Antibodies or antibody fragments targeting b7-h3, and their use in the field of chimeric antigen receptor immune cell therapy

Abstract

This invention relates to an antibody or antibody fragment targeting B7-H3, a chimeric antigen receptor based on the antibody or antibody fragment, and chimeric antigen receptor-immune cells, and their use in the treatment of tumors. Specifically, the antibody or antibody fragment comprises a heavy chain variable region VH and a light chain variable region VL. VH includes VH-CDR1 as in SEQ ID NO:9, VH-CDR2 as in SEQ ID NO:10, and VH-CDR3 as in SEQ ID NO:11, and VL includes VL-CDR1 as in SEQ ID NO:12, VL-CDR2 as in SEQ ID NO:13, and VL-CDR3 as in SEQ ID NO:14; or VH includes VH-CDR1 as in SEQ ID NO:15, VH-CDR2 as in SEQ ID NO:16, and VH-CDR3 as in SEQ ID NO:17, and VL includes VL-CDR1 as in SEQ ID NO:18, VL-CDR2 as in SEQ ID NO:19, and VL-CDR3 as in SEQ ID NO:20.

Description

Technical Field

[0001] This invention relates to an antibody or antibody fragment targeting B7-H3, a chimeric antigen receptor (CAR) based on the antibody or antibody fragment, and CAR-immune cells, as well as their use in the treatment of tumors. Background Technology

[0002] As a form of immunotherapy, monoclonal antibodies have shown great therapeutic potential in treating malignant diseases and have attracted widespread attention. Anti-PD-1 and anti-PD-L1 antibody therapies, such as those targeting the B7-H1 (PD-L1) / PD-1 signaling pathway, have already achieved groundbreaking applications in cancer treatment. The key to the success of a monoclonal antibody therapy lies in selecting appropriate targets or immune checkpoints to reactivate and enhance the subject's own immune response.

[0003] B7-H3 (also known as CD276) is a type I transmembrane protein composed of 316 amino acids. In the human body, based on differences in its extramembrane domains, B7-H3 can be divided into two different forms: 2Ig-B7-H3 and 4Ig-B7-H3. For 2Ig-B7-H3, its extramembrane terminus contains a pair of immunoglobulin V-like domains (IgV-domain) and immunoglobulin C-like domains (IgC-domain). The extramembrane terminus of 4Ig-B7-H3 contains two pairs of IgV- and IgC-domains. B7-H3 belongs to the B7 protein family; normal tissues do not express or express B7-H3 at low levels. However, B7-H3 is expressed in a variety of tumor tissues (more than 60% of tumors express B7-H3), including pancreatic cancer (positive rate 77.8%), colorectal cancer (positive rate 63.8%), gastric cancer (positive rate 69.2%), lung cancer (positive rate 69.5%), prostate cancer (positive rate 93%), ovarian cancer (positive rate 73.1%), etc. It is also highly expressed in a variety of rare tumors, and therefore it has been called "tumor-associated antigen". It is a pan-tumor universal drug target with great potential[1].

[0004] Like other members of the B7 protein family, such as PD-L1, B7-H3 also functions as an immunosuppressive molecule. Blocking the binding of B7-H3 using neutralizing monoclonal antibodies (mAbs) can enhance the cytotoxic function of NK cells and CD8 cytotoxic T cells, promote their infiltration into tumor cells, reduce tumor burden, and improve the survival of tumor-bearing mice. [2] .

[0005] Therefore, given the widespread high expression of B7-H3 in tumor tissues and low expression in healthy tissues, as well as the function of B7-H3 in the tumor microenvironment, the clinical therapeutic potential of B7-H3 has attracted considerable attention. Several clinical trials targeting B7-H3 have already been initiated. Among these, more than 20 clinical trials are based on mAbs, including neutralizing antibodies, antibody-drug conjugates (ADCs), bispecific antibodies, antibody-dependent cell-mediated cytotoxicity (ADCC), and NK cell connector-linked antibodies.

[0006] Neutralizing antibodies can be used to block the binding of ligands and receptors, thereby inhibiting signal transduction and affecting related cellular functions. Blocking B7-H3 signaling with neutralizing antibodies can release the function of immune cells affected by B7-H3 signaling, enhancing their anti-tumor capabilities. This therapeutic potential has been demonstrated in various solid tumors, including ovarian cancer, melanoma, and colorectal cancer. [3-5] .

[0007] Antibody-drug conjugates (ADCs) are drugs that specifically recognize antigens and are conjugated with small molecule drugs with cytotoxic effects. They are characterized by high specificity, good safety, and high efficiency in killing tumor cells. Research teams have demonstrated the effectiveness of ADCs targeting B7-H3 in various solid tumor models and their safety in primates. [6] .

[0008] Bispecific antibodies combine two antibodies that target different sites. Clinically approved bispecific antibodies that simultaneously target B7-H3 and CD3 have been approved for the treatment of solid tumors (clinical trial No: NCT03406949). This bispecific antibody can recruit activated T cells to B7-H3-positive tumor tissue, helping T cells recognize and kill tumor cells.

[0009] Advanced antibody-dependent cell-mediated cytotoxicity (ADCC) targeting B7-H3 has also made progress in solid tumors. A research team designed a monoclonal antibody targeting B7-H3 and engineered its Fc receptor, which can enhance the killing effect of cytotoxic immune cells on B7-H3-positive tumor cells via the Fc receptor. Its safety has also been verified in primates. [7] .

[0010] Linking antibodies targeting B7-H3 with NK cell receptor (CD16) antibodies can enhance the recognition and killing function of NK cells against B7-H3-positive tumor cells. Another team further modified the antibody-protein complex by linking an interleukin-15 (IL15) protein to the CD16 and B7-H3 antibodies. IL15 can further promote NK cell activity. This antibody-protein complex has shown excellent efficacy in the treatment of solid tumors in vitro and in animal models. [8] .

[0011] In addition, chimeric antigen receptor T-cell immunotherapy (CAR-T) targeting B7-H3 has also made some progress in solid tumors, such as glioma. [9] Atypical teratoma-like rhabdomyosarcoma

[10] Anaplastic meningioma

[11] .

[0012] Although several immunotherapies targeting B7-H3 have been developed globally (including B7-H3 monoclonal antibodies and CAR-B7-H3-T therapy), and some early positive therapeutic effects have been achieved, this target immunotherapy still faces challenges such as a limited range of antibody selection and limited clinical efficacy. Further exploration of new antibodies and treatment methods is still needed.

[0013] In addition, CAR-T therapy is a revolutionary immunotherapy that involves gene modification of αβT cells, and it has made significant clinical progress over the past decade. To date, six CAR-T therapies have been approved by the FDA, and two have been approved in China. These eight CAR-T drugs all target CD19 or BCMA, treating B-cell-related hematological malignancies, and have high response and disease remission rates. However, CAR-T therapy also has many drawbacks, such as limited efficacy in solid tumors, the inability to use allogeneic therapy and readily available supply, and high cost.

[0014] γδT cells can address these issues to some extent. Classified by cell type, γδT cells are T cells expressing the γδT cell receptor (TCR). They are a special type of immune cell, exhibiting characteristics of both innate and adaptive immunity. Their recognition of antigens and pathogens is independent of the major histocompatibility complex (MHC), allowing for allogeneic therapy without any modification and without triggering reactions such as GvHD. Numerous studies have reported that γδT cells possess broad-spectrum anti-tumor capabilities, exhibiting stronger infiltration and anti-tumor effects in solid tumors. CAR-modified γδT cells can further enhance their targeting and tumor-killing effects. [12，13]However, there are no studies or reports on the application of B7-H3 antibodies to CAR-γδT.

[0015] Cited literature 1. Kontos, F., et al., B7-H3: An Attractive Target for Antibody-based Immunotherapy. Clin Cancer Res, 2021.27(5): p.1227-1235.

[0016] Cited literature 2. Lee, Y.-H., et al., Inhibition of the B7-H3 immune checkpoint limits tumor growth by enhancing cytotoxic lymphocyte function. Cell research, 2017.27(8): p.1034-1045.

[0017] Cited literature 3. Cai, D., et al., Tumor-expressed B7-H3 mediates the inhibition of antitumor T-cell functions in ovarian cancer insensitive to PD-1 b1ockadetherapy. Cell Mol Immunol, 2020.17(3): p.227-236.

[0018] Cited literature 4. Lee, YH, et al., Inhibition of the B7-H3 immune checkpointlimits tumor growth by enhancing cytotoxic lymphocyte function. Cell Res, 2017.27(8): p.1034-1045.

[0019] Cited literature 5. Lu, H., et al., B7-H3 inhibits the IFN-gamma-dependent cytotoxicity of Vgamma9Vdelta2 T cells against colon cancer cells. Oncoimmunology, 2020.9(1): p.1748991.

[0020] Citation 6. Scribner, J.A., et al., Preclinical Development of MGC018, a Duocarmycin-based Antibody-drug Conjugate Targeting B7-H3 for Solid Cancer. Mol Cancer Ther, 2020. 19(11): p. 2235-2244.

[0021] Citation 7. Loo, D., et al., Development of an Fc-enhanced anti-B7-H3 monoclonal antibody with potent antitumor activity. Clin Cancer Res, 2012. 18(14): p. 3834-45.

[0022] Citation 8. Vallera, D.A., et al., NK-Cell-Mediated Targeting of Various Solid Tumors Using a B7-H3 Tri-Specific Killer Engager In Vitro and In Vivo. Cancers, 2020. 12(9).

[0023] Citation 9. Tang, X., et al., Administration of B7-H3 targeted chimeric antigen receptor-T cells induce regression of glioblastoma. Signal Transduct Target Ther, 2021. 6(1): p. 125.

[0024] Citation 10. Theruvath, J., et al., Locoregionally administered B7-H3-targeted CAR T cells for treatment of atypical teratoid / rhabdoid tumors. Nat Med, 2020. 26(5): p. 712-719.

[0025] Cited literature 11. Tang,

[0026] Cited literature 12. Ang, WX, et al., Electroporation of NKG2D RNA CAR ImprovesVγ9Vδ2 T Cell Responses against Human Solid Tumor Xenografts. MolecularTherapy-Oncolytics, 2020.17: p.421-430.

[0027] Cited literature 13. Rozenbaum, M., et al., Gamma-Delta CAR-T Cells Show CAR-Directed and Independent Activity Against Leukemia. Frontiers in Immunology, 2020.11. Summary of the Invention

[0028] To address the aforementioned issues, this invention provides novel B7-H3 antibodies or antibody fragments, which have been successfully used in the field of CAR-ααβT / CAR-γδT therapy. This brings new options and feasibility to the clinical treatment and detection of the B7-H3 target. Furthermore, this invention also solves problems in CAR immunotherapy such as limited efficacy against solid tumors, inability to use allogeneic therapy and readily available supply, and high cost.

[0029] The first aspect of the present invention provides an isolated antibody or antibody fragment that specifically binds to B7-H3, comprising a heavy chain variable region VH and a light chain variable region VL, wherein the heavy chain variable region VH comprises: VH-CDR1 as shown in the amino acid sequence of SEQ ID NO: 9, VH-CDR2 as shown in the amino acid sequence of SEQ ID NO: 10, and VH-CDR3 as shown in the amino acid sequence of SEQ ID NO: 11; and the light chain variable region VL comprises: VL-CDR1 as shown in the amino acid sequence of SEQ ID NO: 12, VL-CDR2 as shown in the amino acid sequence of SEQ ID NO: 13, and VL-CDR3 as shown in the amino acid sequence of SEQ ID NO: 14.

[0030] SEQ ID NO:9 NYWIN

[0031] SEQ ID NO: 10 RIAPGTISTYYNEKFKG

[0032] SEQ ID NO:11 QDNYFIN

[0033] SEQ ID NO: 12 :SASSSISSSDLH

[0034] SEQ ID NO: 13 :GTSNLAS

[0035] SEQ ID NO: 14 :QQWFSYPFT

[0036] A second aspect of the present invention provides an isolated antibody or antibody fragment that specifically binds to B7-H3, comprising a heavy chain variable region VH and a light chain variable region VL, wherein the heavy chain variable region VH includes VH-CDR1 as shown in the amino acid sequence of SEQ ID NO: 15, VH-CDR2 as shown in the amino acid sequence of SEQ ID NO: 16, and VH-CDR3 as shown in the amino acid sequence of SEQ ID NO: 17; and the light chain variable region VL includes VL-CDR1 as shown in the amino acid sequence of SEQ ID NO: 18, VL-CDR2 as shown in the amino acid sequence of SEQ ID NO: 19, and VL-CDR3 as shown in the amino acid sequence of SEQ ID NO: 20.

[0037] S EQ ID NO: 15 RYDMS

[0038] SEQ ID NO: 16 :TISDDGRHTYDRDSVKG

[0039] SEQ ID NQ: 17 :HRAITTARFDY

[0040] SEQ ID NO: 18 :KASQDIYSNIG

[0041] SEQ ID NQ: 19 HGTNLED

[0042] SEQ ID NO: 20 :LQYVQFPYT

[0043] A third aspect of the present invention provides a separated antibody or antibody fragment that specifically binds to B7-H3 as described in the first and second aspects, wherein the aforementioned heavy chain variable region VH is selected from any one of the following: the amino acid sequence shown in SEQ ID NO: 1; an amino acid sequence having 80% or more, or 85% or more, or 88% or more, or 90% or more, or 93% or more, or 95% or more, or 98% or more homology with the amino acid sequence shown in SEQ ID NO: 2 outside the CDR region; an amino acid sequence shown in SEQ ID NO: 2; and an amino acid sequence having 80% or more, or 85% or more, or 88% or more, or 90% or more, or 93% or more, or 95% or more, or 98% or more homology with the amino acid sequence shown in SEQ ID NO: 2 outside the CDR region.

[0044] SEQ ID NO: 1

[0045]

[0046] SEQ ID NO: 2

[0047]

[0048] The fourth aspect of the present invention provides a separated antibody or antibody fragment that specifically binds to B7-H3 as described in the first or second aspect, wherein the light chain variable region VL is selected from any one of the following: the amino acid sequence shown in SEQ ID NO: 3; an amino acid sequence having 80% or more, 85% or more, 88% or more, 90% or more, 93% or more, 95% or more, or 98% or more homology to the amino acid sequence shown in SEQ ID NO: 4, and an amino acid sequence having 80% or more, 85% or more, 88% or more, 90% or more, 93% or more, 95% or more, or 98% or more homology to the amino acid sequence shown in SEQ ID NO: 4, outside the CDR region.

[0049] SEQ ID NO: 3

[0050]

[0051] S EQ ID NO: 4

[0052]

[0053] The fifth aspect of the present invention provides a specific antibody or antibody fragment that binds to B7-H3, comprising: (i) the heavy chain variable region VH shown in SEQ ID NO: 1 and the light chain variable region VL shown in SEQ ID NO: 3, or (ii) the heavy chain variable region VH shown in SEQ ID NO: 2 and the light chain variable region VL shown in SEQ ID NO: 4.

[0054] The sixth aspect of the present invention provides a specific antibody or antibody fragment that binds to B7-H3 as described in any one of the first to fifth aspects above, wherein the antibody or antibody fragment is a genetically engineered antibody or antibody fragment.

[0055] The seventh aspect of the present invention provides a separated antibody or antibody fragment that specifically binds to B7-H3 as described in any one of the first to sixth aspects above, wherein the antibody or antibody fragment is scFv.

[0056] The eighth aspect of the present invention provides a chimeric antigen receptor comprising: an antibody or antibody fragment that specifically binds to B7-H3 as described in the seventh aspect above.

[0057] The ninth aspect of the present invention provides a chimeric antigen receptor as described in the eighth aspect of the present invention, comprising: the antibody or antibody fragment described in the seventh aspect above, and a transmembrane region fused to the carboxyl terminus of the antibody or antibody fragment.

[0058] The tenth aspect of the present invention provides a chimeric antigen receptor as described in the eighth or ninth aspect above, comprising: an antibody or antibody fragment as described in the seventh aspect above, a transmembrane region fused to the carboxyl terminus of the antibody or antibody fragment, and an immunologically active cell activation signal transduction region fused to the carboxyl terminus of the transmembrane region.

[0059] The eleventh aspect of the present invention provides a chimeric antigen receptor as described in any one of the eighth to tenth aspects above, which further comprises one or more of membrane proteins, secretory proteins, intracellular proteins, small molecule drugs, and cytotoxic drugs.

[0060] The twelfth aspect of the present invention provides a nucleic acid molecule comprising: a nucleotide sequence encoding an antibody or antibody fragment as described in any one of the first to seventh aspects above, or a chimeric antigen receptor as described in any one of the eighth to eleventh aspects above.

[0061] The thirteenth aspect of the present invention provides a nucleic acid molecule as described in the twelfth aspect above, comprising: (i) a nucleotide sequence encoding a heavy chain variable region as shown in SEQ ID NO: 5, and a nucleotide sequence encoding a light chain variable region as shown in SEQ ID NO: 7, or (ii) a nucleotide sequence encoding a heavy chain variable region as shown in SEQ ID NO: 6, and a nucleotide sequence encoding a light chain variable region as shown in SEQ ID NO: 8.

[0062] SEQ ID NO: 5

[0063]

[0064]

[0065] SEQ ID NO: 6

[0066]

[0067] SEQ ID NO: 7

[0068]

[0069] SEQ ID NO: 8

[0070]

[0071] The fourteenth aspect of the present invention provides a carrier comprising the nucleic acid molecules described in the twelfth or thirteenth aspects above.

[0072] The fifteenth aspect of the present invention provides a vector as described in the fourteenth aspect, which is a lentiviral vector, a retroviral vector, an adenovirus vector, or an adeno-associated virus vector, etc.

[0073] The sixteenth aspect of the present invention provides a cell comprising the nucleic acid molecule described in the twelfth or thirteenth aspect above, or the carrier described in the fourteenth or fifteenth aspect.

[0074] The seventeenth aspect of the present invention provides cells as described in the sixteenth aspect, including autologous or allogeneic T cells, B cells, NK cells, macrophages, monocytes, dendritic cells, neutrophils, basophils, eosinophils, mast cells, NK-T cells, MAIT cells, hematopoietic stem cells, embryonic stem cells, induced pluripotent stem cells, and erythrocytes, wherein the T cells include αβT cells, γδT cells, and regulatory T cells.

[0075] The eighteenth aspect of the present invention provides a pharmaceutical composition comprising: an antibody or antibody fragment selected from any one of the first to seventh aspects described above, a chimeric antigen receptor selected from any one of the eighth to eleventh aspects, a nucleic acid molecule selected from the twelfth or thirteenth aspect, a carrier selected from the fourteenth or fifteenth aspect, and a cell selected from the sixteenth or seventeenth aspect; or comprising: an antibody or antibody fragment selected from any one of the first to seventh aspects described above, a chimeric antigen receptor selected from any one of the eighth to eleventh aspects, a nucleic acid molecule selected from the twelfth or thirteenth aspect, a carrier selected from the fourteenth or fifteenth aspect, and a cell selected from the sixteenth or seventeenth aspect, and a pharmaceutically acceptable carrier; or comprising: an antibody or antibody fragment selected from any one of the first to seventh aspects described above, a chimeric antigen receptor selected from any one of the eighth to eleventh aspects, a nucleic acid molecule selected from the twelfth or thirteenth aspect, a carrier selected from the fourteenth or fifteenth aspect, and a cell selected from the sixteenth or seventeenth aspect, and other pharmaceutically active agents or pharmaceuticals.

[0076] The nineteenth aspect of the present invention provides the use of the antibody or antibody fragment described in any one of the first to seventh aspects, the chimeric antigen receptor described in any one of the eighth to eleventh aspects, the nucleic acid molecule described in the twelfth or thirteenth aspect, the carrier described in the fourteenth or fifteenth aspect, or the cell described in the sixteenth or seventeenth aspect in the preparation of a medicament for the treatment or prevention of B7-H3 positive diseases.

[0077] The twentieth aspect of the present invention provides the use as described in the nineteenth aspect, wherein the above-mentioned B7-H3 positive diseases include malignant / benign hematologic malignancies, malignant / benign solid tumors, autoimmune diseases, bacterial infections, viral infections, parasitic infections, bone growth abnormalities, allogeneic transplantation, transplant rejection, etc., and the autoimmune diseases include systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, Sjögren's syndrome, ankylosing spondylitis, etc.

[0078] The twenty-first aspect of the present invention provides the use as described in the twenty-first aspect, wherein the diseases include acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, non-Hodgkin's lymphoma, multiple myeloma, melanoma, lung cancer, colorectal cancer, kidney tumor, bladder cancer, gastrointestinal cancer, prostate cancer, liver cancer, ovarian cancer, pancreatic cancer, endometrial cancer, gastric cancer, prostate cancer, kidney cancer, cervical cancer, thyroid cancer, uterine cancer, neuroendocrine cancer, head and neck cancer, nasopharyngeal carcinoma, testicular cancer, basal cell carcinoma, squamous cell carcinoma, dermatofibrosarcoma protrusion, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma, or myelodysplastic syndrome, etc.

[0079] The twenty-second aspect of the present invention provides a method for detecting B7-H3, comprising the step of using an antibody or antibody fragment described in any one of the first to seventh aspects, a chimeric antigen receptor described in any one of the eighth to eleventh aspects, or a nucleic acid molecule described in the twelfth or thirteenth aspect to detect B7-H3.

[0080] The twenty-third aspect of the present invention provides a kit for detecting B7-H3, which contains an antibody or antibody fragment or a marker thereof as described in any one of the first to seventh aspects, a chimeric antigen receptor or a marker thereof as described in any one of the eighth to eleventh aspects, or a nucleic acid molecule or a marker thereof as described in the twelfth or thirteenth aspects.

[0081] The twenty-fourth aspect of the present invention provides an isolation kit for separating B7-H3 positive cells, which contains the antibody or antibody fragment or its marker as described in any one of the first to seventh aspects, the chimeric antigen receptor or its marker as described in any one of the eighth to eleventh aspects, or the nucleic acid molecule or its marker as described in the twelfth or thirteenth aspects.

[0082] The twenty-fifth aspect of the present invention provides a method for enhancing cell function in vitro, comprising the step of contacting cells with an antibody or antibody fragment or a marker thereof described in any one of the first to seventh aspects, a chimeric antigen receptor or a marker thereof described in any one of the eighth to eleventh aspects, or a nucleic acid molecule or a marker thereof described in the twelfth or thirteenth aspect.

[0083] The twenty-sixth aspect of the present invention provides the use of the antibody or antibody fragment described in any one of the first to seventh aspects above in the preparation of articles for detecting B7-H3 protein.

[0084] The twenty-seventh aspect of the present invention provides the use of the antibody or antibody fragment described in any one of the first to seventh aspects above in the preparation of articles for blocking B7-H3 protein. Attached Figure Description

[0085] Figure 1 The base sequences of the variable regions of the heavy chains of antibodies 1B4 and 2Y31 are shown, among which, Figure 1 A is the base sequence of the variable region of the heavy chain of antibody 1B4 (SEQ ID NO: 5). Figure 1 B is the base sequence of the variable region of the heavy chain of the 2Y31 antibody (SEQ ID NO: 6).

[0086] Figure 2 The base sequences of the variable regions of the light chains of antibodies 1B4 and 2Y31 are shown, among which, Figure 2 A is the base sequence of the variable region of the light chain of antibody 1B4 (SEQ ID NO: 7). Figure 2 B is the base sequence of the variable region of the light chain of the 2Y31 antibody (SEQ ID NO: 8).

[0087] Figure 3 The amino acid sequences of the variable regions of the heavy chains of antibodies 1B4 and 2Y31 are shown, among which, Figure 3 A is the amino acid sequence of the variable region of the heavy chain of antibody 1B4 (SEQ ID NO: 1). Figure 3 B is the amino acid sequence of the variable region of the heavy chain of the 2Y31 antibody (SEQ ID NO: 2).

[0088] Figure 4 The amino acid sequences of the variable regions of the light chains of antibodies 1B4 and 2Y31 are shown, among which, Figure 4 A is the amino acid sequence of the variable region of the light chain of antibody 1B4 (SEQ ID NO: 3). Figure 4 B is the amino acid sequence of the variable region of the light chain of the 2Y31 antibody (SEQ ID NO: 4).

[0089] Figure 5 The results of affinity testing for the humanized 1B4 antibody are shown.

[0090] Figure 6 The results show the binding of the humanized antibody to the glioma cell line U87 and the B7-H3 knockout U87 cell line. Specifically, for... Figure 6 For A, the gray peak represents the negative control, the solid peak represents the binding of the maternal mouse antibody 1B4 to the tumor cell line, and the dashed peak represents the binding of the humanized 1B4 antibody to the tumor cell line. Figure 6 For B, m2Y31 represents the binding of maternal mouse antibody 2Y31 to tumor cell lines, and hu2Y31 represents the binding of humanized antibody 2Y31 to tumor cell lines. 8H9 represents the binding of antibodies recognizing different B7-H3 antigenic epitopes to tumor cells, and NC is the negative control.

[0091] Figure 7 This is a schematic diagram of the CAR structure. From extracellular to intracellular, the segments are scFv (VH-(G4S)3-VL), the CD8 hinge region, the transmembrane region (CD8TM), the co-stimulatory signals CD28 and 4-1BB, and the CD3z signaling activation domain. The scFv includes a variable region of the heavy chain and a variable region of the light chain, linked together by a (GGGGS)3 linker peptide. In addition, at the C-terminus of the CAR structural region, a truncated EGFR structure is linked via a P2A cleavage peptide. This truncated EGFR structure can serve as a selection marker for CAR-positive cells and adds a safety switch for clinical research.

[0092] Figure 8 The binding ability of Jurkat T cells expressing CAR-B7-H3 to the B7-H3 protein was demonstrated.

[0093] Figure 9 The results showed that wild-type colon cancer tumor cells RKO (dark peak, RKO WT) expressed high levels of B7-H3, while B7-H3 knockout RKO tumor cells (light peak, RKO B7-H3KO) did not express B7-H3.

[0094] Figure 10 The expression level of CD69 in CAR-Jurkat T cells constructed using the 1B4 antibody was shown after stimulation with RKO WT or RKO (B7-H3KO) tumor cells.

[0095] Figure 11 A shows the transduction positivity rate of αβT cells expressing CAR-B7-H3 (scFv is the light chain and heavy chain sequence of the 1B4 antibody).

[0096] Figure 11 B shows the expression level of CD69 in αβT or CAR-B7-H3-αβT cells after stimulation by RKO WT or RKO(B7-H3 KO) tumor cells.

[0097] Figure 12 The in vitro killing ability of CAR-B7-H3-αβT cells (scFv is the light chain and heavy chain sequence of the 1B4 antibody) against RKO tumor cells was demonstrated.

[0098] Figure 13 The in vitro killing ability of CAR-B7-H3-αβT cells (scFv is the light chain and heavy chain sequence of the 1B4 antibody) against glioma cells was demonstrated.

[0099] Figure 14 The transduction positivity rate of γδT cells expressing CAR-B7-H3 was shown (scFv represents the light and heavy chain sequences of the 1B4 antibody).

[0100] Figure 15 A shows the killing effect of CAR-αβT and CAR-γδT expressing CAR-B7-H3 on RKO WT tumor cells (scFv is the light chain and heavy chain sequence of the 1B4 antibody).

[0101] Figure 15 B shows the killing effect of CAR-αβT and CAR-γδT expressing CAR-B7-H3 on RKO tumor cells with B7-H3 knockout (scFv is the light chain and heavy chain sequence of 1B4 antibody).

[0102] Figure 16 The in vitro killing ability of CAR-γδT expressing CAR-B7-H3 against B7-H3 positive tumor cells SKOV3 was demonstrated (scFv were the light chain and heavy chain sequences of 1B4 antibody or 2Y31 antibody, respectively).

[0103] Figure 17 The in vivo antitumor effect of CAR-vδT expressing CAR-B7-H3 (scFv is the light and heavy chain sequence of 1B4 antibody) on SKOV3 was demonstrated.

[0104] Figure 18 The growth curve of SKOV3 tumors in model mice is shown.

[0105] Figure 19 The survival curve changes in SKOV3 tumor model mice are shown. Detailed Implementation

[0106] The present invention will be further described below with reference to the accompanying drawings through specific embodiments. However, this is not a limitation of the present invention. Those skilled in the art can make various modifications or improvements based on the basic idea of ​​the present invention, but as long as they do not depart from the basic idea of ​​the present invention, they are all within the scope of the present invention.

[0107] It should be noted that, unless otherwise defined, the technical or scientific terms used in this specification should have the ordinary meaning understood by one of ordinary skill in the art. Furthermore, it should be understood that the terminology used in this application is for the purpose of describing specific embodiments only and is not intended to be restrictive.

[0108] In this specification, "specific binding" means that the antigen recognition region of this invention does not cross-react with, or substantially does not cross-react with, any polypeptide other than the target antigen. The degree of specificity can be determined using immunological techniques, including but not limited to Western blotting, immunoaffinity chromatography, and flow cytometry.

[0109] In this specification, "antibody" refers to an immunoglobulin molecule that specifically binds to an antigen. Antibodies can be complete immunoglobulins derived from natural or recombinant sources, and can be the immunoreactive portion of a complete immunoglobulin. Antibodies are typically tetramers of immunoglobulin molecules. The antibodies of this invention can exist in various forms, including, for example, Fab, Fab′, F(ab′)2, Fv fragments, linear antibodies formed from antibody fragments, scFv antibodies, ADCs-conjugated antibodies, multispecific antibodies, single-chain antibodies, and humanized antibodies.

[0110] In this specification, "antibody fragment" refers to a portion of the complete antibody structure or sequence.

[0111] In this specification, "heavy chain" refers to the larger chain among the two types of polypeptide chains present in the spontaneous conformation of all antibody molecules.

[0112] In this specification, "light chain" refers to the smaller chain among the two types of polypeptide chains present in the spontaneous conformation of all antibody molecules.

[0113] The “variable region of heavy chain (VH)” in this specification refers to the approximately 110 amino acid residues near the amino terminus (N-terminus) of the heavy chain, where the composition and arrangement of these amino acid residues vary considerably.

[0114] The “variable region of light chain (VL)” in this specification refers to the approximately 110 amino acid residues near the amino terminus (N-terminus) of the light chain, the composition and arrangement of which vary greatly.

[0115] In this invention, "CDR" refers to complementarity-determining region or complementarity-determining cluster, which is a discontinuous antigen-binding site found within the variable region of heavy and light chain polypeptides. This invention uses the Kabat numbering system to label the variable region of an antibody with CDR and FR regions.

[0116] In this specification, "homology" refers to a high proportion of amino acid or nucleotide matching between the target amino acid sequence or target nucleotide sequence and a reference sequence. Homology in this specification can be determined using standard software such as BLAST or FASTA.

[0117] In this specification, "homology of 80% or more" means that the homology is 80% or more, preferably 85% or more, more preferably 88% or more, further preferably 90% or more, further preferably 93% or more, particularly preferably 95% or more, particularly more preferably 98% or more, and most preferably 100% homology.

[0118] In this specification, "genetically engineered antibodies or antibody fragments" refers to antibodies or antibody fragments that have been modified and altered through genetic engineering. "Genetically engineered" in this context refers to techniques such as gene splicing and DNA recombination to integrate and express genes from different sources. Specific examples include inserting other proteins or protein fragments into antibodies or antibody fragments, replacing a portion of an antibody or antibody fragment with another protein or protein fragment, or linking different antibodies or antibody fragments with other proteins or protein fragments to form a larger protein complex. "Humanized antibodies" are also a type of "genetically engineered antibodies or antibody fragments," referring to antibodies that are re-expressed after being modified using gene cloning and DNA recombination techniques from non-human monoclonal antibodies (such as murine or rabbit antibodies). Their characteristic is a reduced immunogenicity compared to the corresponding murine antibody, while retaining the affinity and antigen-binding specificity of the murine antibody. The basic implementation involves encoding the constant region or all parts of the non-human antibody using a human antibody gene. Depending on the modification method, humanized antibodies include chimeric antibodies, modified antibodies, and fully humanized antibodies.

[0119] In this specification, "constant region" refers to a region on the antibody near the C-terminal amino acid sequence that is relatively stable. This includes the "light chain constant region" and the "heavy chain constant region," which refer to the relatively stable regions on the antibody light chain and heavy chain near the C-terminal amino acid sequence, respectively.

[0120] In this specification, "scFv" refers to an antibody fragment comprising a recombinant protein containing a heavy chain variable region and a light chain variable region linked by a linker, which associates these two domains to ultimately form an antigen-binding site. The size of an scFv is typically 1 / 6 that of a complete antibody. An scFv is preferably an amino acid sequence encoded by a single nucleotide chain. The scFv used in this invention can be further modified, alone or in combination, using conventional techniques known in the art, such as amino acid deletion, insertion, substitution, addition, and / or recombination, and / or other modification methods. Methods for introducing such modifications into the DNA sequence of an antibody based on its amino acid sequence are well known to those skilled in the art (see, for example, Sambrook Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory (1989) NY). The modifications are preferably performed at the nucleic acid level. The aforementioned scFv may also include its derivatives. scFvs can be expressed as single-chain polypeptides. scFvs retain the specificity of the complete antibody from which they are derived. The light and heavy chains can be in any order, such as VH-linker-VL or VL-linker-VH, as long as the specificity of scFv for the target antigen is preserved. In this specification, a linker may be exemplified as a flexible linker peptide chain rich in glycine and serine.

[0121] The "chimeric antigen receptor" (CAR) in this specification is an artificially engineered receptor that anchors specific molecules (such as antibodies) that recognize tumor cell surface antigens onto immune cells (such as T cells), enabling the immune cells to recognize tumor antigens or viral antigens and kill tumor cells or virus-infected cells. A CAR typically comprises, in sequence, an optional signal peptide, a polypeptide (such as a single-chain antibody) that binds to tumor cell membrane antigens, a hinge region, a transmembrane region, and an immune-active cell activation signal transduction region, which further includes a co-stimulatory domain and a signal transduction domain.

[0122] The "hinge region" refers to the hydrophilic region located between the antigen recognition domain and the transmembrane structural domain. The hinge region can be the hinge region of various antibodies or antigen receptors, particularly the hinge region of the CD molecule. In one specific embodiment, the hinge region can be selected from, for example, CD4, CD8α, CD28, IgG1, and IgG4. In a preferred embodiment of the invention, the CD8α hinge region is employed.

[0123] The term "transmembrane region" simply requires a peptide capable of crossing the cell membrane. Preferably, the transmembrane region is the transmembrane region of the CD molecule. In one embodiment, the transmembrane region can be selected from, for example, the transmembrane regions of CD4, CD8, CD28, CD3ζ, the α and β chains of the T cell receptor, CD3ζ, CD3ε, CD45, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, EGFR (epidermal growth factor receptor), NKG2D, or GITR. In a preferred embodiment of the invention, the CD8α transmembrane region is used.

[0124] The term "co-stimulatory domain" refers to the portion of the CAR that enhances the proliferation, survival, and / or development of memory cells. The CAR of this invention may contain one or more co-stimulatory domains. Each co-stimulatory domain contains, for example, any one or more of the following: CD28, 4-1BB, ICOS, OX40, CD27, CD40, Myd88, HVEM, GITR, Fc receptor-associated γ chain, etc. In a preferred embodiment of this invention, a combination of CD28 and 4-1BB is used.

[0125] The term "signal transduction domain" refers to the portion of CAR that transduces effector functional signals and guides cells to perform their specialized functions. Examples of signal transduction domains for effector functional signals include, but are not limited to, CD3ζ and FcεRIγ. In a preferred embodiment of the invention, the CD3ζ domain is employed.

[0126] In this specification, the terms "membrane proteins, secretory proteins, intracellular proteins, small molecule drugs, and cytotoxic drugs" refer to proteins that are wholly or partially inserted into various membrane structures (including cell membranes, mitochondrial membranes, endoplasmic reticulum membranes, nuclear membranes, etc.); secretory proteins refer to proteins that are secreted outside the cell after synthesis; intracellular proteins refer to all proteins present inside the cell membrane, including free proteins in the cytoplasm and membrane proteins on the membranes of various intracellular organelles; small molecule drugs refer to chemically synthesized drugs with a molecular weight of less than 1000, which can be linked to other proteins or molecular drugs via linker arms; and cytotoxic drugs refer to drugs that have toxic effects on cells or specific cells, including but not limited to alkaloid drugs (such as paclitaxel), metabolites (such as decitabine), antibiotics (such as idarubicin), alkylating agents (such as ifosfamide), and platinum-based drugs (such as cisplatin), which can also be linked to other proteins or molecular drugs via linker arms.

[0127] In the chimeric antigen receptor of the present invention, examples of membrane proteins include CD40L, CXCR5, CXCR3, 4-1BB, ICOS, OX40, CD27, NKG2D, etc.; examples of secretory proteins include IL2, IL15, IL4, IL7, IL10, IL18, IFNy, IL1β, and antibodies (such as anti-PD1 antibodies, anti-CTLA4 antibodies, etc.); examples of small molecule drugs include FITC / folic acid, rapamycin, Rimiducid, PROTAC compounds, Dasatinib, etc.; examples of cytotoxic drugs include paclitaxel, vinorelbine, docetaxel, hydroxycamptothecin, gemcitabine, cytarabine, tegafur, methotrexate, epirubicin, pirarubicin, idarubicin, mitomycin, mitoxantrone, ifosfamide, dacarbazine, cisplatin, oxaliplatin, etc.

[0128] In this specification, "nucleic acid molecule" refers to a biomolecular compound polymerized from deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Its building block is the nucleotide, and a nucleotide monomer consists of a pentose sugar, a phosphate group, and a nitrogenous base.

[0129] In this specification, the term “encoding” when applied to a nucleic acid sequence means that the polynucleotide “encoding” a polypeptide, in its natural state or when manipulated by methods well known to those skilled in the art, can be transcribed and / or translated to produce mRNA for the polypeptide and / or fragments thereof.

[0130] In this specification, "vector" refers to a recombinant vector that retains the ability to infect and transduce non-dividing and / or slowly dividing cells and integrate into the genome of a target cell. In some preferred embodiments, the vector is derived from or based on a wild-type virus. In other, more preferred embodiments, the vector is derived from or based on a wild-type lentivirus. Examples of such vectors include retroviral vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, herpes simplex virus vectors, etc. More specifically, in one preferred embodiment of the invention, the vector is a lentiviral vector; in another preferred embodiment, the vector is a retroviral vector.

[0131] In this instruction manual, "autologous or allogeneic" refers to "autologous cells" or "allogeneic cells." "Autologous cells" refers to cells derived from the same individual and subsequently re-administered to that individual; "allogeneic cells" refers to cells derived from sources other than the autologous cell.

[0132] The cells used in this invention can include, for example, T cells, B cells, NK cells, macrophages, monocytes, dendritic cells, neutrophils, basophils, eosinophils, mast cells, NK-T cells, MA1T cells, hematopoietic stem cells, embryonic stem cells, induced pluripotent stem cells, and erythrocytes. T cells can include, for example, αβT cells, γδT cells, and regulatory T cells. In a preferred embodiment of this invention, γδT cells are used.

[0133] The pharmaceutical compositions of the present invention contain any one of the antibodies, CARs, nucleic acid molecules, carriers, and cells of the present invention, and may further contain a pharmaceutically acceptable carrier. Alternatively, the pharmaceutical compositions of the present invention may contain any two or more of the antibodies, CARs, nucleic acid molecules, carriers, and cells of the present invention, for example, CARs and nucleic acid molecules, and may further contain a pharmaceutically acceptable carrier. Furthermore, the pharmaceutical compositions of the present invention may also contain other pharmaceutically active agents or drugs, such as chemotherapeutic agents, exemplified by asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc. In a preferred embodiment, the pharmaceutical composition comprises the cells of the present invention.

[0134] Furthermore, the pharmaceutically acceptable carrier in the drug composition can be any conventionally used, pharmaceutically acceptable carrier. Those skilled in the art can select a suitable pharmaceutically acceptable carrier based on chemical-physical conditions and route of administration. Examples of pharmaceutically acceptable carriers described in this specification include, for instance, vehicles, adjuvants, excipients, and diluents. Preferably, the pharmaceutically acceptable carrier is a chemically inert carrier to the active reagent and a carrier that does not have harmful side effects or toxicity under the conditions of use.

[0135] In this specification, "treatment or prevention" refers to methods that eliminate a disease after its occurrence or methods that prevent its occurrence. "Treatment" and "prevention" in this specification do not necessarily mean 100% or complete treatment or prevention. Rather, there are different degrees of treatment or prevention that those skilled in the art recognize as having potential beneficial or therapeutic effects. Furthermore, the treatment or prevention provided by the methods of this invention may include treating or preventing one or more symptoms or conditions of the disease being treated or prevented (e.g., cancer). Similarly, when used for the purposes of this invention, "prevention" may include delaying the onset of a disease or its symptoms or conditions.

[0136] The detection kit of the present invention is a kit for detecting B7-H3. The kit typically contains ingredients commonly used in such kits, such as pH buffers, stabilizers, and supplementary materials such as operating instructions and instructions for detecting B7-H3.

[0137] The isolation kit of the present invention is an isolation kit for isolating B7-H3 positive cells. The kit typically contains components commonly used in such kits, such as pH buffers, stabilizers, and supplementary materials such as operating instructions and instructions for isolating B7-H3 positive cells.

[0138] Additionally, the "P2A cleavage peptide" mentioned in this specification refers to a short peptide (18-25 amino acids in length) derived from a virus that, through a process known as "self-cleavage," can generate multiple proteins from a single transcript. The basic principle is that it works by causing the ribosome to skip the synthesis of the glycine and proline peptide bonds at the C-terminus of the 2A sequence, leading to the separation of the 2A sequence terminus and downstream products. P2A is one such peptide; other peptides include T2A, E2A, and F2A, these four 2A peptides originating from different viruses.

[0139] In this instruction manual, "truncated EGFR" is abbreviated as "EGFRt," referring to domains III and IV of wild-type EGFR. This truncated EGFR lacks intracellular signaling and does not transmit other signals to T cells. These two domains are also the recognition epitopes of cetuximab. Expression of these epitopes on T cells not only serves as a selection marker for CAR-T cells but also adds a safety switch for clinical research. EGFRt can be specifically bound by an inducer (such as cetuximab), and antibody-dependent cell-mediated cytotoxicity can induce apoptosis in CAR-T cells, thereby allowing for the timely elimination of CAR-T cells in the body.

[0140] The "antibody-dependent cell-mediated cytotoxicity" mentioned above, abbreviated as ADCC, refers to the binding of the Fab fragment of an antibody to the antigenic epitope of tumor cells, and the binding of its Fc fragment to the FcR on the surface of killer cells (NK cells, macrophages, etc.), mediating the direct killing of target cells by killer cells.

[0141] In this specification, "flexible linker peptide chain" refers to an oligopeptide or polypeptide region of about 1 to 100 amino acids in length that links any domain / region of the CAR of the present invention together. The linker may consist of flexible residues (such as glycine and serine) to allow adjacent protein domains to move freely relative to each other. Longer linkers may be used when it is desirable to ensure that two adjacent domains do not interfere with each other spatially. In a preferred embodiment of the invention, (GGGGS)3 is employed.

[0142] The “transduction positivity rate” in this instruction manual refers to the proportion of exogenous genes transduced on T cells, including the transduction rate of CAR on T cells, which reflects the proportion of CAR-positive T cells to the total number of T cells.

[0143] In this specification, "affinity" or "binding affinity" or "KD" is determined by measuring the equilibrium association constant (ka) and the equilibrium dissociation constant (kd) and calculating the quotient of kd and ka (KD = kd / ka). A smaller KD value indicates a stronger affinity, and a larger KD value indicates a weaker affinity.

[0144] Experimental Materials and Methods

[0145] 1. Tumor cell line culture

[0146] Six adherent tumor cell lines were used in this study: 293T, U87, HTB15, TJ905, RKO, and SKOV3 cells. 293T cells are a human renal epithelial cell line, commonly used to study exogenous gene expression and virus preparation. U87, HTB15, and TJ905 are human glioma cell lines, RKO is a human colon cancer cell line, and SKOV3 is a human ovarian cancer cell line. Except for SKOV3, the other tumor cells were cultured in complete DMEM medium (DMEM medium + 10% FBS + 1% penicillin antibody). SKOV3 cells were cultured in McCoy's 5a complete medium (McCoy's 5a medium + 20% FBS + 1% penicillin antibody).

[0147] JurkatT cells are a human T-lymphoblastic leukemia cell line, and the cells were cultured in RPMI-1640 complete medium (RPMI-1640 medium + 10% FBS + 1% penicillin antibody).

[0148] 2. Lentiviral production

[0149] 1×10 7293T cells were seeded into 10cm cell culture dishes coated with poly-L-lysine, with a culture volume of 10mL. The next day, the expression plasmid (backbone from pCDH-EFl-MCS-T2A-copGFP plasmid (Addgene, Plasmid #72263)), helper plasmid pspAx2 (Addgene, #12260), and helper plasmid pCMV-VSV-G (Addgene, Plasmid #8454) were mixed at 6μg, 4μg, and 2μg respectively in 300μL opti-MEM medium to prepare a plasmid-containing medium. Separately, 30μg of PEI was added to 300μL opti-MEM medium, mixed thoroughly, and then added to the plasmid-containing medium. The mixture was immediately mixed, incubated at room temperature for 15min, and then evenly added to the 293T cell culture dishes (at 0h). After 8h, the medium was replaced with a fresh 10mL DMEM complete medium. The culture supernatant was collected at 48 h and 72 h, respectively. After collecting the supernatant at 48 h, 8 mL of fresh DMEM complete medium was added. Lenti-X was used. TM The supernatant was concentrated using a concentrator (Takara) to obtain the viral fluid, and the viral titer was detected using 293T cells. The viral fluid was then aliquoted and stored at -80°C for long-term preservation.

[0150] 3. Primary cell culture

[0151] (1) γδT cells

[0152] Prepare PBMC cells by washing twice with PBS. Count the cells and assess cell viability using an AO / PI cell counter.

[0153] PBMC cells were cultured in RPMI 1640 medium + 10% FBS + 1% penicillin-drug antibiotics + 200 U / mL rhIL-2, with an initial cell density of 2 × 10⁻⁶ cells / mL. 6 Cells / mL. Simultaneously, 5 μM zoledronic acid (ZOL) was added. After 48 h, an equal volume of fluid was added (without further ZOL addition), and thereafter, cell density was adjusted every 48 h (1 × 10⁻⁶ cells / mL). 6 (cells / mL).

[0154] (2) αβ T cells

[0155] Prepare PBMC cells by washing twice with PBS. Count the cells and assess cell viability using an AO / PI cell counter.

[0156] Antibody coating treatment for 6-well plates: CD3 and CD28 antibodies were diluted to 200 ng / mL with Opti-MEM medium. 2 mL of antibody dilution solution was added to each well of the 6-well plate and incubated overnight at 4°C or for 2 h at 37°C.

[0157] Before adding PBMC cells to the 6-well plate, aspirate the antibody dilution buffer and wash once with 2 mL of PBS.

[0158] Then, PBMCs were added, and the PBMC cells were resuspended in RPMI 1640 medium + 10% FBS + 1% penicillin antibiotics + 200 U / mL rhIL-2 culture system, and the cell density was adjusted to 2 × 10⁶ cells / mL. 6 Cells / mL. Subsequently, every 48 hours, the cell density was adjusted to 1×10⁻⁶ cells / mL using a culture system of RPMI 1640 medium + 10% FBS + 1% penicillin-dextrose antibody + 200 U / mL rhIL-2. 6 (cells / mL).

[0159] 4. Viral transduction of αβT and γδT

[0160] 1×10 7 TU lentivirus was diluted in 200 μL PBS and added to 24-well plates. The 24-well plates were pre-coated with RetroNectin. The plates were centrifuged at 2000g and 32°C for 2 h. After centrifugation, the virus dilution was aspirated, and the plates were washed three times with 1 mL PBS before adding cells.

[0161] Regarding the transduction of αβT or γδT cells, αβT or γδT cells were counted after 24 hours of in vitro activation and culture, with 1×10⁻⁶ cells... 6 Total cells were resuspended in 1 mL RPMI 1640 medium + 10% FBS + 1% penicillin-dextrose antibody + 200 U / mL rhIL-2 culture system, and then added to virus-treated 24-well plates. After centrifugation at 800g and 32℃ for 10 min, the plates were incubated in a cell culture incubator. Cell counting was performed every two to three days, and the density was adjusted to 1 × 10⁶ cells / well. 6 cells / mL.

[0162] 5. Flow cytometry staining analysis

[0163] Wash the cells to be analyzed by flow cytometry once with PBS. 2×1 0 Five cells were stained using a 50 μL staining system. After resuspending the cells in the staining system, they were incubated at 4°C for 30 min. After incubation, the cells were washed twice with flow cytometry buffer, resuspended in 200 μL of flow cytometry buffer, and then analyzed.

[0164] The staining system was prepared as follows: 50 μL flow cytometry buffer + antibody.

[0165] The flow cytometry buffer formulation is: PBS + 1% FBS + 2.5mM EDTA.

[0166] The amount of antibody used depends on the antibody concentration and the actual situation. For most antibodies (concentration of 0.5-1 mg / mL), a dilution of 1:50 to 1:100 is sufficient.

[0167] The antibodies used in this experiment mainly include: anti-human CD3, anti-human αβTCR, anti-human γδTCR, and anti-human EGFR.

[0168] 6. CD69 expression detection

[0169] Effector cells and tumor cells were incubated at an effector-to-target ratio of 1:1 in 24-well plates, with 0.5 × 10⁻⁶ cells added. 6 Effector cells and 0.5 × 10 6 Tumor cells. Flow cytometry analysis was performed after 24 hours of incubation.

[0170] For Jurkat T cells, the flow cytometry staining system was: anti-human EGFR, anti-human CD69. For αβ T cells, the flow cytometry staining system was: anti-human CD3, anti-human αβTCR, anti-human EGFR, anti-human CD69.

[0171] 7. In vitro cell killing experiment

[0172] In vitro cell killing assays were performed using Luciferase fluorescence detection. The tumor cells to be tested stably expressed Luciferase. Killing assays were conducted in 24-well plates, with different effector-to-target ratios set according to experimental requirements. If the effector-to-target ratio was 1:1, tumor cells and effector cells were counted separately, and both cell types were resuspended in their respective culture media to 0.5 × 10⁻⁶. 6 cells / mL. Add 500 μL of tumor cells and 500 μL of effector cells to the same well, and mix thoroughly. Set up a separate control well containing only tumor cells, adding 500 μL of tumor cell culture medium and 500 μL of effector cell culture medium. Incubate in a cell culture incubator, and detect Luciferase fluorescence signal after 24 h.

[0173]

[0174] 8. Detection of binding ability between B7-H3 and CAR-B7-H3

[0175] The binding affinity of B7-H3 to CAR-B7-H3 was detected by flow cytometry. Specifically, CAR-B7-H3 was stably expressed on Jurkat T cells. Different concentrations of biotin-B7-H3 antigen (ranging from 0.03125 μg / mL to 16 μg / mL) were mixed with 1×10⁻⁶ CAR-B7-H3 in a 100 μL flow cytometry buffer staining system. 5 CAR-B7-H3-Jurkat T cells were incubated at 4°C for 45 min. After incubation, the cells were centrifuged at 500g for 5 min, the supernatant was discarded, and the cells were resuspended in 500 μL of flow cytometry buffer and centrifuged again at 500g for 5 min, discarding the supernatant. The cells were then resuspended in 100 μL of flow cytometry buffer, and 0.5 μL of fluorescently labeled streptavidin secondary antibody and 0.5 μL of fluorescently labeled anti-human EGFR antibody were added. The cells were incubated at 4°C for 30 min. After incubation, the cells were centrifuged at 500g for 5 min, the supernatant was discarded, and the cells were resuspended in 300 μL of flow cytometry buffer. The fluorescence signal of the streptavidin secondary antibody in the EGFR-positive population was analyzed by flow cytometry.

[0176] 9. Antibody Affinity Detection

[0177] The B7-H3 antigen protein was prepared into seven serially diluted concentrations of 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.13 nM, and 1.56 nM, and sampled into 96-well plates. Human Protein G probe was selected, the antigen was used as the stationary phase, and the B7-H3 antibody was used as the mobile phase. The binding time was set to 180 s, and the dissociation time to 300 s. The binding and dissociation constants of the human monoclonal antibody were measured, and the affinity was calculated.

[0178] 10. ELISA method for detecting the binding ability of B7-H3 antibody to antigen.

[0179] The antigens to be tested, including B7-H1, B7-H3, B7-H4, and B7-H5, were diluted with PBS to five concentrations: 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL, and 0.5 mg / mL, respectively. 100 μL of each solution was plated into a 96-well ELISA plate and incubated at room temperature for 1 hour. The plates were then washed twice with PBST (100 μL per well) and incubated for 5 minutes. Next, 0 μg / mL, 2 μg / mL, or 4 μg / mL of B7-H3 antibody was added, diluted with PBS, and 100 μL was added to each well. The plates were incubated at room temperature for 1 hour. The plates were washed three times with PBST (100 μL per well) and incubated for 5 minutes. Finally, HRP-anti-human Fc antibody was diluted 1:2000 (antibody concentration: 0.5 mg / mL) with skim milk, and 100 μL was added to each well. The plates were incubated at room temperature for 1 hour. Wash three times with PBST, 100 μL per well, and let stand for 5 min. Add 100 μL of TMB substrate solution to each well in the dark, and let stand at room temperature in the dark for 40 min. Stop the reaction by adding 100 μL of 2M sulfuric acid to each well. Place the plate in a microplate reader and measure the OD value at 450 nm.

[0180] 11. Maternal antibody blocking experiment

[0181] Add 100 μL of 10 μg / mL B7-H3 protein diluted in PBS to a 96-well microplate and incubate overnight at 4°C. Wash twice with PBST, 100 μL per well, and let stand for 5 min after addition. Add 100 μL of 0.625, 1.25, 2.5, 5, 10, 20, and 40 μg / mL mouse maternal antibody diluted in PBS, respectively, and react for 1 h. Wash twice with PBST, 100 μL per well, and let stand for 5 min after addition. Then add 100 μL of 2 μg / mL B7-H3 antibody diluted in PBS and 2 μg / mL 8H9 antibody recognizing different B7-H3 antigenic epitopes and incubate. Detect by ELISA (see Experimental Materials and Methods, Section 10).

[0182] Example

[0183] The following specific embodiments further describe the technical solutions provided by the present invention, and are intended merely to illustrate the present invention and not to limit the scope of the present invention.

[0184] In addition, unless otherwise specified, all experimental methods used in the examples are conventional methods. All reagents and materials used in the examples are commercially available products unless otherwise specified.

[0185] Example 1: Affinity test of humanized 1B4 antibody

[0186] To determine the affinity of the humanized 1B4 antibody for antigen B7-H3, seven serially diluted concentrations of B7-H3 antigen protein (100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.13 nM, and 1.56 nM) were prepared and loaded onto 96-well plates. Human Protein G was selected as the probe, the antigen was used as the stationary phase, and 1B4 was used as the mobile phase. The binding time was set to 180 s, and the dissociation time to 300 s. The binding and dissociation constants of the humanized monoclonal antibody were measured, and the affinity was calculated.

[0187] The results are shown in Figure 5 The results showed that the binding constant ka was 2.81E+05, the dissociation constant kd was 7.38E-05, and the equilibrium dissociation constant KD was 2.625E-10.

[0188] Example 2: Maternal Antibody Blocking Experiment

[0189] Add 100 μL of 10 μg / mL B7-H3 protein diluted in PBS to a 96-well ELISA plate and incubate overnight at 4°C. Wash twice with PBST, 100 μL per well, and let stand for 5 min after addition. Add 100 μL of mouse maternal B7-H3 antibody (mlB4 or m2Y31) diluted in PBS at concentrations of 0.625, 1.25, 2.5, 5, 10, 20, and 40 μg / mL, respectively, to each well and react for 1 h. Wash twice with PBST, 100 μL per well, and let stand for 5 min after addition. Then add 100 μL of 2 μg / mL humanized B7-H3 antibody (hu1B4 or hu2Y31) diluted in PBS or 100 μL of 2 μg / mL 8H9 antibody recognizing different B7-H3 epitopes diluted in PBS and incubate. Detect by ELISA.

[0190] The results are shown in Table 1.

[0191] Table 1 Results of maternal antibody blocking experiment

[0192]

[0193] Experiments showed that the addition of either 1B4 or 2Y31 maternal antibodies significantly reduced the binding ability of the corresponding humanized 1B4 or 2Y31 antibodies to the B7-H3 antigen, while having no significant effect on 8H9 antibodies recognizing different epitopes. The results indicate that 0.625 μg / mL of m1B4 can block the binding of hu1B4 to the antigen molecule, suggesting that hu1B4 and m1B4 recognize the same site. Neither low nor high concentrations of m1B4 could block the binding of 8H9 to the antigen molecule, further verifying that 1B4 and 8H9 recognize different sites. Similarly, 0.625 μg / mL of m2Y31 can block the binding of hu2Y31 to the antigen molecule, also suggesting that hu2Y31 and m2Y31 recognize the same site, but a different site than 8H9.

[0194] Example 3: Determination of B7-H3 expression level in wild-type U87 tumor cells and U87 tumor cells with B7-H3 knockout (U87-B7-H3CAS9)

[0195] A B7-H3 knockout U87 tumor cell line was constructed using CRISPR-Cas9 technology. One million human glioma cells (U87) were washed once with PBS and resuspended in 100 μL of PBS. Then, 1 μg of reagent was added to each group (NC for negative control, B7-H3 antibody 8H9 for positive control, and humanized 1B4 antibody, humanized 2Y31 antibody, maternal mouse 1B4 antibody, and maternal mouse 2Y31 antibody, respectively). The cells were incubated at 4°C for 30 min, washed once with PBS, and resuspended in 500 μL of PBS. The binding ability of the cells to the B7-H3 antigen was then detected.

[0196] Except for replacing the 1 million human glioma cells U87 in the above procedure with 1 million U87 cells with B7-H3 knocked out, the other procedures were performed in the same manner.

[0197] The results are shown in Figure 6 ,like Figure 6 As shown in Figure A, the signal (dashed peak) of the humanized 1B4 antibody binding to the glioma cell line U87 was significantly higher than that of the maternal mouse-derived 1B4 antibody (solid peak). Furthermore, knocking out B7-H3 in the U87 cell line resulted in no binding activity from either antibody. This indicates that the 1B4 antibody specifically binds to the B7-H3 antigen.

[0198] Figure 6Results B showed that the humanized 2Y31 antibody (hu2Y31) showed a significantly higher binding signal to U87 cell lines than the maternal mouse 2Y31 antibody (m2Y31), and also higher than the 8H9 antibody. No binding signal was observed between any of the antibodies and B7-H3 knockout U87 tumor cells. This indicates that the 2Y31 antibody is specific for the B7-H3 antigen, and that the humanized antibody has a higher binding affinity to the B7-H3 antigen than the maternal mouse antibody.

[0199] Example 4: Binding ability experiment of 1B4 antibody with other proteins in the B7-H3 family

[0200] To verify the specificity of the 1B4 antibody in binding to the B7-H3 protein and exclude its binding to other members of the B7 family, this patent utilizes an ELISA method for testing. The antigens to be tested, including B7-H1, B7-H3, B7-H4, and B7-H5, were diluted with PBS to five concentrations: 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL, and 4 mg / mL, respectively. These were then plated onto a 96-well microplate, 100 μL per well, and incubated at room temperature for 1 hour. The plates were washed twice with PBST (100 μL per well) and incubated for 5 minutes. Then, 0 μg / mL, 2 μg / mL, or 4 μg / mL of B7-H3 antibody, diluted with PBS, were added to each well (100 μL per well). The plates were incubated at room temperature for 1 hour. Finally, the plates were washed three times with PBST (100 μL per well) and incubated for 5 minutes. The HRP-anti-human Fc antibody was diluted 1:2000 with skim milk (antibody concentration: 0.5 mg / mL), and 100 μL was added to each well. The mixture was incubated at room temperature for 1 hour. After washing three times with PBST (100 μL per well), the mixture was incubated for 5 minutes. TMB substrate solution was added to each well in the dark, and the mixture was incubated at room temperature in the dark for 40 minutes. The reaction was stopped by adding 100 μL of 2M sulfuric acid to each well. The plate was then placed in a microplate reader, and the OD value was measured at 450 nm. The results are shown in Table 2.

[0201]

[0202] As shown in Table 2, 2 μg / mL of 1B4 antibody exhibits a very high binding affinity to 0.5 mg / mL of B7-H3 antigen, with an OD450 value exceeding 3.6, reaching saturation. Further increases in either the 1B4 antibody or B7-H3 antigen concentration do not further increase the OD450 value. However, 1B4 shows no binding affinity to B7-H1, B7-H4, or B7-H5; increasing either the 1B4 antibody or B7-H3 antigen concentration does not improve the OD450 value, which remains around the baseline value of 0.04. This indicates that the binding of 1B4 antibody to the B7-H3 protein is specific, and it does not bind to other members of the B7 family.

[0203] Example 5: Construction of CAR Structure

[0204] The CAR structure used in this embodiment is a third-generation CAR structure, such as... Figure 7 As shown, the specific structure consists of an scFv sequence at the 5' end, followed by the CD8 hinge region, CD8 transmembrane region, CD28 and 4-1BB intracellular co-stimulatory domains, and the CD3ζ signal transduction domain. The scFv sequence, starting from the 5' end, comprises the antibody heavy chain variable region, the GGGGS GGGGS GGGGS flexible linker peptide, and the antibody light chain variable region. Following the CAR structure, a truncated EGFR is linked via a P2A cleavage peptide.

[0205] Example 6: Experiment on the binding ability of Jurkat T cells expressing CAR-B7-H3 to B7-H3 protein

[0206] The binding affinity of B7-H3 to CAR-B7-H3 was detected by flow cytometry. Specifically, CAR-B7-H3 was stably expressed on Jurkat T cells. Different concentrations of biotin-B7-H3 antigen (ranging from 0.03125 μg / mL to 16 μg / mL) were mixed with 1×10⁻⁶ CAR-B7-H3 in a 100 μL flow cytometry buffer staining system. 5 CAR-B7-H3-Jurkat T cells were incubated at 4°C for 45 min. After incubation, the cells were centrifuged at 500g for 5 min, the supernatant was discarded, and the cells were resuspended in 500 μL of flow cytometry buffer and centrifuged again at 500g for 5 min, discarding the supernatant. The cells were then resuspended in 100 μL of flow cytometry buffer, and 0.5 μL of fluorescently labeled streptavidin secondary antibody and 0.5 μL of fluorescently labeled anti-human EGFR antibody were added. The cells were incubated at 4°C for 30 min. After incubation, the cells were centrifuged at 500g for 5 min, the supernatant was discarded, and the cells were resuspended in 300 μL of flow cytometry buffer. The fluorescence signal of the streptavidin secondary antibody in the EGFR-positive population was analyzed by flow cytometry.

[0207] The results are shown in Figure 8 The results show that both CAR-B7-H3(1B4) and CAR-B7-H3(2Y31) have the ability to bind to the B7-H3 protein, with CAR-B7-H3(1B4) having a higher binding ability to B7-H3.

[0208] Example 7: Functional assay of Jurkat T cells expressing CAR-B7-H3(1B4)

[0209] A B7-H3 knockout RKO tumor cell line was constructed using gene editing technology, and its validation was performed using flow cytometry with 1B4 antibody. The results are as follows: Figure 9 As shown, wild-type RKO tumor cells (dark peak, RKO WT) express high levels of B7-H3, while B7-H3 knockout RKO tumor cells do not express B7-H3 (light peak, RKO B7-H3 KO).

[0210] The CD69 signaling activation level of CAR-B7-H3 was then further examined. Effector cells (including JurkatT-control cells without CAR-B7-H3 expression and CAR-B7-H3-JurkatT cells expressing CAR-B7-H3) were incubated with tumor cells (RKO WT and RKO B7-H3 KO) at an effector-target ratio of 1:1. Specifically, incubation was performed in 24-well plates with 0.5 × 10⁻⁶ cells added. 6 Effector cells and 0.5 × 10 6 Tumor cells. Flow cytometry analysis was performed after 24 hours of incubation. The flow cytometry staining system consisted of anti-human EGFR and anti-human CD69.

[0211] The results are as follows Figure 10 As shown, Jurkat T cells expressing CAR-B7-H3(1B4) (CAR-B7-H3-JurkatT) exhibited significantly upregulated CD69 signaling upon stimulation by wild-type RKO (RKO WT) tumor cells, while no enhancement of CD69 signaling was observed in RKO tumor cells lacking B7-H3. Furthermore, expression of CAR-B7-H3(1B4) did not upregulate the baseline CD69 signal, ruling out its activating cytotoxic effect on T cells.

[0212] Example 8: CAR transduction positivity rate and functional assay of αβT cells expressing CAR-B7-H3(1B4)

[0213] (1) CAR transduction positivity rate

[0214] CAR-B7-H3(1B4)-αβT cells were labeled and stained with fluorescently labeled EGFR antibody, and the results were detected by flow cytometry. Figure 11 As shown in Figure A, the EGFR positivity rate is 89.4%, which means the CAR transduction positivity rate is 89.4%.

[0215] (2) CD69 expression level in CAR-B7-H3(1B4)-αβT cells

[0216] To analyze the CD69 expression level of CAR-B7-H3(1B4)-αβT cells, effector cells (including αβT-control cells without CAR-B7-H3 expression and CAR-B7-H3-αβT cells expressing CAR-B7-H3) were incubated with tumor cells (RKO WT and RKO B7-H3 KO) at an effector-target ratio of 1:1. Specifically, incubation was performed in 24-well plates with 0.5 × 10⁻⁶ cells added. 6 Effector cells and 0.5 × 10 6 Tumor cells. Flow cytometry analysis was performed after 24 hours of incubation. The flow cytometry staining system consisted of anti-human CD3, anti-human αβTCR, anti-human EGFR, and anti-human CD69.

[0217] The results are as follows Figure 11 As shown in Figure B, RKO WT tumor cells significantly increased CD69 expression in CAR-B7-H3(1B4)-αβT cells, while RKO tumor cells lacking B7-H3 did not affect CD69 expression in CAR-B7-H3(1B4)-αβT cells. Furthermore, CAR-B7-H3 expression in αβT cells did not induce upregulation of their own CD69 baseline signal.

[0218] (3) In vitro killing ability of CAR-B7-H3(1B4)-αβT cells against RKO tumor cells

[0219] In vitro cell killing assays were performed using Luciferase fluorescence detection. Tumor cells to be tested (including RKO WT and B7-H3 knockout RKO cells (RKO B7-H3 KO)) stably expressed Luciferase. Killing assays were performed in 24-well plates with different effector-to-target ratios of 0.5:1, 1:1, and 3:1. For an effector-to-target ratio of 0.5:1, tumor cells and effector cells were counted separately, and both cell types were resuspended in their respective culture media. Effector cells were resuspended to a density of 0.25 × 10⁻⁶. 6 Cells / mL, tumor cells resuspended to 0.5 × 10⁻⁶. 6 Cells / mL. For an effector-to-target ratio of 1:1, both effector cells and tumor cells were resuspended to 0.5 × 10⁻⁶ cells / mL. 6 Cells / mL. For an effector-to-target ratio of 3:1, effector cells were resuspended to 1.5 × 10⁻⁶ cells / mL. 6 Cells / mL, tumor cells resuspended to 0.5 × 10⁻⁶. 6Cells / mL. Add 500 μL of tumor cells and 500 μL of effector cells to the same well, and mix thoroughly. Set up a separate control well containing only tumor cells, adding 500 μL of tumor cell culture medium and 500 μL of effector cell culture medium. Incubate in a cell culture incubator, and detect Luciferase fluorescence signal after 24 h.

[0220]

[0221] The results are shown in Figure 12 As can be seen, compared with the control group αβT cells (αβT), CAR-B7-H3-expressing αβT cells have a good in vitro killing effect on RKO tumor cells, and their killing ability gradually increases with the increase of effector-target ratio. However, they have no killing effect on RKO tumor cells lacking B7-H3. This further verifies that the killing effect of CAR-B7-H3-αβT cells on RKO tumor cells is achieved by recognizing B7-H3.

[0222] (4) In vitro killing ability of CAR-B7-H3(1B4)-αβT cells against glioma cells

[0223] Using the same experimental method as in (3), this patent further examined the killing ability of αβT cells expressing CAR-B7-H3 against glioma tumor cells (including HTB1 5 and TJ905).

[0224] The results are as follows Figure 13 As shown, αβT cells expressing CAR-B7-H3 also exhibit a strong killing effect on glioma tumor cells (including HTB15 and TJ905), with their killing ability gradually increasing with the effector-to-target ratio. In contrast, αβT cells not expressing CAR-B7-H3 (αβT) have no killing ability against these two glioma tumor cell lines. This further verifies that the killing effect of CAR-B7-H3-αβT cells on the two glioma tumor cell lines is mediated by the recognition of B7-H3.

[0225] Example 9: CAR transduction positivity rate and functional assay of γδT cells expressing CAR-B7-H3(1B4)

[0226] (1) CAR transduction positivity rate

[0227] CAR-B7-H3(1B4)-γδT cells were labeled and stained with fluorescently labeled EGFR antibody, and the results were detected by flow cytometry. The results are shown below. Figure 14 As can be seen, the EGFR positivity rate is 45.9%, which means the CAR transduction positivity rate is 45.9%.

[0228] (2) Killing effects of CAR-αβT and CAR-γδT expressing CAR-B7-H3(1B4) on RKO WT tumor cells

[0229] Using the experimental method in Example 8 (3), the killing effects of CAR-αβT and CAR-γδT expressing CAR-B7-H3 on RKO WT tumor cells were further analyzed and compared.

[0230] The results are shown in Figure 15 It can be seen that the killing ability of CAR-B7-H3-αβT cells is significantly higher than that of αβT cells that do not express CAR-B7-H3. Figure 15 A) The killing ability of CAR-B7-H3-αβT cells against B7-H3 knockout RKO tumor cells is comparable to that of αβT cells that do not express CAR-B7-H3. Figure 15 B); CAR-B7-H3-γδT cells showed significantly higher killing ability against RKO tumor cells than γδT cells that did not express CAR-B7-H3. Figure 15 A) The tumor-killing ability of CAR-B7-H3-γδT cells against B7-H3 knockout RKO tumor cells is comparable to that of γδT cells that do not express CAR-B7-H3. Figure 15 B). This further confirms that CAR-B7-H3-αβT cells and CAR-B7-H3-γδT cells enhance their killing effect on RKO tumor cells by recognizing B7-H3.

[0231] Furthermore, it can be seen that under the same transduction positivity rate (by adding αβT cells that do not express CAR to CAR-B7-H3-αβT cells to lower their transduction positivity rate to be consistent with that of CAR-B7-H3-γδT) and the same effector-target ratio, CAR-B7-H3-γδT cells have a significantly higher killing ability against RKO tumor cells than CAR-B7-H3-αβT cells.

[0232] Furthermore, γδT cells that do not express CAR-B7-H3 also have a certain killing ability against RKO tumor cells. Figure 15 A) and significantly higher than αβT cells that do not express CAR-B7-H3. This further indicates that γδT cells have a higher anti-tumor capacity than αβT cells.

[0233] Example 10: Examination of the functional differences of CAR-γδT from two different antibody sources

[0234] Using the experimental method in Example 8 (3), the killing effect of γδT expressing CAR-B7-H3 (1B4) and CAR-B7-H3 (2Y31) on RKO WT tumor cells was further analyzed and compared.

[0235] The results are shown in Figure 16 As can be seen, CAR-B7-H3-γδT, regardless of whether it is scFv derived from 1B4 or 2Y31 antibodies, exhibits tumor-killing ability against SKOV3, and its killing ability is significantly higher than that of unmodified γδT. There is no significant difference in the killing ability of CAR-B7-H3-γδT against SKOV3 between the two antibody sources. Under all killing conditions, the antitumor ability of CAR-B7-H3-γδT from both antibody sources increases with increasing effector-to-target ratio.

[0236] Example 11: In vivo anti-tumor ability experiment

[0237] This experiment used an animal model of peritoneal tumor formation. Each mouse was intraperitoneally inoculated with 1×10⁻⁶ mol / L. 6 SKOV3 tumor cells (stable expression of Luciferase), of which 1×10 6 SKOV3 tumor cells were resuspended in 200 μL PBS and injected intraperitoneally (the day of inoculation was designated as day 0). On day 4 after SKOV3 tumor cell inoculation, body weight was measured and tumor in vivo imaging analysis was performed. Based on body weight and tumor size data, the patients were randomly divided into 3 groups: control group, γδT treatment group, and CAR-B7-H3(1B4)-γδT treatment group.

[0238] Cellular intervention therapy was administered on day 5 after inoculation. In the control group, each mouse received an intraperitoneal injection of 200 μL PBS, while in the γδT treatment group, each mouse received an intraperitoneal injection of 1 × 10⁻⁶ PBS. 6 γδT cells (resuspended in 200 μL PBS) were administered intraperitoneally to each mouse in the CAR-B7-H3-γδT treatment group at a dose of 1 × 10⁻⁶ cells. 6 CAR-B7-H3(1B4)-γδT cells (resuspended in 200 μL PBS). Tumor in vivo imaging analysis was performed periodically to observe mouse survival and to statistically analyze survival curves.

[0239] For in vivo tumor imaging: Mice were anesthetized with isoflurane and injected intraperitoneally with 100 μL of luciferin substrate (2 mg) using an insulin syringe. After 10 minutes, images were captured using an IVIS fluorescence imaging system, the images were saved, and the fluorescence values ​​were counted.

[0240] Experimental results

[0241] (1) Tumor growth in mice

[0242] refer to Figure 17 and Figure 18 It was found that, in the SKOV3 tumor model with peritoneal tumor formation, intraperitoneal infusion of CAR-B7-H3-γδT cells significantly inhibited tumor growth. Unmodified γδT cells showed some inhibitory ability against tumors, but the effect was not significant.

[0243] (2) Survival status of tumor model mice

[0244] refer to Figure 19 It can be seen that all tumor-bearing mice in the control group died at around 85 days; the survival time of mice in the γδT treatment group was extended by about 10 days, to 95 days; while the survival time of mice in the CAR-B7-H3-γδT treatment group was significantly increased, and they were still able to maintain an 80% survival rate at 125 days.

Claims

1. An isolated antibody or antibody fragment that specifically binds to B7-H3, comprising a heavy chain variable region VH and a light chain variable region VL. The heavy chain variable region VH includes: VHCDR1 as shown in SEQ ID NO:9, VHCDR2 as shown in SEQ ID NO:10, and VHCDR3 as shown in SEQ ID NO:11; The light chain variable region VL includes: VLCDR1 as shown in the amino acid sequence of SEQ ID NO:12, VLCDR2 as shown in the amino acid sequence of SEQ ID NO:13, and VLCDR3 as shown in the amino acid sequence of SEQ ID NO:

14.

2. An isolated antibody or antibody fragment that specifically binds to B7-H3, comprising a heavy chain variable region VH and a light chain variable region VL. The heavy chain variable region VH includes VHCDR1 as shown in the amino acid sequence of SEQ ID NO:15, VHCDR2 as shown in the amino acid sequence of SEQ ID NO:16, and VHCDR3 as shown in the amino acid sequence of SEQ ID NO:

17. The light chain variable region VL includes: VLCDR1, as shown by the amino acid sequence of SEQ ID NO:18; VLCDR2, as shown by the amino acid sequence of SEQ ID NO:19; and VLCDR3, as shown by the amino acid sequence of SEQ ID NO:

20.

3. The isolated antibody or antibody fragment that specifically binds to B7-H3 as described in claim 1 or 2, wherein, The heavy chain variable region VH is selected from any one of the following: the amino acid sequence shown in SEQ ID NO:1, an amino acid sequence having more than 80% homology with the amino acid sequence shown in SEQ ID NO:1 outside the CDR region, the amino acid sequence shown in SEQ ID NO:2, and an amino acid sequence having more than 80% homology with the amino acid sequence shown in SEQ ID NO:2 outside the CDR region.

4. The isolated antibody or antibody fragment that specifically binds to B7-H3 as described in claim 1 or 2, wherein, The light chain variable region VL is selected from any one of the following: the amino acid sequence shown in SEQ ID NO:3, an amino acid sequence having more than 80% homology with the amino acid sequence shown in SEQ ID NO:3 outside the CDR region, the amino acid sequence shown in SEQ ID NO:4, and an amino acid sequence having more than 80% homology with the amino acid sequence shown in SEQ ID NO:4 outside the CDR region.

5. An isolated antibody or antibody fragment that specifically binds to B7-H3, comprising: (i) the heavy chain variable region VH shown in SEQ ID NO:1 and the light chain variable region VL shown in SEQ ID NO:3, or (ii) The heavy chain variable region VH shown in SEQ ID NO:2 and the light chain variable region VL shown in SEQ ID NO:

4.

6. The isolated antibody or antibody fragment that specifically binds to B7-H3 as described in any one of claims 1 to 2 or 5, wherein, The antibody or antibody fragment is a genetically engineered antibody or antibody fragment.

7. The isolated antibody or antibody fragment that specifically binds to B7-H3 as described in any one of claims 1 to 2 or 5, wherein, The antibody or antibody fragment is scFv.

8. A chimeric antigen receptor comprising: the antibody or antibody fragment that specifically binds to B7-H3 as described in claim 7.

9. The chimeric antigen receptor of claim 8, comprising: the antibody or antibody fragment of claim 7, and a transmembrane region fused to the carboxyl terminus of the antibody or antibody fragment.

10. The chimeric antigen receptor of claim 8 or 9, comprising: the antibody or antibody fragment of claim 7, a transmembrane region fused to the carboxyl terminus of the antibody or antibody fragment, and an immunologically active cell activation signal transduction region fused to the carboxyl terminus of the transmembrane region.

11. The chimeric antigen receptor according to any one of claims 8 to 9, further comprising one or more of membrane proteins, secretory proteins, intracellular proteins, small molecule drugs, and cytotoxic drugs.

12. A nucleic acid molecule comprising: a nucleotide sequence encoding an antibody or antibody fragment of any one of claims 1 to 7 or a chimeric antigen receptor of any one of claims 8 to 9.

13. The nucleic acid molecule of claim 12, comprising: (i) The nucleotide sequence encoding the heavy chain variable region as shown in SEQ ID NO:5, and the nucleotide sequence encoding the light chain variable region as shown in SEQ ID NO:7, or, (ii) The nucleotide sequence encoding the heavy chain variable region as shown in SEQ ID NO:6, and the nucleotide sequence encoding the light chain variable region as shown in SEQ ID NO:

8.

14. A vector comprising the nucleic acid molecule of claim 12 or 13.

15. The vector as described in claim 14, wherein it is a lentiviral vector, a retroviral vector, an adenovirus vector, or an adeno-associated virus vector.

16. A cell comprising the nucleic acid molecule of claim 12 or 13 or the vector of claim 14 or 15; said cell being selected from autologous or allogeneic T cells, B cells, NK cells, macrophages, monocytes, dendritic cells, neutrophils, basophils, eosinophils, mast cells, NK-T cells or MAIT cells.

17. The cell of claim 16, wherein the T cell is selected from αβT cells, γδT cells, or regulatory T cells.

18. A pharmaceutical composition, It comprises: an antibody or antibody fragment selected from any one of claims 1 to 7, a chimeric antigen receptor selected from any one of claims 8 to 11, a nucleic acid molecule selected from claim 12 or 13, a vector selected from claim 14 or 15, and any one or more of the cells selected from claim 16 or 17, or... It comprises: an antibody or antibody fragment selected from any one of claims 1 to 7, a chimeric antigen receptor selected from any one of claims 8 to 11, a nucleic acid molecule selected from claim 12 or 13, a vector selected from claim 14 or 15, a cell-derived molecule selected from claim 16 or 17, and a pharmaceutically acceptable carrier, or, It comprises: an antibody or antibody fragment selected from any one of claims 1 to 7, a chimeric antigen receptor selected from any one of claims 8 to 11, a nucleic acid molecule selected from claim 12 or 13, a vector selected from claim 14 or 15, a cell selected from claim 16 or 17, and other pharmaceutically active reagents or drugs.

19. The use of the antibody or antibody fragment of any one of claims 1 to 7, the chimeric antigen receptor of any one of claims 8 to 11, the nucleic acid molecule of claim 12 or 13, the vector of claim 14 or 15, or the cell of claim 16 or 17 in the preparation of a medicament for treating B7-H3 positive diseases; wherein the B7-H3 positive diseases are selected from acute myeloid leukemia, chronic myeloid leukemia, melanoma, lung cancer, colorectal cancer, bladder cancer, prostate cancer, liver cancer, ovarian cancer, pancreatic cancer, endometrial cancer, gastric cancer, kidney cancer, cervical cancer, head and neck cancer, nasopharyngeal carcinoma, basal cell carcinoma of the skin, squamous cell carcinoma of the skin, glioblastoma, or glioma.

20. A non-diagnostic detection method for B7-H3, comprising: The step of using the antibody or antibody fragment of any one of claims 1 to 7, the chimeric antigen receptor of any one of claims 8 to 11, or the nucleic acid molecule of claim 12 or 13 to detect B7-H3.

21. A kit for detecting B7-H3, comprising the antibody or antibody fragment or its marker as described in any one of claims 1 to 7, the chimeric antigen receptor as described in any one of claims 8 to 11, or the nucleic acid molecule as described in claim 12 or 13.

22. An isolation kit for separating B7-H3 positive cells, comprising an antibody or antibody fragment or a marker thereof as described in any one of claims 1 to 7, a chimeric antigen receptor or a marker thereof as described in any one of claims 8 to 11, or a nucleic acid molecule or a marker thereof as described in claim 12 or 13.

23. A method for enhancing cell function in vitro, comprising: The step of contacting cells with the antibody or antibody fragment or its marker as described in any one of claims 1 to 7, wherein the cells are selected from T cells, B cells, NK cells, macrophages, monocytes, dendritic cells, neutrophils, basophils, eosinophils, mast cells, NK-T cells or MAIT cells.

24. Use of the antibody or antibody fragment of any one of claims 1 to 7 in the preparation of articles for detecting B7-H3 protein.

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