAACAAAGACCTCCCATCACCCATCGAGAGAACCATCTCAAAAAT

TAAAGGGCTAGTCAGAGCTCCACAAGTATACATCTTGCCGCCACCAGCAGAGCAGTT

GTCCAGGAAAGATGTCAGTCTCACTTGCCTGGTCGTGGGCTTCAACCCTGGAGACATC

AGTGTGGAGTGGACCAGCAATGGGCATACAGAGGAGAACTACAAGGACACCGCACC

AGTCCTGGACTCTGACGGTTCTTACTTCATATACAGCAAGCTCGATATAAAAACAAGC

AAGTGGGAGAAAACAGATTCCTTCTCATGCAACGTGAGACACGAGGGTCTGAAAAATTACTACCTGAAGAAGACCATCTCCCGGTCTCCGGGTAAAtga.

Humanized antibody light chain amino acid sequence (SEQ ID NO.7, double underlined indicates the framework regions in the variable region, wavy line indicates the complementarity determining regions in the variable region):

MMSSAQFLGLLLLCFQGTRC-DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGQGTKVEIK-RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLN SWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC.

The variable region of the humanized antibody light chain consists of complementarity-DETERMINING REGION (CDR) and Framework Regions (FR) in the order:

Light chain framework region 1 (VL_FR1): DIQMTQSPSSLSASVGDRVTITCRAS;

Light chain complementarity determining region 1 (VL_CDR1): QSISSYLN;

light chain framework region 2 (VL_FR2): WYQQKPGKAPKLLIY;

light chain complementarity determining region 2 (VL_CDR2): AASSLQS;

light chain framework region 3 (VL_FR3): GVPSRFSGSGSGTDFTLTISSLQPEDFATYY;

light chain complementarity determining region 3 (VL_CDR3): CQQSYSTPLTF;

light chain framework region 4 (VL_FR4): GQGTKVEIK;

The sequence of the complementarity determining region and the framework region is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

Human antibody light chain nucleotide sequence (SEQ ID NO.8, double underlined indicates the framework regions in the variable region, wavy lines indicate complementarity determining regions in the variable region):

Sequence comparison analysis showed that the humanized antibody has a high degree of similarity to the spatial structure (fig. 3), and that the humanized antibody is highly similar to the human IgG2B gene sequence, and differs significantly from known HER2 antibodies (e.g., trastuzumab, pertuzumab, HER2 antibody of applicant's prior patent CN 118580359B). The humanized antibody of this scheme is shown in FIGS. 4 and 5 in detail in comparison with the heavy and light chains of the HER2 antibody of prior patent CN118580359B, and it can be seen that there are large sequence differences between the heavy chain CDRs and between the light chain CDRs. The novel antibody with the sequence structure different from that of the prior patent is screened out by the technical scheme, and the ideal antigen binding efficiency and the treatment effect can be obtained (see the efficacy experimental data later in detail). The humanized antibody obtained by the scheme enriches the diversity of the anti-HER 2 antibody and can be used as an effective means for coping with drug resistance. After the cancer cells develop resistance to a portion of the monoclonal antibodies, treatment can be continued with other antibodies having different CDR sequences. In addition, the novel humanized antibody provided by the patent can be used as a candidate of antibody cocktail therapy aiming at the antibody cocktail therapy. The inventors next further studied the combined effect of antibodies targeting murine cell lines expressing HER2 protein antigen.

Example 4 expression, purification and functional verification of humanized antibody against HER2

The construction, expression and purification of the anti-HER 2 humanized antibody expression vector are carried out by referring to the prior patent method, which is a conventional mode in the prior art, and are not repeated here. According to biological principles, the heavy chain and the light chain are assembled under the control of the respective signal peptide after in vivo synthesis, so that the complete antibody molecule is formed. The complete antibody molecule will be used for subsequent experimental validation.

(1) Humanized antibody detection of tumor cell surface HER2 protein

Different antibodies (humanized antibody, trastuzumab, control human IgG (Invitrogen, cat. No. 02-7102)) at the same concentration (10 μg/mL) were incubated with human breast cancer cell line SK-BR-3,4 ℃ for 30min, respectively, which highly expressed HER2 protein, using flow cytometry, followed by 1:500 dilution of the solution647 Goat anti-human IgG (h+l) antibodies, the mean fluorescence intensity MFI of each antibody bound to the cell surface HER2 molecule was determined. The detection results are shown in fig. 6 (sample size n=5), and the average fluorescence intensities of the humanized group and the trastuzumab group are not significantly different and are significantly stronger than those of the control group, so that the results indicate that the humanized antibody can specifically bind to HER2 protein on the surface of tumor cells.

(2) Humanized antibody mediated immune cell killing breast cancer cells

In order to prove that the humanized Antibody can effectively mediate the killing effect of immune cell tumor cells, human breast cancer cell lines AU565 and BT-474 which highly express HER2 protein are selected for in vitro Antibody-dependent cell-mediated cytotoxicity (ADCC-DEPENDENT CELL-Mediated Cytotoxicity) experiments. In vitro experiments adopt a luciferase method to detect the cell viability, NK cells and the AU565 and BT-474 cells are used for co-culture according to the proportion of 1:1, humanized antibodies, trastuzumab and control IgG (0.5 mug/sample) are respectively used for incubating the co-cultured cells for 48 hours, and then an enzyme-labeled instrument is used for detecting the cell viability and counting the cell killing rate. The test results are shown in fig. 7 (sample size n=5), indicating that humanized antibodies can effectively mediate immune cell killing of tumor cells and act similarly to trastuzumab.

Example 5 construction of CAR-T cells based on anti-HER 2 humanized antibody sequences

The CAR-T cells are conventional T cells modified by genetic engineering in the prior art, and the expression plasmid containing the CAR gene is transferred into the T cells mainly by a transgenic technology, so that the T cells express the CAR protein, and the immunotherapy effect of the T cells is improved.

(1) Construction of CAR-expressing lentiviral vector plasmids and lentiviral packaging

The humanized antibody sequences of the present protocol are integrated into a HER 2-targeting chimeric antigen receptor CAR (HER 2-CAR) of the present protocol. The HER2-CAR of this embodiment comprises, in order from extracellular to intracellular, a signal peptide (SIGNAL PEPTIDE), a light chain variable region (Iglv), a hinge region (HINGE CHAIN), a heavy chain variable region (Ighv), a CD28 extracellular region (CD 28 Extra), a CD28 transmembrane region (CD 28 TM), a CD28 intracellular region (CD 28 Cyto), a CD3zeta intracellular region (CD 3zeta Cyto). The original lentiviral expression vector (empty vector) used was pLVX-IRES-mCherry (Takara doctor technologies Co., ltd., product number: 631237). The technical scheme adopts the light chain variable region and the heavy chain variable region of the humanized antibody to construct an anti-HER 2-CAR, and expresses the HER2-CAR in T cells to form CAR-T cells (HER 2-CAR-T) for T cell therapy of targeting HER 2. HER2-CAR-T can recognize and bind to HER 2-expressing cells (cancer cells), and T cells further achieve a killing effect on HER 2-expressing cells. 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 underlined indicates the framework regions in the variable regions, wavy lines indicate the complementarity determining regions in the variable regions, the light chain variable region is forward and the heavy chain variable region is rearward):

GGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCC

GAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGG

CGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCAC

GATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGgccctgccccctcgc。

and (3) carrying out conventional lentivirus packaging on the constructed vector to obtain the lentivirus for subsequent experiments. The construction process can be found in applicant's prior patent CN118580359B.

(2) Isolated culture of primary mouse T lymphocytes

The spleen T lymphocytes of the mice are isolated, namely, spleen of C57BL/6 mice is taken and is ground by a filter screen, and spleen single cell suspension is obtained. Treating the cell suspension by using erythrocyte lysate, incubating on ice for 5-10min, adding PBS (phosphate buffer solution) with 3-5 times of volume, diluting at 1500rpm, centrifuging for 5min, washing cells once by using PBS, incubating anti-CD3 magnetic beads for 20min, separating CD3 ⁺ T cells by using a magnetic separation column, counting cells, and washing the cells 1-2 times by using PBS. CD3 ⁺ T lymphocytes represent whole T lymphocytes, including all types of T lymphocytes, including helper/inducer T lymphocytes (CD 3 ⁺CD4⁺), suppressor/cytotoxic T lymphocytes (CD 3 ⁺CD8⁺), and the like.

T cell in vitro stimulation culture, preparing T lymphocyte stimulation liquid, and the components and the proportion are shown in Table 5. Cells were resuspended in 12-well plates, 2 mL/well, with the prepared stimulation solution. Cell density was observed under the microscope, the stimulation solution was changed every day, stimulation was continued for three days, and after the third day the medium was changed to one containing only IL-2. The T lymphocyte stimulating liquid comprises 90% of lymphocyte culture medium, 10% of Fetal Bovine Serum (FBS), 100U/mL of green/streptomycin double antibody, 10ng/mL of IL-2 (mouse), 0.6 mug/mL of Anti-CD3 (mouse) and 0.3 mug/mL of Anti-CD28 (mouse).

(3) T cell viral infection

The third day stimulated T cells were plated in six well plates at a cell density of 2 x 10 ⁶ cells/well and virus-infected solutions were prepared and the reagents are shown in table 6. Six-well plate centrifugation (1000 g,37 ℃ C., 90 min) was then carried out, after 8-10h in a cell incubator, to change to normal medium (containing IL-2 in the medium). After 48h of T cell infection, the cells are subjected to fluorescence detection to detect whether transfection is successful. Thus, the construction of CAR-T cells was completed. For a single sample, the viral infection solution composition was 2mL of virus solution, 2mL of cell solution, 8. Mu.l of polybrene (1 mg/mL), 40. Mu.l of HEPES (1M; gibco).

In the process, the processes of constructing a lentiviral expression vector, packaging the lentivirus, acquiring T lymphocytes, infecting the T cell viruses and the like all adopt the conventional mode in the prior art. The difference of the embodiment compared with the prior art is that the heavy chain variable region and the light chain variable region of the humanized antibody obtained by the scheme are added on the basis of the sequence of the chimeric antigen receptor CAR of the prior art, so that the constructed CAR-T cell has the capability of targeting HER 2.

Example 6 in vitro tumor killing Capacity detection of CAR-T cells

In order to prove the in vitro tumor killing capability of the CAR-T cells designed and constructed based on the humanized antibody sequence of the scheme, the embodiment utilizes in vitro tumor cells and CAR-T cells (experimental group) co-culture experiments to detect the in vitro tumor killing capability of the CAR-T cells. And normal T cells were used as a control group. On day 0, the prepared CAR-T is inoculated into a 24-well plate at 1.0X10 ⁶/well, breast cancer SK-BR-3 and cervical cancer SK-OV-3 tumor cell lines expressing HER2 antigen and thyroid cancer cell line BHT-101 not expressing HER2 are respectively co-cultured with CAR-T cells according to the effective target ratio of 1:1, the number of tumor cells is detected after 48 hours, and the tumor lysis rate of the CAR-T cells is calculated. Experimental results are shown in fig. 8 (sample size n=5), which indicates that CAR-T cells designed and constructed based on the humanized antibody sequence of the present scheme have the capability of specifically killing tumor cells in vitro.

The foregoing is merely exemplary of the present application, and specific technical solutions and/or features that are well known in the art have not been described in detail herein. It should be noted that, for those skilled in the art, several variations and modifications can be made without departing from the technical solution of the present application, and these should also be regarded as the protection scope of the present application, which does not affect the effect of the implementation of the present application and the practical applicability of the patent. The protection scope of the present application is subject to the content of the claims, and the description of the specific embodiments and the like in the specification can be used for explaining the content of the claims.

Claims (8)

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 respectively: QVQLVQSGSELKKPGASVKVSCKAS, AMNWVRQAPGQGLEWMGWI, PTYAQGFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYY, 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. Use of a humanized antibody against HER2 antigen according to any one of claims 1 to 4 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. 6. Use of a humanized antibody against HER2 antigen according to any one of claims 1 to 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 to 4 in preparing chimeric antigen receptor T cells, characterized in that the nucleotide sequence of the chimeric antigen receptor of the chimeric antigen receptor T cells 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.