Bispecific chimeric antigen receptors targeting her2 and hla-g and uses thereof

Abstract

The application discloses a bispecific chimeric antigen receptor targeting HER2 and HLA-G and application thereof, and belongs to the tumor immunotherapy field.The chimeric antigen receptor comprises an extracellular domain, a hinge domain, a transmembrane domain and at least one intracellular domain, and the extracellular domain comprises an anti-HER2 single-chain antibody and an anti-HLA-G single-chain antibody.The bispecific chimeric antigen receptor of the application can effectively improve the killing signal of immune cells, and can effectively avoid the inhibition of the immunosuppressive signal on the function of immune cells, thereby delaying the recurrence of tumors, and has very important clinical application value.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of tumor immunotherapy, and particularly relates to a bispecific chimeric antigen receptor targeting HER2 and HLA-G and application thereof. BACKGROUND

[0002] In recent years, with the continuous development and cross penetration of oncology, immunology and molecular biology and other related disciplines, tumor immunotherapy has made rapid development in basic and clinical research, and has brought revolutionary changes to cancer treatment. The tumor cell immunotherapy represented by chimeric antigen receptor T-cell immunotherapy (CAR-T) has achieved great success in the treatment of hematological tumors, and the overall remission rate (ORR) for drug-resistant B-cell acute lymphoblastic leukemia can be as high as 93%. At the same time, CAR-T cell therapy for the treatment of solid tumors is also gradually advancing. There are as many as 22 different targets (even more than the number of hematological tumors) being tried to be applied to the treatment of solid tumors.

[0003] However, the preliminary results of most clinical trials are not satisfactory, and the effectiveness is much lower than that of the treatment of hematological tumors. The main reason is that the heterogeneity of solid tumors and the immunosuppressive tumor microenvironment lead to the inability of CAR-T cells to effectively kill all tumor cells.

[0004] How to make a breakthrough in this regard to improve the treatment effect of solid tumors has become the top priority in the research of immune cell therapy in recent years. SUMMARY

[0005] In view of the current situation that the tumor microenvironment affects the treatment effect of CAR-T and other immune cell therapies, the application aims to provide a bispecific chimeric antigen receptor targeting HER2 and HLA-G, a preparation method and application thereof. The bispecific chimeric antigen receptor can effectively improve the killing signal of NK or T cells and can effectively avoid the inhibition of immunosuppressive signals on the function of NK or T cells.

[0006] To achieve the above-mentioned purpose, the application specifically adopts the following scheme:

[0007] The first aspect of the present application provides a bispecific chimeric antigen receptor targeting HER2 and HLA-G, comprising an extracellular domain, a hinge domain, a transmembrane domain and at least one intracellular domain, wherein the extracellular domain comprises an anti-HER2 single-chain antibody and an anti-HLA-G single-chain antibody, the amino acid sequences of the CDRs of the heavy chain variable region of the anti-HLA-G single-chain antibody are shown in SEQ ID No. 16-SEQ ID No. 18, and the amino acid sequences of the CDRs of the light chain variable region are shown in SEQ ID No. 21-SEQ ID No. 23.

[0008] HER2 overexpression can promote tumor cell cycle, induce expression of pro-angiogenic related factors and expression of DNA damage repair genes, thereby promoting tumor progression. In view of the significant effect of trastuzumab in the treatment of breast cancer, gastric cancer and other tumors, the surface antigen HER-2 is considered as a relatively ideal target for immune cell therapy.

[0009] HLA-G is an atypical MHC class I molecule, which mainly functions to inhibit the function of cytotoxic immune cells, especially as a ligand for inhibitory NK cell receptors. HLA-G molecules can bind to NK cell surface receptors to inhibit NK cell killing activity. In addition to inhibiting NK cell killing activity, HLA-G molecules can inhibit NK cell migration. HLA-G can highly express various isoforms on the surface of solid tumor (such as melanoma, sarcoma and lymphoma) cells, and these HLA-G molecules enable tumor cells to escape the killing and lysis of NK cells and CTL cells, which is a new mechanism for tumor cells to escape immune surveillance. The occurrence of non-solid tumors, such as choriocarcinoma, may also be related to the inhibition of NK cell killing by HLA-G molecules.

[0010] Therefore, the bispecific chimeric antigen receptor targeting HER2 and HLA-G of the present application not only effectively improves the killing effect of NK and T cells, but also effectively avoids target escape, and can provide a more effective treatment option for various tumors.

[0011] In some embodiments of the present application, the amino acid sequence of the heavy chain variable region of the anti-HLA-G single-chain antibody is shown in SEQ ID No. 15, and the amino acid sequence of the light chain variable region is shown in SEQ ID No. 20.

[0012] In some more specific embodiments of the present application, the heavy chain variable region and the light chain variable region of the anti-HLA-G single chain antibody are connected by (G4S)3. Among them, the amino acid sequence of (G4S)3 is shown as SEQ ID No. 11, and thus the amino acid sequence of the anti-HLA-G single chain antibody is composed of SEQ ID No. 15, SEQ ID No. 11 and SEQ ID No. 20, as shown in SEQ ID No. 25.

[0013] In some embodiments of the present application, the amino acid sequences of the CDRs of the heavy chain variable region of the anti-HER2 single chain antibody are shown as SEQ ID No. 2~SEQ ID No. 4, and the amino acid sequences of the CDRs of the light chain variable region are shown as SEQ ID No. 7~SEQ ID No. 9, respectively.

[0014] In some specific embodiments of the present application, the amino acid sequence of the heavy chain variable region of the anti-HER2 single chain antibody is shown as SEQ ID No. 1, and the amino acid sequence of the light chain variable region is shown as SEQ ID No. 6.

[0015] In some more specific embodiments of the present application, the heavy chain variable region and the light chain variable region of the anti-HER2 single chain antibody are connected by (G4S)3. Thus, the amino acid sequence of the anti-HER2 single chain antibody is composed of SEQ ID No. 1, SEQ ID No. 11 and SEQ ID No. 6, as shown in SEQ ID No. 13.

[0016] In some preferred embodiments of the present application, the anti-HER2 single chain antibody and the anti-HLA-G single chain antibody are connected by the linker (G4S)3or (EAAAK)3to form a bispecific antibody against HER2 and HLA-G. Among them, the order of the anti-HER2 single chain antibody and the anti-HLA-G single chain antibody does not affect the effect of the chimeric antigen receptor, and the connection order can be anti-HER2 single chain antibody-linker-anti-HLA-G single chain antibody, or anti-HLA-G single chain antibody-linker-anti-HER2 single chain antibody.

[0017] When the anti-HER2 single chain antibody and the anti-HLA-G single chain antibody are connected by (G4S)3, the amino acid sequence of the obtained bispecific antibody is composed of SEQ ID No. 13, SEQ ID No. 11 and SEQ ID No. 25, as shown in SEQ ID No. 27, and of course, the skilled in the art can also connect in the order of SEQ ID No. 25, SEQ ID No. 11 and SEQ ID No. 13.

[0018] In some embodiments of the present application, the amino acid sequence of (EAAAK)3 is as shown in SEQ ID No. 29, when the anti-HER2 single-chain antibody and the anti-HLA-G single-chain antibody are connected by (EAAAK)3, the amino acid sequence of the obtained bispecific antibody consists of SEQ ID No. 13, SEQ ID No. 29 and SEQ ID No. 25, as shown in SEQ ID No. 31. Of course, the skilled in the art can also connect in the order of SEQ ID No. 25, SEQ ID No. 29 and SEQ ID No. 13.

[0019] In some embodiments of the present application, the extracellular domain further comprises a signal peptide, preferably, the signal peptide is CD8a, the amino acid sequence of the CD8a signal peptide is as shown in SEQ ID No. 33.

[0020] In some optional embodiments of the present application, the hinge domain is selected from one of CD8a, CD28, IgG1 and IgG4. Preferably, the hinge domain is CD8a or IgG1, wherein the amino acid sequence of the CD8a hinge domain is as shown in SEQ ID No. 35, the amino acid sequence of the IgG1 hinge domain is as shown in SEQ ID No. 37; more preferably, the hinge domain is CD8a.

[0021] In some optional embodiments of the present application, the transmembrane domain is selected from one of NKG2D, CD8, CD28, 2B4 and DNAM1. Preferably, the transmembrane domain is NKG2D or CD8, wherein the amino acid sequence of the NKG2D transmembrane domain is as shown in SEQ ID No. 39, the amino acid sequence of the CD8 transmembrane domain is as shown in SEQ ID No. 41; more preferably, the transmembrane domain is NKG2D.

[0022] In some embodiments of the present application, the intracellular domain comprises a costimulatory domain and a signaling domain.

[0023] Optionally, the co-stimulatory domain is selected from at least one of CD28, ICOS, OX40, CD27, CD137 (4-1BB), DAP10, DAP12 and 2B4. Preferably, the co-stimulatory domain is selected from one or two of CD28, CD137, DAP10 and 2B4, wherein the amino acid sequence of the DAP10 co-stimulatory domain is set forth in SEQ ID No. 43; the amino acid sequence of the CD137 (4-1BB) co-stimulatory domain is set forth in SEQ ID No. 45; the amino acid sequence of the 2B4 co-stimulatory domain is set forth in SEQ ID No. 47; the amino acid sequence of the CD28 co-stimulatory domain is set forth in SEQ ID No. 49.

[0024] In some embodiments of the present application, the signaling domain is CD3 zeta, the amino acid sequence of which is set forth in SEQ ID No. 51.

[0025] In some specific embodiments of the present application, the bispecific chimeric antigen receptor is GS·DAP10·BBz, the structure of which is as follows:

[0026] CD8a SP-HGH-CD8a Hinge-NKG2D TM-DAP10-CD137 CD-CD3 zeta SD,

[0027] wherein "-" is a connecting peptide or a peptide bond; "CD8a SP" represents a CD8a signal peptide; "HGH" represents a bispecific antibody obtained by connecting HER2 scFV and HLA-G scFV with (G4S)3; "CD8a Hinge" represents a CD8a hinge domain; "NKG2D TM" represents a NKG2D transmembrane domain; "DAP10-CD137 CD" represents a DAP10 co-stimulatory domain and a CD137 (4-1BB) co-stimulatory domain; "CD3 zeta SD" represents a CD3 zeta signaling domain. Preferably, the amino acid sequence of the GS·DAP10·BBz is a sequence obtained by connecting SEQ ID No. 33, SEQ ID No. 27, SEQ ID No. 35, SEQ ID No. 39, SEQ ID No. 43, SEQ ID No. 45 and SEQ ID No. 51 in order.

[0028] In some specific embodiments of the present application, the bispecific chimeric antigen receptor is GS·DAP10·BBz, the structure of which is as follows:

[0029] CD8a SP-HGH-IgG1 Hinge-NKG2D TM-2B4 CD-CD3 zeta SD,

[0030] wherein "-" is a connecting peptide or a peptide bond; "CD8a SP" represents a CD8a signal peptide; "HGH" represents a bispecific antibody obtained by connecting HER2 scFV and HLA-G scFV with (G4S)3; "IgG1 Hinge" represents an IgG1 hinge domain; "NKG2D TM" represents an NKG2D transmembrane domain; "2B4 CD" represents a 2B4 costimulatory domain; "CD3 zeta SD" represents a CD3 zeta signaling domain. Preferably, the amino acid sequence of the GS·2B4z is a sequence obtained by connecting SEQ ID No. 33, SEQ ID No. 27, SEQ ID No. 37, SEQ ID No. 39, SEQ ID No. 47 and SEQ ID No. 51 in order.

[0031] In still some specific embodiments of the present application, the bispecific chimeric antigen receptor is GS·28·BBz, which has the following structure:

[0032] CD8a SP-HGH-CD8a Hinge-CD28 TM-CD28-CD137 CD-CD3 zeta SD,

[0033] wherein "-" is a connecting peptide or a peptide bond; "CD8a SP" represents a CD8a signal peptide; "HGH" represents a bispecific antibody obtained by connecting HER2 scFV and HLA-G scFV with (G4S)3; "CD8a Hinge" represents a CD8a hinge domain; "CD28 TM" represents a CD28 transmembrane domain; "CD28-CD137 CD" represents a CD28 costimulatory domain and a CD137 (4-1BB) costimulatory domain; "CD3 zeta SD" represents a CD3 zeta signaling domain. Preferably, the amino acid sequence of the GS·28·BBz is a sequence obtained by connecting SEQ ID No. 33, SEQ ID No. 27, SEQ ID No. 35, SEQ ID No. 41, SEQ ID No. 49, SEQ ID No. 45 and SEQ ID No. 51 in order.

[0034] In still some specific embodiments of the present application, the bispecific chimeric antigen receptor is EA·DAP10·BBz, which has the following structure:

[0035] CD8a SP-HEH-CD8a Hinge-NKG2D TM-DAP10-CD137 CD-CD3 zeta SD,

[0036] wherein "-" is a connecting peptide or a peptide bond; "CD8a SP" represents a CD8a signal peptide; "HEH" represents a bispecific antibody obtained by connecting HER2 scFV and HLA-G scFV with (EAAAK)3; "CD8a Hinge" represents a CD8a hinge domain; "NKG2D TM" represents a NKG2D transmembrane domain; "DAP10-CD137 CD" represents a DAP10 costimulatory domain and a CD137 (4-1BB) costimulatory domain; "CD3 zeta SD" represents a CD3 zeta signaling domain. Preferably, the amino acid sequence of the EA·DAP10·BBz is a sequence obtained by connecting SEQ ID No. 33, SEQ ID No. 31, SEQ ID No. 35, SEQ ID No. 39, SEQ ID No. 43, SEQ ID No. 45 and SEQ ID No. 51 in order.

[0037] The second aspect of the present application provides a nucleic acid molecule encoding the bispecific chimeric antigen receptor of any one of the first aspect of the present application.

[0038] In some embodiments of the present application, the nucleotide sequence encoding the heavy chain variable region of HER2 scFV is shown as SEQ ID No. 5; the nucleotide sequence encoding the light chain variable region of HER2 scFV is shown as SEQ ID No. 10, preferably, the nucleotide sequence encoding HER2 scFV consists of SEQ ID No. 5, SEQ ID No. 12 and SEQ ID No. 10, which is shown as SEQ ID No. 14.

[0039] In some embodiments of the present application, the nucleotide sequence encoding the heavy chain variable region of HLA-G scFV is shown as SEQ ID No. 19; the nucleotide sequence encoding the light chain variable region of HLA-G scFV is shown as SEQ ID No. 24, preferably, the nucleotide sequence encoding HLA-G scFV consists of SEQ ID No. 19, SEQ ID No. 12 and SEQ ID No. 24, which is shown as SEQ ID No. 26.

[0040] In some embodiments of the present application, the nucleotide sequence encoding HGH consists of SEQ ID No. 14, SEQ ID No. 12 and SEQ ID No. 26, which is shown as SEQ ID No. 28.

[0041] In some embodiments of the application, the nucleotide sequence of the HEH consists of SEQ ID No. 14, SEQ ID No. 30, and SEQ ID No. 26, as set forth in SEQ ID No. 32.

[0042] In some embodiments of the application, the nucleotide sequence encoding the CD8a signal peptide is as set forth in SEQ ID No. 34.

[0043] In some embodiments of the application, the nucleotide sequence encoding the CD8a hinge domain is as set forth in SEQ ID No. 36.

[0044] In some embodiments of the application, the nucleotide sequence encoding the IgGl hinge domain is as set forth in SEQ ID No. 38.

[0045] In some embodiments of the application, the nucleotide sequence encoding the NKG2D transmembrane domain is as set forth in SEQ ID No. 40.

[0046] In some embodiments of the application, the nucleotide sequence encoding the CD8 transmembrane domain is as set forth in SEQ ID No. 42.

[0047] In some embodiments of the application, the nucleotide sequence encoding the DAP10 costimulatory domain is as set forth in SEQ ID No. 4.

[0048] In some embodiments of the application, the nucleotide sequence encoding the 4-1BB costimulatory domain is as set forth in SEQ ID No. 46.

[0049] In some embodiments of the application, the nucleotide sequence encoding the 2B4 costimulatory domain is as set forth in SEQ ID No. 48.

[0050] In some embodiments of the application, the nucleotide sequence encoding the CD28 costimulatory domain is as set forth in SEQ ID No. 50.

[0051] In some embodiments of the application, the nucleotide sequence encoding the CD3 zeta signaling domain is as set forth in SEQ ID No. 52.

[0052] In some embodiments of the application, the nucleotide sequence encoding the CAR is obtained by sequentially linking the nucleotide sequences encoding the individual parts.

[0053] For example, the nucleotide sequence encoding the GS·DAP10·BBz is obtained by sequentially connecting SEQ ID No. 34, SEQ ID No. 28, SEQ ID No. 36, SEQ ID No. 40, SEQ ID No. 44, SEQ ID No. 46 and SEQ ID No. 52.

[0054] For another example, the nucleotide sequence encoding the GS·2B4z is obtained by sequentially connecting SEQ ID No. 34, SEQ ID No. 28, SEQ ID No. 38, SEQ ID No. 40, SEQ ID No. 48 and SEQ ID No. 52.

[0055] For another example, the nucleotide sequence encoding the GS·28·BBz is obtained by sequentially connecting SEQ ID No. 34, SEQ ID No. 28, SEQ ID No. 36, SEQ ID No. 42, SEQ ID No. 50, SEQ ID No. 46 and SEQ ID No. 52.

[0056] For another example, the nucleotide sequence encoding the EA·DAP10·BBz is obtained by sequentially connecting SEQ ID No. 34, SEQ ID No. 32, SEQ ID No. 36, SEQ ID No. 40, SEQ ID No. 44, SEQ ID No. 46 and SEQ ID No. 52.

[0057] The third aspect of the present application provides a viral vector comprising the nucleic acid molecule of any one of the second aspect of the present application.

[0058] In some embodiments of the present application, the viral vector is a lentiviral vector and / or a retroviral vector, preferably a lentiviral vector.

[0059] The fourth aspect of the present application provides a recombinant lentivirus obtained by co-transfecting a mammalian cell with the viral vector of the third aspect of the present application and a packaging helper plasmid.

[0060] In some embodiments of the present application, the packaging helper plasmid comprises gag / pol, Rev and BAEV.

[0061] In some embodiments of the present application, the mammalian cell is selected from any one of 293 cell, 293T cell or 293F cell.

[0062] The fifth aspect of the present application provides an engineered immune cell expressing the bispecific chimeric antigen receptor of any one of the first aspect of the present application.

[0063] In the present application, the engineered immune cell is a T cell or an NK cell, preferably an NK cell, which is infected by the recombinant lentivirus of the fourth aspect of the present application, so that the nucleic acid molecule of the second aspect of the present application is expressed in the T cell or the NK cell. The engineered immune cell has good targeted killing effect, while being able to avoid the inhibition of HLA-G on its activity.

[0064] The sixth aspect of the present application provides the bispecific chimeric antigen receptor of any one of the first aspect of the present application, the nucleic acid molecule of the second aspect of the present application, the viral vector of the third aspect of the present application, the recombinant lentivirus of the fourth aspect of the present application, or the engineered immune cell of the fifth aspect of the present application for use in the preparation of a medicament for treating a tumor.

[0065] In the present application, the tumor is a solid tumor, preferably a HER2 expression positive solid tumor, including but not limited to breast cancer, ovarian cancer, endometrial cancer, fallopian tube cancer, gastric cancer and prostate cancer.

[0066] Advantages of the present application

[0067] Compared with the prior art, the present application has the following advantages:

[0068] The bispecific chimeric antigen receptor of the present application not only effectively improves the killing signal of immune cells (such as NK cells or T cells), but also effectively avoids the inhibition of immune suppression signal on the function of immune cells, thereby delaying the recurrence of tumor, and has very important clinical application value.

[0069] Using the CAR sequence of the present application to prepare CAR-NK cells, the CAR-NK cells can effectively kill cells expressing HER2 and / or HLA-G in vitro experiments, and can reduce the IFN-γ cytokine level of cells expressing HER2 and / or HLA-G.

[0070] The present application can significantly improve the killing power of CAR-NK cells by optimizing the linker of single-chain antibody and the structure of CAR. BRIEF DESCRIPTION OF DRAWINGS

[0071] Figure 1 The structure schematic diagrams of different anti-HER2 and HLA-G bispecific chimeric antigen receptors of Example 3 of the present application are shown.

[0072] Figure 2 The vector map of pCDH-SFFV-Luc-T2A-EGFP is shown.

[0073] Figure 3(a) shows a schematic diagram of the lentiviral expression vector pCDH-GS·DAP10·BBz element; Figure 3(b) shows a plasmid map of the lentiviral expression vector pCDH-GS·DAP10·BBz.

[0074] Figure 4 Figure 3(a) shows a schematic diagram of the lentiviral expression vector pCDH-GS·DAP10·BBz element; Figure 3(b) shows a plasmid map of the lentiviral expression vector pCDH-GS·DAP10·BBz.

[0075] Figure 5 Figure 3(a) shows a schematic diagram of the lentiviral expression vector pCDH-GS·DAP10·BBz element; Figure 3(b) shows a plasmid map of the lentiviral expression vector pCDH-GS·DAP10·BBz.

[0076] Figure 6 Figure 3(a) shows a schematic diagram of the lentiviral expression vector pCDH-GS·DAP10·BBz element; Figure 3(b) shows a plasmid map of the lentiviral expression vector pCDH-GS·DAP10·BBz.

[0077] Figure 7 Figure 3(a) shows a schematic diagram of the lentiviral expression vector pCDH-GS·DAP10·BBz element; Figure 3(b) shows a plasmid map of the lentiviral expression vector pCDH-GS·DAP10·BBz.

[0078] Figure 8 Figure 3(a) shows a schematic diagram of the lentiviral expression vector pCDH-GS·DAP10·BBz element; Figure 3(b) shows a plasmid map of the lentiviral expression vector pCDH-GS·DAP10·BBz.

[0079] Figure 9 Figure 3(a) shows a schematic diagram of the lentiviral expression vector pCDH-GS·DAP10·BBz element; Figure 3(b) shows a plasmid map of the lentiviral expression vector pCDH-GS·DAP10·BBz. DETAILED DESCRIPTION

[0080] In order to make the technical problems solved by the present application, technical solutions and beneficial effects more clearly understood, the present application will be further described in detail below with reference to the embodiments.

[0081] The following examples are put forth so as to demonstrate preferred embodiments of the application. Those in the art will recognize that the examples set forth herein demonstrate techniques that can be used in carrying out the application, and that the present application is capable of making use of other techniques for carrying out the application. The examples set forth herein will be appreciated and understood by those of ordinary skill in the art in view of the description of the application. Modifications of the examples set forth herein can readily be made by those with ordinary skill in the art, and are encompassed within the spirit of the application as disclosed herein.

[0082] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs, and the materials described herein will be understood to be incorporated by reference to the extent consistent with the description.

[0083] Those of ordinary skill in the art will appreciate and understand that many equivalents can be substituted for the specific embodiments of the application described herein without departing from the spirit or scope of the application. Accordingly, the application is not limited to the specific embodiments described herein.

[0084] The experimental methods in the following examples are routine unless otherwise specified. The instruments and equipment used in the following examples are routine laboratory instruments and equipment unless otherwise specified. The test materials used in the following examples are commercially available from routine biochemical reagent stores unless otherwise specified.

[0085] Example 1 Construction of Anti-HER2 Single Chain Antibody and Anti-HLA-G Single Chain Antibody

[0086] This example designs an anti-HER2 single chain antibody (scFv) (HER2 scFV) and an anti-HLA-G single chain antibody (HLA-G scFV).

[0087] The HER2 scFV is formed by connecting the heavy chain variable region and the light chain variable region via (G4S)3, wherein:

[0088] The amino acid sequence of the heavy chain variable region of the HER2 scFV is as follows:

[0089] QVQLVQSGHEVKQPGASVKVSCKASGYPFTNYGMNWVPQAPGQGLEWMGWINTSTGESTFADDFKGRFVFSMDTSASTAYLQISSLKAEDMAMYYCARWEVYHGYVPYWGQGTTVTVSS (SEQ ID No. 1)

[0090] The amino acid sequences of the CDRs of the heavy chain variable region of the HER2 scFV are as follows, respectively:

[0091] CDR-H1 : NYGMN (SEQ ID No. 2);

[0092] CDR-H2: WINTSTGESTFADDFKG (SEQ ID No. 3);

[0093] CDR-H3: WEVYHGYVPY (SEQ ID No. 4)

[0094] The nucleotide sequence of the HER2 scFV heavy chain variable region is as follows:

[0095] CAGGTGCAGCTGGTGCAGTCTGGCCATGAGGTGAAGCAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACCCTTTCACAAACTATGGAATGAACTGGGTGCCACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATTAACACCTCCACTGGAGAGTCAACATTTGCTGATGACTTCAAGGGACGGTTTGTCTTCTCCATGGACACCTCTGCCAGCACAGCATACCTGCAGATCAGCAGCCTAAAGGCTGAGGACATGGCCATGTATTACTGTGCGAGATGGGAGGTTACCACGGCTACGTTCCTTACTGGGGCCAAGGGACCACGGTCACCGTTTCCTCT (SEQ ID No. 5)

[0096] The amino acid sequence of the HER2 scFV light chain variable region is as follows:

[0097] DIVMTQSPDSLAVSLGERATNTCKASQDVYNAVAWYQQKPGQPPKLLIYSASSRYTGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHFRTPFTFGSGTKLEIK (SEQ ID No. 6)

[0098] The amino acid sequences of the CDRs of the HER2 scFV light chain variable region are as follows, respectively:

[0099] CDR-L1 : KASQDVYNAVA (SEQ ID No. 7);

[0100] CDR-L2: SASSRYT (SEQ ID No. 8);

[0101] CDR-L3: QQHFRTPFT (SEQ ID No. 9)

[0102] The nucleotide sequence of the HER2 scFV heavy chain variable region is as follows:

[0103] GACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCAACACCTGCAAGGCCAGTCAGGATGTGTATAATGCTGTTGCCTGGTACCAGCAGAAACCAGGACAGCCTCCTAAGCTGCTCATTTACTCGGCATCCTCCCGGTACACTGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTGTCAGCAACATTTTCGTACTCCATTCACGTTCGGCTCGGGGACAAAATTGGAGATCAAA (SEQ ID No. 10)

[0104] The amino acid sequence of (G4S)3 is as follows:

[0105] GGGGSGGGGSGGGGS (SEQ ID No. 11)

[0106] The nucleotide sequence of (G4S)3 is as follows:

[0107] GGCGGTGGCGGTTCTGGTGGCGGTGGCTCCGGCGGTGGCGGTTCT (SEQ ID No. 12)

[0108] Thus, the amino acid sequence of the HER2 scFV consists of SEQ ID No. 1, SEQ ID No. 11, and SEQ ID No. 6, as follows:

[0109] QVQLVQSGHEVKQPGASVKVSCKASGYPFTNYGMNWVPQAPGQGLEWMGWINTSTGES TFADDFKGRFVFSMDTSASTAYLQISSLKAEDMAMYYCARWEVYHGYVPYWGQGTTVT VSSGGGGSGGGGSGGGGSDIVMTQSPDSLAVSLGERATNTCKASQDVYNAVAWYQQKPG QPPKLLIYSASSRYTGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHFRTPFTFGSG TKLEIK (SEQ ID No. 13)

[0110] The nucleotide sequence of the HER2 scFV consists of SEQ ID No. 5, SEQ ID No. 12 and SEQ ID No. 10, as follows:

[0111] CAGGTGCAGCTGGTGCAGTCTGGCCATGAGGTGAAGCAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACCCTTTCACAAACTATGGAATGAACTGGGTGCCACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATTAACACCTCCACTGGAGAGTCAACATTTGCTGATGACTTCAAGGGACGGTTTGTCTTCTCCATGGACACCTCTGCCAGCACAGCATACCTGCAGATCAGCAGCCTAAAGGCTGAGGACATGGCCATGTATTACTGTGCGAGATGGGAGGTTTACCACGGCTACGTTCCTTACTGGGGCCAAGGGACCACGGTCACCGTTTCCTCTGGCGGTGGCGGTTCTGGTGGCGGTGGCTCCGGCGGTGGCGGTTCTGACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCAACACCTGCAAGGCCAGTCAGGATGTGTATAATGCTGTTGCCTGGTACCAGCAGAAACCAGGACAGCCTCCTAAGCTGCTCATTTACTCGGCATCCTCCCGGTACACTGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTGTCAGCAACATTTTCGTACTCCATTCACGTTCGGCTCGGGGACAAAATTGGAGATCAAA (SEQ ID No. 14)

[0112] The HLA-G scFv is also composed of a heavy chain variable region and a light chain variable region connected by (G4S)3, wherein:

[0113] The amino acid sequence of the HLA-G scFV heavy chain variable region is as follows:

[0114] QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYWMHWVRQAPGQGLEWMGWIYPSDSSTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCAREGLAGVFYFDYWGQGTTLTVSS (SEQ ID No. 15)

[0115] The amino acid sequences of the CDRs of the HLA-G scFV heavy chain variable region are as follows, respectively:

[0116] CDR-H1: DYWMH (SEQ ID No. 16);

[0117] CDR-H2: WIYPSDSSTNYAQKFQG (SEQ ID No. 17);

[0118] CDR-H3: EGLAGVFYFDY (SEQ ID No. 18)

[0119] The nucleotide sequence of the HLA-G scFV heavy chain variable region is as follows:

[0120] CAGGTGCAGCTGGTGCAGTCTGGCGCGGAAGTGAAGAAGCCGGGCGCCAGCGTGAAAGTCAGCTGCAAAGCGAGCGGCTATACCTTTACCGATTATTGGATGCACTGGGTGCGACAGGCCCCGGGCCAGGGCCTGGAATGGATGGGCTGGATTTATCCGAGCGATAGCAGCACCAACTATGCACAGAAGTTTCAGGGCAGGGTCACCATGACCAGGGATACGAGCATCAGCACCGCGTATATGGAGCTGAGCAGGCTGAGAAGCGACGATACGGCGGTGTATTATTGCGCGCGCGAAGGCCTGGCGGGCGTGTTTTATTTTGATTATTGGGGCCAGGGCACCACCCTGACCGTGAGCAGC (SEQ ID No. 19)

[0121] The amino acid sequences of the CDRs of the HLA-G scFV light chain variable region are as follows, respectively:

[0122] DIVMTQTPLSLSVTPGQPASISCKSSQSIVHRSGNTYLYWYLQKPGQSPQLLIYKVSSRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHLPPTFGGTTLEIK (SEQ ID No. 20)

[0123] The amino acid sequences of the CDRs of the HLA-G scFV heavy chain variable region are as follows, respectively:

[0124] CDR-H1 : KSSQSIVHRSGNTYLY (SEQ ID No. 21 );

[0125] CDR-H2: KVSSRFS (SEQ ID No. 22);

[0126] CDR-H3: FQGSHLPPT (SEQ ID No. 23)

[0127] The nucleotide sequence of the HLA-G scFV heavy chain variable region is as follows:

[0128] GATATTGTGATGACCCAGACTCCGCTCTCTCTGTCCGTGACTCCTGGCCAGCCGGCGAGCATTAGCTGCAAGAGCAGCCAGAGCATTGTGCATCGCAGCGGCAACACCTATCTGTATTGGTATCTGCAGAAACCGGGCCAGAGCCCGCAGCTGCTGATTTATAAAGTGAGCAGCCGCTTTAGCGGCGTGCCGGATCGCTTTAGCGGCAGCGGCAGCGGCACCGATTTTACCCTGAAAATTAGCCGCGTGGAAGCGGAAGATGTTGGCGTGTATTATTGCTTTCAGGGCAGCCATCTGCCGCCGACCTTTGGCGGCACCACCCTGGAAATTAAA (SEQ ID No. 24)

[0129] Thus, the amino acid sequence of the HLA-G scFV is composed of SEQ ID No. 15, SEQ ID No. 11 and SEQ ID No. 20, as follows:

[0130] QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYWMHWVRQAPGQGLEWMGWIYPSDSSTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCAREGLAGVFYFDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIVMTQTPLSLSVTPGQPASISCKSSQSIVHRSGNTYLYWYLQKPGQSPQLLIYKVSSRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHLPPTFGGTTLEIK (SEQ ID No. 25)

[0131] The nucleotide sequence of HLA-G scFV consists of SEQ ID No. 19, SEQ ID No. 12 and SEQ ID No. 24, as follows:

[0132] CAGGTGCAGCTGGTGCAGTCTGGCGCGGAAGTGAAGAAGCCGGGCGCCAGCGTGAAAGTCAGCTGCAAAGCGAGCGGCTATACCTTTACCGATTATTGGATGCACTGGGTGCGACAGGCCCCGGGCCAGGGCCTGGAATGGATGGGCTGGATTTATCCGAGCGATAGCAGCACCAACTATGCACAGAAGTTTCAGGGCAGGGTCACCATGACCAGGGATACGAGCATCAGCACCGCGTATATGGAGCTGAGCAGGCTGAGAAGCGACGATACGGCGGTGTATTATTGCGCGCGCGAAGGCCTGGCGGGCGTGTTTTATTTTGATTATTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCGGCGGTGGCGGTTCTGGTGGCGGTGGCTCCGGCGGTGGCGGTTCTGATATTGTGATGACCCAGACTCCGCTCTCTCTGTCCGTGACTCCTGGCCAGCCGGCGAGCATTAGCTGCAAGAGCAGCCAGAGCATTGTGCATCGCAGCGGCAACACCTATCTGTATTGGTATCTGCAGAAACCGGGCCAGAGCCCGCAGCTGCTGATTTATAAAGTGAGCAGCCGCTTTAGCGGCGTGCCGGATCGCTTTAGCGGCAGCGGCAGCGGCACCGATTTTACCCTGAAAATTAGCCGCGTGGAAGCGGAAGATGTTGGCGTGTATTATTGCTTTCAGGGCAGCCATCTGCCGCCGACCTTTGGCGGCACCACCCTGGAAATTAAA (SEQ ID No. 26)

[0133] Example 2 Construction of bispecific antibodies against HER2 and HLA-G

[0134] This example makes use of the single chain antibodies HER2 scFV and HLA-G scFV of Example 1 to construct bispecific antibodies against HER2 and HLA-G. Specifically, the HER2 scFV and HLA-G scFV are linked together using two different linkers to obtain two different bispecific antibodies.

[0135] (1) HER2 scFV and HLA-G scFV are connected by (G4S)3 to obtain HGH bispecific antibody

[0136] The amino acid sequence and nucleotide sequence of (G4S)3 are as described above, and thus the amino acid sequence of HGH consists of SEQ ID No. 13, SEQ ID No. 11 and SEQ ID No. 25, as follows:

[0137] QVQLVQSGHEVKQPGASVKVSCKASGYPFTNYGMNWVPQAPGQGLEWMGWINTSTGESTFADDFKGRFVFSMDTSASTAYLQISSLKAEDMAMYYCARWEVYHGYVPYWGQGTTVTVSSGGGGSGGGGSGGGGSDIVMTQSPDSLAVSLGERATNTCKASQDVYNAVAWYQQKPGQPPKLLIYSASSRYTGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHFRTPFTFGSGTKLEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYWMHWVRQAPGQGLEWMGWIYPSDSSTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCAREGLAGVFYFDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIVMTQTPLSLSVTPGQPASISCKSSQSIVHRSGNTYLYWYLQKPGQSPQLLIYKVSSRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHLPPTFGGTTLEIK (SEQ ID No. 27)

[0138] The nucleotide sequence of HGH consists of SEQ ID No. 14, SEQ ID No. 12 and SEQ ID No. 26, as follows:

[0139]

[0140] (2) HER2 scFV and HLA-G scFV are connected by (EAAAK)3 to obtain HEH bispecific antibody

[0141] The amino acid sequence of (EAAAK)3 is as follows:

[0142] EAAAKEAAAKEAAAK (SEQ ID No. 29)

[0143] The nucleotide sequence of (EAAAK)3 is as follows:

[0144] GAGGCAGCCGCCAAAGAGGCCGCCGCAAAGGAGGCTGCCGCCAAA (SEQ ID No. 30)

[0145] Thus, the amino acid sequence of HEH is composed of SEQ ID No. 13, SEQ ID No. 29 and SEQ ID No. 25, as follows:

[0146] QVQLVQSGHEVKQPGASVKVSCKASGYPFTNYGMNWVPQAPGQGLEWMGWINTSTGESTFADDFKGRFVFSMDTSASTAYLQISSLKAEDMAMYYCARWEVYHGYVPYWGQGTTVTVSSGGGGSGGGGSGGGGSDIVMTQSPDSLAVSLGERATNTCKASQDVYNAVAWYQQKPGQPPKLLIYSASSRYTGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHFRTPFTFGSGTKLEIKEAAAKEAAAKEAAAKQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYWMHWVRQAPGQGLEWMGWIYPSDSSTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCAREGLAGVFYFDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIVMTQTPLSLSVTPGQPASISCKSSQSIVHRSGNTYLYWYLQKPGQSPQLLIYKVSSRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHLPPTFGGTTLEIK (SEQ ID No. 31)

[0147] The nucleotide sequence of HEH consists of SEQ ID No. 14, SEQ ID No. 30 and SEQ ID No. 26, as follows:

[0148]

[0149] Example 3 Construction of Anti-HER2 and HLA-G Bispecific Chimeric Antigen Receptors

[0150] This example utilizes the bispecific antibody of Example 2 to construct different anti-HER2 and HLA-G bispecific chimeric antigen receptors (CAR), each of which includes a signal peptide (SP) sequence, an anti-HER2 and HLA-G bispecific antibody (HGH or HEH), a hinge sequence, a transmembrane (TM) sequence, a costimulatory domain (CD) sequence, and a signaling domain (SD) sequence. The specific selection of each part is as follows: Figure 1 and Table 1.

[0151] Table 1 Structure of each anti-HER2 and HLA-G bispecific chimeric antigen receptor

[0152]

[0153] The specific amino acid sequences and nucleotide sequences of each part other than the bispecific antibody are as follows:

[0154] (1) Signal peptide

[0155] Amino acid sequence of CD8a signal peptide:

[0156] MALPVTALLLPLALLLHAARP (SEQ ID No. 33)

[0157] Nucleotide sequence of CD8a signal peptide:

[0158] ATGGCACTGCCAGTGACAGCCCTGCTGCTGCCACTGGCCCTGCTGCTGCACGCAGCACGCCCT (SEQ ID No. 34)

[0159] (2) Hinge domain

[0160] Amino acid sequence of CD8a hinge domain:

[0161] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID No. 35)

[0162] Nucleotide sequence of CD8a hinge domain:

[0163] ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT (SEQ ID No. 36)

[0164] Amino acid sequence of IgG1 hinge domain:

[0165] PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID No. 37)

[0166] Nucleotide sequence of IgG1 hinge domain:

[0167] CCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA (SEQ ID No. 38)

[0168] (3) Transmembrane domain

[0169] Amino acid sequence of NKG2D transmembrane domain:

[0170] PFFFCCFIAVAMGIRFIIMVTIWS (SEQ ID No. 39)

[0171] Nucleotide sequence of NKG2D transmembrane domain:

[0172] CCATTTTTTTTCTGCTGCTTCATCGCTGTAGCCATGGGAATCCGTTTCATTATTATGGTAACAATATGGAGT (SEQ ID No. 40)

[0173] Amino acid sequence of the CD28 transmembrane domain:

[0174] FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID No. 41)

[0175] Nucleotide sequence of the CD28 transmembrane domain:

[0176] TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTG (SEQ ID No. 42)

[0177] (4) Costimulatory domain

[0178] Amino acid sequence of the DAP10 costimulatory domain:

[0179] LCARPRRSPAQDGKVYINMPGRG (SEQ ID No. 43)

[0180] Nucleotide sequence of the DAP10 costimulatory domain:

[0181] CTGTGCGCACGCCCACGCCGCAGCCCCGCCCAAGATGGCAAAGTCTACATCAACATGCCAGGCAGGGGC (SEQ ID No. 44)

[0182] Amino acid sequence of the CD137 (4-1BB) costimulatory domain:

[0183] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID No. 45)

[0184] Nucleotide sequence of the 4-1BB costimulatory domain:

[0185] AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG (SEQ ID No. 46)

[0186] Amino acid sequence of the 2B4 costimulatory domain:

[0187] WRRKRKEKQSETSPKEFLTIYEDVKDLKTRRNHEQEQTFPGGGSTIYSMIQSQSSAPTSQEPAYTLYSLIQPSRKSGSRKRNHSPSFNSTIYEVIGKSQPKAQNPARLSRKELENFDVYS (SEQ ID No. 47)

[0188] Nucleotide sequence of the 2B4 costimulatory domain:

[0189] TGGAGGAGAAAGAGGAAGGAGAAGCAGTCAGAGACCAGTCCCAAGGAATTTTTGACAATTTACGAAGATGTCAAGGATCTGAAAACCAGGAGAAATCACGAGCAGGAGCAGACTTTTCCTGGAGGGGGGAGCACCATCTACTCTATGATCCAGTCCCAGTCTTCTGCTCCCACGTCACAAGAACCTGCATATACATTATATTCATTAATTCAGCCTTCCAGGAAGTCTGGTTCCAGGAAGAGGAACCACAGCCCTTCCTTCAATAGCACTATCTATGAAGTGATTGGAAAGAGTCAACCTAAAGCCCAGAACCCTGCTCGATTGAGCCGCAAAGAGCTGGAGAACTTTGATGTTTATTCC (SEQ ID No. 48)

[0190] Amino acid sequence of the CD28 costimulatory domain:

[0191] RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID No. 49)

[0192] Nucleotide sequence of the CD28 costimulatory domain:

[0193] AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCC (SEQ ID No. 50)

[0194] (5) Signaling domain

[0195] Amino acid sequence of the CD3 zeta signaling domain:

[0196] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID No. 51)

[0197] Nucleotide sequence of the CD3 zeta signaling domain:

[0198] AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC (SEQ ID No. 52)

[0199] Example 4 Construction of expression vectors of anti-HER2 and HLA-G bispecific chimeric antigen receptors

[0200] Each CAR in Example 3 was subjected to full gene synthesis and cloned into a pUC57 vector, and the corresponding plasmid was extracted and subjected to enzyme digestion with QuickCut restriction endonuclease BamHI and XbaI (Takara Co.). The enzyme digestion system was as follows: 1 μg of pUC57 plasmid, 1 μL of each of BamHI and XbaI, 2 μL of 10x QuickCut Green Buffer, and water was added to 20 μL. The enzyme-digested product was separated by agarose gel electrophoresis and subjected to DNA fragment recovery with an agarose gel DNA fragment recovery kit (Takara Co.).

[0201] The pCDH-SFFV-Luc-T2A-EGFP vector was also subjected to enzyme digestion with BamHI and XbaI in the same way. Figure 2), the vector was obtained by replacing the original EF1a promoter sequence with the SFFV promoter sequence by treating pCDH-EF1a-eFFly-eGFP (addgene, Plasmid #104834) with double endonuclease ClaI and XbaI, and the digested vector fragment was separated and recovered by agarose gel electrophoresis. The recovered CAR fragment and the digested vector were ligated by T4 ligase (Takara).

[0202] The connection reaction system and conditions are as follows:

[0203] The CAR fragment and the pCDH-SFFV-Luc-T2A-EGFP vector digested by BamHI and XbaI were quantified, and then added to the system according to the molar ratio of vector: target band (CAR fragment) = 1:3, 1 μL of 10×T4 DNA Ligase Buffer, 1 μL of ligase, and water to 10 μL, and then connected at 16°C for 4 h or overnight. The ligation product was transformed into Escherichia coli After overnight culture at 37°C, single colonies were picked and expanded, and the plasmid of the positive clone was extracted using a plasmid extraction kit (Axygene) according to the kit operation manual. The correct vector was named according to the CAR name, specifically, the vectors were named as pCDH-GS·DAP10·BBz, pCDH-GS·2B4z, pCDH-GS·28·BBz and pCDH-EA·DAP10·BBz, respectively.

[0204] Take pCDH-GS·DAP10·BBz as an example, the elements of the lentiviral expression vector are shown in FIG. 3(a), and the vector map is shown in FIG. 3(b).

[0205] Example 5 Lentivirus packaging

[0206] HEK293T cells with 90% confluence were passaged into 10 mm cell culture dishes, and DMEM high glucose medium containing 10% fetal bovine serum was added, and the cells were cultured at 37°C, 5% CO2 for 24 h. When the cell confluence reached 30%-40%, the fresh serum-containing medium was replaced, and the cells were cultured for another 2 h. Then, the three packaging plasmids pBAEV-LESS (3.24 μg), pMDL-G / P-RRE (4.5 μg), and pRSV-REV (2.25 μg) and the pCDH-GS·DAP10·BBz plasmid (11.52 μg) were co-transfected into HEK293T cells by the calcium phosphate precipitation method. After 16 h of culture, the fresh medium was replaced. After 24 h, the HEK293T cells were observed under a fluorescence microscope, and the supernatant was collected after 48 h. The cell debris was removed by filtration with a 0.45 μm filter. The filtered supernatant was centrifuged at 20,000 r / min, 4°C for 3 h in an ultracentrifuge, and the virus particle precipitate was resuspended with serum-free 1640 medium, and stored in a –70°C refrigerator for standby use.

[0207] Example 6 NK cell purification and expansion

[0208] Fresh anticoagulated blood of 20 mL from healthy volunteers was used to isolate peripheral blood mononuclear cells (PBMCs) by using lymphocyte separation medium (GE Company). After counting the isolated cells, the cells were stimulated and cultured in a 6-well plate coated with CD16 at a density of 2.5×10 6 After 72 h of stimulation and culture, the cells were transferred to a normal 6-well plate for further expansion for 72 h. The cells were purified by using NK magnetic beads (Miltenyi Biotec Company), and induced to culture in 1640 (Thermo Scientific) medium containing 10% FBS and 200 IU / mL IL-2 to obtain purified NK cells. The proportion of CD3 and CD56 phenotypes was detected by flow cytometry.

[0209] The detection results are shown in FIG. 2. Figure 4 In FIG. 2, the horizontal axis represents CD3, and the vertical axis represents CD56. The cell population represented by CD3-negative and CD56-positive is NK cells, indicating that the proportion of NK cells prepared in this example is greater than 90%.

[0210] Example 7 Preparation of target HER2 / HLA-G bispecific CAR-NK cells

[0211] The NK cells prepared in Example 6 were taken, and about 2.5×10 6NK cells were inoculated into 24-well plates and mixed with an appropriate amount of virus supernatant prepared in Example 5 in the presence of Protamine sulfate (Sigma-Aldrich) at a final concentration of 8 μg / mL and BX795 (Sigma-Aldrich) at 1.5 μM, with a final volume of no more than 1 mL; supplemented with cytokines and centrifuged at 1000 g for 1 h at room temperature; after centrifugation, without removing the virus supernatant, the plates were incubated at 37°C, 5% CO2 for 4-6 h; after incubation, a second centrifugation was performed at 1000 g for 1 h at room temperature, and then 1 mL of fresh NK cell growth medium was added; the cells were maintained in the medium supplemented with cytokines for 2 days, thereby obtaining CAR-NK cells expressing a HER2 / HLA-G bispecific chimeric antigen receptor (HER2 / HLA-G CAR-NK), and further transfection efficiency identification was performed; the transfected NK cells were incubated with recombinant human Her-2-Fc protein or for 15 min at 4°C, washed and incubated with PE-labeled mouse anti-human anti-IgG1 antibody for 15 min at 4°C, and further subjected to flow detection.

[0212] The results are shown in Table 1, wherein the abscissa represents the scFv expression, indicating that the scFv is effectively expressed. Figure 5

[0213] Example 8 Killing of tumor cells by HER2 / HLA-G CAR-NK

[0214] K562 cells transfected with HER2 and HLA-G K562 HER2+ , K562 HLA-G+ , K562 HER2+HLA-G+ and K562 cells were adjusted to 1×10 6 / mL with culture medium, labeled with Calcein-AM at a final concentration of 5 μg / mL, incubated at 37°C for 1 h, washed with PBS three times, resuspended with phenol-free 1640 complete medium, and counted; 10000 tumor cells were added to each well of a 96-well round-bottom plate; Mock NK cells and HER2 / HLA-G CAR-NK cells were added at E:T ratios of 5:1, 2.5:1, 1.25:1 and 0.625:1; an additional group of tumor cells was treated with 2% Triton X-100 and an untreated group, centrifuged at 100 g for 5 min, and incubated at 37°C for 3 h, then centrifuged at 300 g for 5 min, and 100 μL was transferred to each well of a 96-well flat-bottom plate for detection of OD values.

[0215] The detection results are shown in Table 2. Figure 6 ​As shown, it indicates that HER2 / HLA-G CAR-NK cells can effectively kill cells expressing HER2 and / or HLA-G, and have no killing effect on double-negative cells K562.

[0216] Example 9 ELISA detection of IFN-γ levels in supernatant of cell lines co-cultured with HER2 / HLA-G CAR-NK cells

[0217] K562 cells transfected with HER2 and HLA-G K562 HER2+ , K562 HLA-G+ , K562 HER2+HLA-G+ After co-culturing K562 cells transfected with HER2 and HLA-G K562 HER2+HLA-G+ and K562 cells with Mock NK cells transfected with empty vector and HER2 / HLA-G CAR-NK cells at an E:T ratio of 2.5:1 for 12h, the supernatant was taken, and the concentration of IFN-γ in the supernatant was detected by ELISA.

[0218] The detection results are shown in Table 1. Figure 7 As shown, the IFN-γ cytokine levels in the supernatant of bispecific CAR-NK cells HER2 / HLA-G CAR-NK co-cultured with HER2 and HLA-G single-positive or double-positive target cells were significantly higher than that of double-negative cells K562.

[0219] Example 10 Killing effect of HER2 / HLA-G CAR-NK with different CAR structures on tumor cells and detection of IFN-γ levels

[0220] K562 cells transfected with HER2 and HLA-G K562 HER2+HLA-G+ and K562 cells were adjusted to 1x10 6 / mL with culture medium, and were labeled with Calcein-AM at a final concentration of 5μg / mL, incubated at 37℃ for 1h, washed with PBS for three times, and resuspended with complete culture medium without phenol red 1640, and counted; 10000 tumor cells were added to each well of a 96-well round-bottom plate; Mock NK cells transfected with empty vector and HER2 / HLA-G CAR-NK cells expressing different CARs were added at an E:T ratio of 5:1, 2.5:1, 1.25:1 and 0.625:1; another group of tumor cells was treated with 2% Triton X-100, and another group was not treated; after centrifugation at 100g for 5min and co-culturing at 37℃ for 3h, centrifugation was performed at 300g for 5min, and 100μL was transferred to a 96-well flat-bottom plate for detection of OD value. 2HER2+HLA-G+ After co-culturing K562 cells transfected with HER2 and HLA-G K562 HER2+HLA-G+ and K562 cells with Mock NK cells transfected with empty vector and HER2 / HLA-G CAR-NK cells with different CAR structures at an E:T ratio of 2.5:1 for 12h, the supernatant was taken, and the concentration of IFN-γ in the supernatant was detected by ELISA.

[0221] Test results such as Figure 8 As shown in the results, CAR-NK expressing GS·DAP10·BBz CAR structure had the most significant cell killing effect, and the level of IFN-γ cytokine in the supernatant was also the highest, indicating that the CAR structure composed of CD8α hinge domain, NKG2D transmembrane domain, DAP10 and 4-1BB (CD137) costimulatory domain and CD3ζ signaling domain in series had significantly better effect.

[0222] Example 11 Detection of the killing effect of HER2 / HLA-G CAR-NK cells with different linkers on tumor cells and IFN-γ levels

[0223] K562 cells transfected with HER2 and HLA-G HER2+HLA-G+ The K562 cells were adjusted to 1×10 6 Cells were labeled with Calcein-AM at a final concentration of 5 μg / mL, incubated at 37°C for 1 hour, washed three times with PBS, and resuspended in phenol red-free 1640 complete medium. The cells were counted and adjusted to 10,000 tumor cells / well and added to a 96-well round-bottom plate. Mock NK cells transfected with empty vectors and bispecific HER2 / HLA-G CAR-NK cells constructed with different linkers (G4S)3 and (EAAAK)3 were added at E:T ratios of 5:1, 2.5:1, 1.25:1, and 0.625:1. Tumor cells were then added to a group of 2% Triton X-100 and an untreated group, centrifuged at 100g for 5 minutes, co-cultured at 37°C for 3 hours, and centrifuged at 300g for 5 minutes. 100 μL of each well was transferred to a 96-well flat-bottom plate to measure the OD value. At the same time, K562 cells transfected with HER2 and HLA-G were also added. HER2+HLA-G+ K562 cells were co-cultured with empty vector-transfected MockNK cells and HER2 / HLA-G CAR-NK cells with different linkers at an E:T ratio of 2.5:1 for 12 h, and the supernatant was collected to detect the concentration of IFN-γ in the supernatant by ELISA.

[0224] Test results such as Figure 9 As shown in the results, the GS·DAP10·BBz CAR-NK cells with (G4S)3 linker were significantly better at killing target cells than the EA·DAP10·BBz CAR-NK cells with (EAAAK)3 linker, and the IFN-γ cytokine level in the supernatant was also higher, indicating that the anti-HER2 and HLA-G bispecific antibody was significantly better connected with the (G4S)3 linker.

[0225] All documents referred to in the present application are incorporated herein by reference as if each were individually incorporated. In addition, it is to be understood that the application can be carried out by specifically different embodiments and that embodiments can be practiced without the specific details that are set forth herein. Further, it should be noted that, in this application, "exemplary" or "for example" is used on a purely illustrative basis to aid the reader in understanding the application. Any process steps, or sequences of steps, or examples, or embodiments, or materials, or components, or compounds, or elements, or techniques described herein are understood not to be limiting, but are exemplary.

Claims

1. A bispecific chimeric antigen receptor targeting HER2 and HLA-G, characterized in that: The chimeric antigen receptor comprises an extracellular domain, a hinge domain, a transmembrane domain, and at least one intracellular domain. The extracellular domain comprises an anti-HER2 single-chain antibody and an anti-HLA-G single-chain antibody. The amino acid sequences of the heavy chain variable regions (CDRs) of the anti-HLA-G single-chain antibodies are shown in SEQ ID No. 16 to SEQ ID No. 18, respectively, and the amino acid sequences of the light chain variable regions (CDRs) are shown in SEQ ID No. 21 to SEQ ID No. 23, respectively.

2. The bispecific chimeric antigen receptor targeting HER2 and HLA-G according to claim 1, wherein: The amino acid sequence of the heavy chain variable region of the anti-HLA-G single-chain antibody is shown in SEQ ID No. 15, and the amino acid sequence of the light chain variable region is shown in SEQ ID No.

20.

3. The bispecific chimeric antigen receptor according to claim 1 or 2, wherein The amino acid sequences of the heavy chain variable region CDRs of the anti-HER2 single-chain antibodies are shown in SEQ ID No. 2 to SEQ ID No. 4, respectively, and the amino acid sequences of the light chain variable region CDRs are shown in SEQ ID No. 7 to SEQ ID No. 9, respectively.

4. The bispecific chimeric antigen receptor according to claim 3, wherein The amino acid sequence of the heavy chain variable region of the anti-HER2 single-chain antibody is shown in SEQ ID No. 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID No.

6.

5. The bispecific chimeric antigen receptor according to claim 1 or 2, characterized in that The anti-HER2 single-chain antibody and the anti-HLA-G single-chain antibody are linked using (G4S)3 or (EAAAK)3.

6. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the bispecific chimeric antigen receptor according to any one of claims 1 to 5.

7. A viral vector, characterized in that Comprising the nucleic acid molecule of claim 6.

8. A recombinant lentivirus, characterized in that The viral vector according to claim 7 is co-transfected with a packaging helper plasmid into mammalian cells to obtain the virus vector.

9. An engineered immune cell, characterized in that: The immune cell expresses the bispecific chimeric antigen receptor according to any one of claims 1 to 5, and the engineered immune cell is a T cell or a NK cell.

10. Use of the bispecific chimeric antigen receptor according to any one of claims 1 to 5, the nucleic acid molecule according to claim 6, the viral vector according to claim 7, the recombinant lentivirus according to claim 8, or the engineered immune cell according to claim 9 in the preparation of a medicament for treating a tumor, wherein the tumor is breast cancer, ovarian cancer, endometrial cancer, or gastric cancer.

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