Chimeric antigen receptors carrying DNase I or DNase IL3 and related uses thereof

Abstract

The invention discloses a chimeric antigen receptor carrying DNase I or DNase IL3 and related application thereof, the invention finds that NETs can significantly inhibit the anti-tumor function of B7H3-CAR-T for the first time, and targeted removal of NETs can effectively improve the anti-lung cancer curative effect of B7H3-CAR-T. Based on this, the anti-lung cancer effect of B7H3-CAR-T can be effectively improved. The invention provides a brand-new B7H3-targeted enhanced chimeric antigen receptor carrying DNase I or DNase IL3, an enhanced chimeric antigen receptor immune cell, a pharmaceutical composition, a biological preparation and other immunotherapy products for the field, provides a new insight and thought for CAR-T clinical treatment of lung cancer or other types of solid tumors, and has a wide clinical application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular, relates to a chimeric antigen receptor carrying DNase I or DNase IL3 and related uses thereof. Background Art

[0002] Chimeric antigen receptor T (CAR-T) cell therapy has been proven to be an effective means of controlling tumor progression, but its efficacy in solid tumors has not met clinical expectations. So far, more than 50 clinical trials have explored the possibility of CAR-T cell treatment of lung cancer, with targets mainly focused on tumor-associated antigens such as MSLN, CEA, MUC1, and EGFR. However, from the data currently published, the efficacy is not significant. The reason is that immunosuppressive factors in the tumor microenvironment (TME), such as immunosuppressive cells (regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs), and tumor-associated macrophages, etc.) and inhibitory cytokines (IL-10, IL-17, TGF-β), restrict the efficacy of CAR-T in solid tumors. Studies have found that neutrophils are the largest immune cell subset in lung cancer TME. Under the influence of TME, neutrophils can release extracellular traps (NETs) to participate in the progression of lung cancer.

[0003] Recent studies have confirmed that NETs can participate in the regulation of the body's immune function. + NETs can metabolize tumor-infiltrating CD4 + and CD8 + The expression of multiple inhibitory receptors on T cells is upregulated, such as PD-1, Tim3, and Lag3, inducing dysfunction. In addition, NETs contain PD-L1 protein components, which inhibit the anti-tumor effect of infiltrating T cells in the tumor by binding to PD-1. Recent studies have found that NETs can act as a "physical barrier" to limit T cell infiltration in the tumor and antagonize CD8 + T cell function and promote the progression of various tumors. However, it is still unclear whether NETs affect the efficacy of CAR-T cells against solid tumors. This study mainly explored whether the elimination of NETs can improve the anti-tumor function of B7H3-CAR-T cells and provide new strategies and ideas for improving the therapeutic efficacy of CAR-T cells.

[0004] B7H3 protein belongs to the type I transmembrane protein of the B7 family. Although its specific function in tumor development is still unclear, studies have continuously confirmed that it is overexpressed in many tumors and is almost not expressed in normal tissues. Therefore, it is expected to become an ideal target for CAR-T cell therapy of solid tumors. Studies have shown that lung cancer tissues highly express B7H3 protein, while normal tissues hardly express it; high expression of B7H3 is closely related to the metastasis and poor prognosis of lung cancer, suggesting that B7H3 is a potential target for CAR-T cell therapy of lung cancer. Studies have confirmed that endonucleases in serum can effectively degrade the chromatin components of NETs and remove the DNA backbone structure of NETs. Based on DNase I treatment, the removal of NETs can significantly inhibit local tumor progression and reduce tumor recurrence and metastasis. In addition, the removal of NETs by DNase I can effectively improve the efficacy of anti-PD-1 treatment of colorectal cancer. DNase I is a type of nuclease produced by the human body that digests single-stranded or double-stranded DNA. Family members include DNase I, DNase IL1, DNase IL2 and DNase IL3, among which DNase I and DNase IL3 play a key role in the degradation of NETs. Summary of the invention

[0005] In order to overcome the research gap in the prior art as to whether NETs affect the efficacy of CAR-T cells against solid tumors, the purpose of the present invention is to provide a chimeric antigen receptor targeting B7H3 and carrying DNase I or DNase IL3 and related uses thereof.

[0006] In the present invention, based on the B7H3 target, the inventors evaluated the effect of NETs on the efficacy of CAR-T cells in treating lung cancer; at the same time, the possibility of using DNase I and DNase IL3 to remove NETs to improve the efficacy of CAR-T in lung cancer was explored. The results of this study confirmed that NETs can significantly inhibit the anti-tumor function of B7H3-CAR-T, and targeted removal of NETs can effectively improve the anti-lung cancer efficacy of B7H3-CAR-T, providing new insights and ideas for the clinical treatment of lung cancer or other types of solid tumors with CAR-T.

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

[0008] In a first aspect, the present invention provides a chimeric antigen receptor carrying DNase I or DNase IL3 targeting B7H3, wherein the chimeric antigen receptor comprises an antibody targeting B7H3, and DNase I and / or DNase IL3;

[0009] The antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1-3, and the light chain variable region comprises LCDR1-3; the HCDR1-3 are the HCDR1-3 in the heavy chain variable region as shown in SEQ ID NO:4; and the LCDR1-3 are the LCDR1-3 in the light chain variable region as shown in SEQ ID NO:9;

[0010] The sequences of HCDR1-3 and LCDR1-3 corresponding to the heavy chain variable region and light chain variable region of the antibody of the present invention can be obtained based on the above heavy chain variable region and light chain variable region sequences according to the Kabat, IMGT, Chothia, AbM or Contact numbering system. The CDR sequences defined according to any one or any combination of the Kabat, IMGT, Chothia, AbM, Contact numbering systems or other numbering systems are also included in the protection scope of the present invention.

[0011] The amino acid sequence of the DNase I is as shown in SEQ ID NO:27 or an amino acid sequence having at least 80% homology with SEQ ID NO:27; the amino acid sequence of the DNase IL3 is as shown in SEQ ID NO:29 or an amino acid sequence having at least 80% homology with SEQ ID NO:29. Further, the amino acid sequences of the HCDR1-3 are as shown in SEQ ID NO:1-3, respectively; alternatively, the amino acid sequences of the LCDR1-3 are as shown in SEQ ID NO:6-8, respectively; alternatively, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:4 or an amino acid sequence having at least 80% homology with SEQ ID NO:4; alternatively, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:9 or an amino acid sequence having at least 80% homology with SEQ ID NO: NO:9 has an amino acid sequence with at least 80% homology; optionally, the chimeric antigen receptor further comprises a transmembrane domain, an intracellular signaling domain, a hinge region, and a co-stimulatory signaling domain; optionally, the chimeric antigen receptor further comprises any one or more of a signal peptide, a self-cleaving peptide, a safety switch, an immunomodulatory molecule or a cytokine; optionally, the transmembrane domain is selected from the transmembrane domain of any one of the following molecules: CD8, 4-1BB, CD28, CD34, CD3ε, PD-1, IgG1, IgG4, OX40, IL-2 receptor, IL-11 receptor; optionally, the intracellular signaling domain is selected from the intracellular signaling domain of any one of the following molecules: CD3ζ, FcRγ, FcRβ, TCRζ, CD3γ, CD3δ, CD3ε, CD4, CD5, CD8, CD21, CD22, CD79a, CD79b, CD278, FcεRI, DAP10, DAP12 ; Optionally, the hinge region is selected from the hinge region of any one of the following molecules: CD8, 4-1BB, CD28, CD34, OX40, CD3ε, IgG1, IgG4, PD-1, IL-2 receptor, IL-7 receptor, IL-11 receptor; Optionally, the costimulatory signaling domain is selected from the costimulatory signaling domain of any one of the following molecules: CD28, 4-1BB, CD19, CD4, CD27, ICOS, CD8α, CD8β, HVEM, LIGHT, NKp30, NKp46, CD40, CD30, TIM1, CD2, CD7, CD226; Optionally, the signal peptide is selected from the signal peptide of any one of the following molecules: α chain and β chain of T cell receptor, CD3ζ, CD33, CD3ε, CD16, CD22, CD28, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, GITR;Optionally, the self-cleaving peptide is selected from any one of the following self-cleaving peptides: T2A, P2A, E2A, F2A; Optionally, the safety switch is selected from any one of the following safety switches: tEGFR, iCaspase-9, RQR8; Optionally, the immunomodulatory molecule or cytokine is selected from any one of the following immunomodulatory molecules or cytokines: B7.1, CCL19, CCL21, CD40L, CD137L, GITRL, GM-CSF, IL-2, IL-15, IL-18, IL-12, IL-21, LEC, OX40L; Optionally, the chimeric antigen receptor is selected from any one of the following groups:;

[0012] (1) A chimeric antigen receptor obtained by sequentially connecting a signal peptide, a heavy chain variable region of the antibody, a linker, a light chain variable region of the antibody, a CD8 hinge region and a transmembrane domain, a CD28 co-stimulatory signal domain, a CD3ζ intracellular signal transduction domain, a T2A self-cleaving peptide, and DNAseI;

[0013] (2) a chimeric antigen receptor obtained by sequentially connecting a signal peptide, a heavy chain variable region of the antibody, a linker, a light chain variable region of the antibody, a CD8 hinge region and a transmembrane domain, a CD28 co-stimulatory signal domain, a CD3ζ intracellular signal transduction domain, a T2A self-cleaving peptide, and DNAseIL3;

[0014] (3) a derivative chimeric antigen receptor formed by replacing, deleting or adding one or more amino acids based on the amino acid sequence of the chimeric antigen receptor described in (1) or (2);

[0015] Optionally, the Linker is (G4S)3; optionally, the amino acid sequence of the signal peptide is as shown in SEQ ID NO:11; optionally, the amino acid sequence of the Linker is as shown in SEQ ID NO:15; optionally, the amino acid sequence of the CD8 hinge region and transmembrane domain is as shown in SEQ ID NO:19; optionally, the amino acid sequence of the CD28 co-stimulatory signal domain is as shown in SEQ ID NO:21; optionally, the amino acid sequence of the CD3ζ intracellular signaling domain is as shown in SEQID NO:23; optionally, the amino acid sequence of the T2A self-cleaving peptide is as shown in SEQ ID NO:25; optionally, the amino acid sequence of the chimeric antigen receptor is as shown in SEQ ID NO:31 or SEQ ID NO:33.

[0016] In some embodiments, those skilled in the art can change the combination category and sequence of signal peptides, hinge regions and transmembrane regions, co-stimulatory signal domains and intracellular signal transduction domains according to actual conditions or needs. Regardless of the form of the change, as long as the chimeric antigen receptor has the antibody targeting B7H3 as described in the first aspect of the present invention, and the sequence of DNAse I and / or DNAse IL3, it is included in the protection scope of the present invention.

[0017] In some embodiments, the chimeric antigen receptor provided by the present invention includes not only the chimeric antigen receptor whose amino acid sequence is as described in the first aspect of the present invention, but also the mutant of the chimeric antigen receptor described in the first aspect of the present invention. These mutants include: an amino acid sequence having at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 97% sequence identity or homology with the amino acid sequence of the chimeric antigen receptor described in the first aspect of the present invention and retaining the biological activity (such as activation of T cells) of the chimeric antigen receptor. For example, NCBI's BLASTp can be used to calculate the sequence identity between the two aligned sequences. That is, the mutants obtained by mutation based on the amino acid sequence / nucleotide sequence corresponding to the chimeric antigen receptor described in the first aspect of the present invention are also included in the protection scope of the present invention.

[0018] In a second aspect, the present invention provides a nucleic acid molecule encoding the chimeric antigen receptor described in the first aspect of the present invention;

[0019] Optionally, the nucleotide sequence encoding the heavy chain variable region of the antibody in the chimeric antigen receptor is as shown in SEQ ID NO: 14; optionally, the nucleotide sequence encoding the light chain variable region of the antibody in the chimeric antigen receptor is as shown in SEQ ID NO: 18; optionally, the nucleotide sequence encoding the signal peptide in the chimeric antigen receptor is as shown in SEQ ID NO: 12; optionally, the nucleotide sequence encoding the Linker in the chimeric antigen receptor is as shown in SEQ ID NO: 16; optionally, the nucleotide sequence encoding the CD8 hinge region and transmembrane domain in the chimeric antigen receptor is as shown in SEQ ID NO: 20; optionally, the nucleotide sequence encoding the CD28 co-stimulatory signal domain in the chimeric antigen receptor is as shown in SEQ ID NO: 22; optionally, the nucleotide sequence encoding the CD3ζ intracellular signaling domain in the chimeric antigen receptor is as shown in SEQ ID NO: 24; optionally, the nucleotide sequence encoding the T2A self-cleaving peptide in the chimeric antigen receptor is as shown in SEQ ID NO: 26; optionally, the nucleotide sequence encoding DNAse I in the chimeric antigen receptor is as shown in SEQ ID NO:28; optionally, the nucleotide sequence encoding the DNAse IL3 in the chimeric antigen receptor is shown in SEQ ID NO:30; optionally, the nucleotide sequence encoding the chimeric antigen receptor is shown in SEQ ID NO:32 or SEQ ID NO:34.

[0020] Nucleic acid molecules of the present invention generally refer to polymers of DNA or RNA, which can be single-stranded or double-stranded, synthesized or obtained, and can include natural, non-natural or changed nucleotides, and can include natural, non-natural or changed internucleotide bonding, such as phosphoramidate bonding or thiophosphate bonding. In some embodiments, nucleic acid molecules do not include any insertion, deletion, inversion and / or substitution. However, as described herein, in some cases, nucleic acid molecules may be suitable to include one or more insertions, deletions, inversions and / or substitutions. In some embodiments, nucleic acid molecules can encode other amino acid sequences that do not affect the function of polypeptides, proteins, chimeric antigen receptors and can be translated or not translated by host cells after nucleic acid expression.

[0021] In a third aspect, the present invention provides an expression vector comprising the nucleic acid molecule described in the second aspect of the present invention; optionally, the vector is a DNA vector, an RNA vector, a plasmid, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, a retroviral vector or a transposon system.

[0022] In a fourth aspect, the present invention provides a genetically engineered immune cell comprising the nucleic acid molecule described in the second aspect of the present invention or the expression vector described in the third aspect of the present invention; alternatively, the genetically engineered immune cell expresses the chimeric antigen receptor described in the first aspect of the present invention; alternatively, the immune cell is a T cell, a B cell, a NK cell, an iNKT cell, a CTL cell, a dendritic cell, a myeloid cell, a monocyte, a macrophage, or any combination thereof. In some embodiments, the vector described in the present invention can be any suitable vector and can be used to transform or transfect any suitable host cell. Those skilled in the art can make conventional selections according to actual needs.

[0023] In a fifth aspect, the present invention provides a pharmaceutical composition, comprising the chimeric antigen receptor described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, the expression vector described in the third aspect of the present invention and / or the genetically engineered immune cell described in the fourth aspect of the present invention; optionally, the pharmaceutical composition comprises DNase I and / or DNase IL3 as described above, and a chimeric antigen receptor immune cell targeting B7H3; optionally, the chimeric antigen receptor in the chimeric antigen receptor immune cell targeting B7H3 is obtained by sequentially connecting the signal peptide described in the first aspect of the present invention, the heavy chain variable region of the antibody, Linker, the light chain variable region of the antibody, the CD8 hinge region and transmembrane domain, the CD28 co-stimulatory signal domain, and the CD3ζ intracellular signal transduction domain in series; optionally, the immune cell is a T cell, a B cell, a NK cell, an iNKT cell, a CTL cell, a dendritic cell, a myeloid cell, a monocyte, a macrophage or any combination thereof.

[0024] In some embodiments, the present invention discovers for the first time that the combination of DNase I and / or DNase IL3 as described above, and chimeric antigen receptor immune cells targeting B7H3 has a synergistic therapeutic effect on B7H3-positive tumors.

[0025] In a sixth aspect, the present invention provides a biological preparation, which comprises the pharmaceutical composition described in the fifth aspect of the present invention. In some embodiments, the present invention has no particular restrictions on the specific dosage form, administration route and administration dose of the pharmaceutical composition or pharmaceutical preparation, and those skilled in the art can make routine selections according to the actual conditions of the subject. In some embodiments, the administration route includes, but is not limited to, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intranasal, intrapulmonary, rectal, intraperitoneal, etc.

[0026] In a seventh aspect, the present invention provides a kit, which comprises the nucleic acid molecule described in the second aspect of the present invention and / or the expression vector described in the third aspect of the present invention; optionally, the kit also includes a reagent for introducing the nucleic acid molecule and expression vector into immune cells.

[0027] In an eighth aspect, the present invention provides a method for preparing the genetically engineered immune cells described in the fourth aspect of the present invention, the method comprising the following steps: introducing the nucleic acid molecule described in the second aspect of the present invention or the expression vector described in the third aspect of the present invention into the immune cells; optionally, the introduction method is calcium phosphate transfection, DEAE-dextran-mediated transfection, microinjection, electroporation, TALEN, ZFN method, liposome-mediated transfection, lentiviral infection, retroviral infection, adenoviral infection, transposon technology or CRISPR-Cas9 technology.

[0028] In addition, the present invention also provides a method for treating and / or preventing B7H3-positive related diseases, the method comprising the following steps: administering a therapeutically and / or preventively effective amount of the genetically engineered immune cells, pharmaceutical compositions and / or biological preparations as described above of the present invention to a subject in need.

[0029] In some embodiments, the subject includes humans and non-human animals. Non-human animals include all vertebrates (e.g., mammals and non-mammals) such as non-human primates (e.g., cynomolgus monkeys), sheep, dogs, cows, chickens, amphibians and reptiles. In some embodiments, the subject is preferably a human.

[0030] In a ninth aspect, the present invention provides any of the following applications:

[0031] (1) Use of the chimeric antigen receptor according to the first aspect of the present invention, the nucleic acid molecule according to the second aspect of the present invention, the expression vector according to the third aspect of the present invention, the genetically engineered immune cell according to the fourth aspect of the present invention, the pharmaceutical composition according to the fifth aspect of the present invention, the biological preparation according to the sixth aspect of the present invention and / or the kit according to the seventh aspect of the present invention in the preparation of a drug for treating and / or preventing a B7H3-positive related disease;

[0032] (2) Use of the chimeric antigen receptor described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, the expression vector described in the third aspect of the present invention, the genetically engineered immune cell described in the fourth aspect of the present invention, the pharmaceutical composition described in the fifth aspect of the present invention, and / or the kit described in the seventh aspect of the present invention in the preparation of a biological agent for treating and / or preventing a B7H3-positive related disease;

[0033] (3) Use of the chimeric antigen receptor described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, and / or the expression vector described in the third aspect of the present invention in the preparation of a kit for preparing genetically engineered immune cells for treating and / or preventing B7H3-positive related diseases;

[0034] (4) Use of the chimeric antigen receptor described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, the expression vector described in the third aspect of the present invention, and / or the kit described in the seventh aspect of the present invention in the preparation of genetically engineered immune cells for treating and / or preventing B7H3-positive related diseases;

[0035] (5) Use of the kit according to the sixth aspect of the present invention in the preparation of genetically engineered immune cells for treating and / or preventing B7H3-positive related diseases;

[0036] (6) Use of NETs degraders in combination with chimeric antigen receptor immune cells targeting B7H3 in the preparation of drugs for treating and / or preventing B7H3-positive related diseases;

[0037] (7) Use of NETs degraders in the preparation of drugs for enhancing the anti-tumor efficacy of chimeric antigen receptor immune cells;

[0038] (8) Use of NETs degraders in the preparation of drugs for increasing the number of intratumoral infiltrating chimeric antigen receptor immune cells;

[0039] Optionally, the NETs degrader is DNase I, DNase IL3, DNase IL1 and / or DNase IL2; Optionally, the NETs degrader is DNase I and / or DNase IL3; Optionally, the chimeric antigen receptor immune cell is a chimeric antigen receptor immune cell targeting B7H3 as described in the fifth aspect of the present invention; Optionally, the B7H3-positive related disease is lung cancer, ovarian cancer, prostate cancer, liver cancer, kidney cancer, breast cancer, colorectal cancer, esophageal cancer, oral cancer, gastric cancer, pancreatic cancer, endometrial cancer, bladder cancer, osteosarcoma, glioma or myeloid leukemia. The B7H3-positive related diseases described in the present invention are not limited to the above diseases, and any disease and / or condition related to the expression of B7H3 is within the scope of protection of the present invention.

[0040] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0041] The present invention discovers for the first time that NETs can significantly inhibit the anti-tumor function of B7H3-CAR-T, and targeted clearance of NETs can effectively improve the anti-lung cancer efficacy of B7H3-CAR-T. Based on this, the present invention provides a novel immunotherapy product in the art, including an enhanced chimeric antigen receptor (CAR) targeting B7H3 and carrying DNase I or DNase IL3, an enhanced chimeric antigen receptor immune cell (CAR-T), a pharmaceutical composition, a biological preparation, etc., which provides new insights and ideas for the clinical treatment of lung cancer or other types of solid tumors with CAR-T, and has broad prospects for clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 : Targeting NETs can inhibit the progression of LLC subcutaneous transplanted tumors, where A: subcutaneous transplanted tumor entity image; B: subcutaneous transplanted tumor volume change curve; C: subcutaneous transplanted tumor weight statistical chart, *P < 0.05;

[0043] Figure 2 : Targeting NETs increases the number of tumor-infiltrating T cells and improves their effector function in C57BL / 6 mice, where A: absolute value of tumor-infiltrating CD8+ T cells; B: absolute value of tumor-infiltrating CD4 + Absolute value of T cells; Figure C: Flow cytometry detection of infiltrating IFN-γ in tumor tissue + / CD8 + T cell ratio; Figure D: Flow cytometry detection of infiltrated IL-2 in tumor tissue + / CD8 + T cell ratio; Figure E: Flow cytometry detection of TNF-α infiltrating in tumor tissue + / CD8 + T cell ratio. *P<0.05, **P<0.01;

[0044] Figure 3 : Expression of B7H3 antigen in lung cancer tissues and lung cancer cells, where A: Immunohistochemistry to detect B7H3 expression in clinical lung cancer samples; B: Flow cytometry to detect B7H3 expression in lung cancer cells;

[0045] Figure 4 :B7H3-CAR-T cells were successfully prepared, where A: Schematic diagram of B7H3-CAR-T structure; B: CAR positivity of B7H3-CAR-T cells detected by flow cytometry; C: Proliferation curves of T cells and B7H3-CAR-T cells, n=3; ns, no statistical significance;

[0046] Figure 5: B7H3-CAR-T inhibits the proliferation of lung cancer cells, wherein, Figure A: RTCA detection of B7H3-CAR-T killing of A549; Figure B: RTCA detection of B7H3-CAR-T killing of H1299, n=3; *P<0.05, **P<0.01, ***P<0.001;

[0047] Figure 6 : NETs inhibit B7H3-CAR-T cells from secreting cytokines, where A: Flow cytometry detection of the effect of NETs on the secretion of TNF-α by B7H3-CAR-T cells; B: Flow cytometry detection of the effect of NETs on the secretion of IFN-γ by B7H3-CAR-T cells, n=3; *P<0.05;

[0048] Figure 7 :NETs inhibited the targeted killing of H1299 cells by B7H3-CAR-T, where A: RTCA detected the effect of NETs on CAR-T killing of lung cancer cells at different effector-target ratios; B: Cell index statistics at 80h, n=3; ***P<0.001;

[0049] Figure 8 : NETs inhibit the proliferation and migration of B7H3-CAR-T cells, where A: CFSE detection of the effect of NETs on the proliferation of B7H3-CAR-T cells; B: Transwell detection of the effect of NETs on the migration of B7H3-CART cells, n = 3; *P < 0.05, **P < 0.01;

[0050] Fig. 9 :NETs promote apoptosis of B7H3-CAR-T cells, where A: Flow cytometry detection of the effect of NETs on apoptosis of B7H3-CAR-T cells; B: Statistical chart of apoptosis ratio, n=3; *P<0.05;

[0051] Fig.10 : NETs induce changes in the memory phenotype of B7H3-CAR-T cells, where A: Flow cytometry detection of the effect of NETs on the differentiation of the memory phenotype of B7H3-CAR-T cells; B: Corresponding statistical chart of the proportion of each subtype, n=3; *P<0.05, **P<0.01, ***P<0.001, ns, no statistical significance;

[0052] Fig.11 :NETs promote B7H3-CAR-T cell exhaustion;

[0053] Fig.12: DNase I did not affect the proliferation, survival and migration of B7H3-CAR-T cells, where A: CFSE detection of the effect of DNase I on the proliferation of B7H3-CAR-T cells; B: Flow cytometry detection of the effect of DNase I on the apoptosis of B7H3-CAR-T cells; C: Transwell detection of the effect of DNase I on the migration of B7H3-CAR-T cells, n = 3; ns, no statistical significance;

[0054] Fig.13 : DNase I does not affect the secretion of cytokines and memory phenotype differentiation of B7H3-CAR-T cells, where A: Flow cytometry detection of the effect of DNase I on the secretion of IFN-γ by B7H3-CAR-T cells; B: Flow cytometry detection of the effect of DNase I on the memory phenotype of B7H3-CAR-T cells, n = 3; ns, no statistical significance;

[0055] Fig.14 : DNase I does not affect the targeted killing of lung cancer cells by B7H3-CAR-T;

[0056] Fig.15 : To evaluate the anti-tumor efficacy of targeting NETs, ​​among which, Figure A: Schematic diagram of NCG mouse lung cancer subcutaneous transplant tumor model and treatment plan; Figure B: Subcutaneous transplant tumor entity; Figure C: Subcutaneous transplant tumor volume change curve; Figure D: Subcutaneous transplant tumor weight statistical chart, n = 5; *P < 0.05, **P < 0.01, ns, not statistically significant;

[0057] Fig.16 :Immunofluorescence detection of NETs content in tumor tissues, blue fluorescence DAPI represents cell nuclei; green fluorescence Ly6G represents mouse neutrophils; red fluorescence CitH3 represents citrullinated histones, which are used as detection markers for NETs;

[0058] Fig.17 : DNase I and DNase IL3 promote B7H3-CAR-T tumor infiltration, where A: flow cytometry detection of tumor-infiltrating T cells; B: immunohistochemistry detection of tumor-infiltrating T cells; **P < 0.01;

[0059] Fig.18 : DNase I has no obvious toxicity to lung cancer cells, among which, Figure A: CCK-8 assay to detect the effect of DNase I on the activity of A549 and H1299 cells; Figure B: RTCA assay to detect the effect of DNase I on the proliferation of A549 and H1299 cells; Figure C: Transwell assay to detect the effect of DNase I on the migration and invasion of A549 and H1299 cells, n=3; ns, no statistical significance;

[0060] Fig.19 : DNase I and B7H3-CAR-T treatment had no obvious toxicity to the main organs of mice;

[0061] Fig. 20 :B7H3-CAR-T cells carrying DNase I or DNase IL3 were successfully constructed, where: Figure A: Schematic diagram of the CAR-T structure carrying DNase I or DNase IL3; Figure B: PCR detection of DNase I and DNase IL3 bands; Figure C: Western Blot detection of DNase I and DNase IL3 proteins;

[0062] Fig.21 : Detection of DNA enzyme function secreted by CAR-T cells carrying DNase I or DNase IL3, among which, Figure A: Gel electrophoresis detection of B7H3-CAR-T DNase I and B7H3-CAR-T DNase IL3 Degradation of plasmid DNA by cell culture supernatant; Figure B: Sytoxgreen staining to detect B7H3-CAR-T DNase I and B7H3-CAR-T DNase IL3 Degradation of mouse and human NETs by cell culture supernatant;

[0063] Fig. 22 :Flow cytometry detection of the positive rate of B7H3-CAR-T carrying DNase I or DNase IL3;

[0064] Fig.23 :RTCA detection of B7H3-CAR-T DNase I and B7H3-CAR-T DNase IL3 Cell killing of lung cancer cells;

[0065] Fig.24 : Detection of CAR-T cell function carrying DNase I or DNase IL3, among which, Figure A: CFSE detection of B7H3-CAR-T DNase I and B7H3-CAR-T DNase IL3 Cell proliferation level; Figure B: Flow cytometry detection of B7H3-CAR-T DNase I and B7H3-CAR-T DNase IL3 Cell apoptosis ratio; Figure C: Transwell assay to detect B7H3-CAR-T DNase I and B7H3-CAR-T DNase IL3 Cell migration ability, n = 3; ns, not statistically significant;

[0066] Fig.25: Detection of CAR-T cell function carrying DNase I or DNase IL3, among which, Figure A: Flow cytometry detection of B7H3-CAR-T DNase I and B7H3-CAR-T DNase IL3 IFN-γ secretion level of cells; Figure B: Flow cytometry detection of B7H3-CAR-T DNase I and B7H3-CAR-T DNase IL3 Cell memory phenotype, n = 3; **P < 0.01, ns, not statistically significant;

[0067] Fig.26 : Evaluation of the anti-tumor efficacy of CAR-T cells carrying DNase I or DNase IL3, including: Figure A: Schematic diagram of the subcutaneous transplanted tumor model and treatment plan of NCG mice with lung cancer; Figure B: Entity diagram of subcutaneous transplanted tumor; Figure C: Subcutaneous transplanted tumor volume change curve; Figure D: Subcutaneous transplanted tumor weight statistical diagram. n = 6; *P < 0.05, **P < 0.01, **P < 0.001, ns, not statistically significant;

[0068] Fig. 27 :Immunofluorescence detection of NETs content in tumor tissues, blue fluorescence DAPI represents cell nuclei; green fluorescence Ly6G represents mouse neutrophils; red fluorescence CitH3 represents citrullinated histones, which are used as detection markers for NETs;

[0069] Fig.28 : Carrying DNase I or DNase IL3 promotes B7H3-CAR-T tumor infiltration, where A: flow cytometry detection of tumor-infiltrating T cells; B: immunohistochemistry detection of tumor-infiltrating T cells; n = 5; *P < 0.05, **P < 0.01;

[0070] Fig.29 : B7H3-CAR-T cells carrying DNase I or DNase IL3 had no obvious toxicity to the major organs of mice. DETAILED DESCRIPTION

[0071] The present invention is further described below in conjunction with specific embodiments. The specific embodiments are only used to explain the present invention and cannot be construed as limiting the present invention. It will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and purposes of the present invention, and the scope of the present invention is defined by the claims and their equivalents. The experimental methods for which specific conditions are not specified in the following examples are usually tested under conventional conditions or under conditions recommended by the manufacturer. The reagents, biomaterials, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

[0072] Example 1 Monoclonal antibody AHF23050 targeting B7H3

[0073] The present invention constructs a fully human monoclonal antibody AHF23050 that specifically targets B7H3. The sequence information of the AHF23050 antibody is shown in Table 1 below. The amino acid sequence of its heavy chain variable region is shown in SEQ ID NO: 4, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO: 9. In addition, this example demonstrates through experiments that the AHF23050 antibody has good affinity and specificity for B7H3.

[0074] Table 1 Sequence of AHF23050 antibody

[0075]

[0076] Example 2 NETs impair anti-tumor immunity and promote lung cancer progression

[0077] 1. Targeting NETs can delay the progression of subcutaneous transplanted lung cancer tumors

[0078] NETs (neutrophil extracellular traps) are a network of DNA and protein complexes released by neutrophils after external stimulation. Their DNA backbone structure can be digested by deoxyribonucleases (DNase) and rapidly degraded. In order to clarify the effect of NETs on the progression of lung cancer, we first constructed an LLC subcutaneous transplant tumor model (lung cancer mouse model) in C57BL / 6 mice, and promoted NETs degradation by intraperitoneal injection of DNase I (Sigma, USA) to evaluate its role in the progression of lung cancer. Mice were randomly divided into groups before treatment: The tumor volume of mice was monitored, and the volume formula = length × width 2 / 2, wait until the average value reaches 50-100mm 3 The mice were divided into a PBS group and a DNase I treatment group. The mice in the PBS group were intraperitoneally injected with 200 μL PBS daily, and the mice in the DNase I treatment group were intraperitoneally injected with 200 μL DNase I working solution (1000 U / mL) daily. The results showed that compared with the control group, the subcutaneous transplanted tumor volume and tumor weight in the DNase I treatment group were significantly reduced, suggesting that targeting NETs can delay the progression of lung cancer ( Figure 1 AC).

[0079] 2. Targeting NETs promotes T cell infiltration and enhances its effector function

[0080] Next, we further detected the infiltrating T cells in the tumor tissues of the above C57BL / 6 mice by flow cytometry. The results of further detecting the infiltrating T cells in the tumor tissues by flow cytometry showed that compared with the control group, the CD4 +T cells and CD8 + The number of T cells increased significantly ( Figure 2 AB), suggesting that targeting NETs can promote T cell infiltration into tumor tissue. Figure 2 As shown in CE, CD8 + T cells expressed higher levels of cytokines (IFN-γ, IL-2, and TNF-α), and the difference was statistically significant, suggesting that NETs can impair antitumor immunity in lung cancer by inhibiting T cell function.

[0081] Example 3 NETs impair B7H3-CAR-T targeted killing function in vitro

[0082] 1. B7H3 is a potential target for CAR-T therapy of lung cancer

[0083] B7H3 protein is a type I transmembrane protein. Studies have shown that B7H3 is highly expressed in a variety of malignant tumors and is closely related to poor prognosis of patients. We detected the expression level of B7H3 in clinical lung cancer tissues and lung cancer cells (A549 and H1299) by immunohistochemistry and flow cytometry, respectively. The results showed that compared with paracancerous tissues, lung cancer tissues highly expressed B7H3 protein, while paracancerous tissues hardly expressed B7H3 protein ( Figure 3 A); In addition, B7H3 was highly expressed on the cell membrane surface of A549 and H1299 cells ( Figure 3 B). The above research results confirm that lung cancer tissues and cells highly express B7H3 protein, suggesting that B7H3 can be used as a target for CAR-T therapy.

[0084] 2. Successfully constructed CAR-T cells targeting B7H3 (B7H3-CAR-T)

[0085] Based on B7H3-scFv, CD8α hinge region, transmembrane region, CD28 co-stimulatory domain and CD3ζ signaling domain, the Figure 4 A schematic diagram of the construction of CAR-T cells targeting B7H3 (B7H3-CAR-T), wherein the B7H3-CAR is a signal peptide, a heavy chain variable region of the antibody, a linker, a light chain variable region of the antibody, a CD8 hinge region and a transmembrane domain, a CD28 costimulatory signal domain, and a CD3ζ intracellular signal transduction domain, which are sequentially connected in series. The flow cytometry results showed that compared with the Control T control group, the CAR structure was detected on the surface of the B7H3-CAR-T cell membrane, and the CAR positive rate was 87.8%, confirming that the above vectors can be successfully constructed B7H3-CART ( Figure 4 B). To clarify whether carrying the CAR structure affects T cell proliferation, continuous monitoring after infection found that there was no significant difference in T cell proliferation between the two groups ( Figure 4C), indicating that carrying the B7H3-CAR structure has no significant effect on the proliferation of T cells.

[0086] 3. B7H3-CAR-T inhibits lung cancer cell proliferation

[0087] In order to clarify the targeted killing ability of B7H3-CAR-T on lung cancer cells A549 and H1299, B7H3-CAR-T was co-cultured with A549 and H1299 cells at a specific effector-target ratio (E:T = 1:1, 1:2 and 1:4), and the changes in tumor cell index were monitored in real time by RTCA. The results showed that compared with the Blank group, the cell index of the B7H3-CAR-T group was significantly reduced. At around 90h, the cell index of the B7H3-CAR-T group was close to 0 ( Figure 5 AB). The above results show that B7H3-CAR-T can significantly kill lung cancer A549 and H1299 cells.

[0088] 4. NETs weaken the targeted killing ability of B7H3-CAR-T cells in vitro

[0089] Previous in vivo experiments have confirmed that targeting NETs can promote the release of effector cytokines by tumor-infiltrating T cells, suggesting that NETs can damage the effector function of T cells. To determine whether NETs affect the secretion of B7H3-CAR-T cytokines, we co-incubated B7H3-CAR-T cells with in vitro purified NETs. Intracellular cytokine detection confirmed that NETs can significantly inhibit the secretion of IFN-γ and TNF-α by B7H3-CAR-T cells ( Figure 6 AB). The above experimental results further confirmed that NETs can inhibit the effector function of CAR-T cells. To further clarify whether NETs damage the targeted killing ability of B7H3-CAR-T cells, B7H3-CAR-T cells were co-incubated with NETs for 24 hours and then co-cultured with H1299 cells at effector-target ratios of 2:1, 1:1 and 1:2. RTCA experiments confirmed that compared with the untreated group, the ability of B7H3-CAR-T cells in the NETs-treated group to target lung cancer cells was significantly weakened, and the difference was significant ( Figure 7 AB). The above results indicate that NETs can inhibit the secretion of effector cytokines IFN-γ and TNF-α by B7H3-CAR-T cells, antagonizing their targeted killing ability, thereby weakening the anti-tumor function of B7H3-CAR-T cells.

[0090] Example 4 NETs inhibit B7H3-CAR-T cell proliferation and migration and induce apoptosis

[0091] 1. NETs inhibit B7H3-CAR-T proliferation and migration

[0092] To clarify the effect of NETs on the proliferation and migration of B7H3-CAR-T cells, in vitro purified human neutrophil NETs were co-cultured with B7H3-CAR-T cells, and then CFSE staining was used to evaluate the effect of NETs on the proliferation of B7H3-CAR-T cells. The CFSE fluorescence intensity gradually decreased with cell proliferation and division. Flow cytometry results showed that compared with the untreated group, the division and proliferation of B7H3-CAR-T cells in the NETs-treated group was significantly weakened ( Figure 8 A), indicating that NETs inhibit CAR-T cell proliferation. Transwell migration assay found that adding NETs to the upper chamber can significantly inhibit CAR-T cell migration. Compared with the control group, the number of CAR-T cells in the lower chamber was significantly reduced, suggesting that NETs can limit CAR-T cell migration ( Figure 8 B) The above results indicate that NETs can limit the function of CAR-T in terms of cell proliferation and migration.

[0093] 2. NETs induce apoptosis of B7H3-CAR-T cells

[0094] To clarify the effect of NETs on B7H3-CAR-T cell apoptosis, B7H3-CAR-T cells were co-incubated with NETs for 24 h in vitro, and then Annexin V and PI staining were used to evaluate the effect of NETs on B7H3-CAR-T apoptosis. Flow cytometry results showed that the proportion of Annexin V-positive cells in the NETs treatment group increased significantly ( Fig. 9 AB), the difference was significant, suggesting that NETs can induce apoptosis of B7H3-CAR-T cells.

[0095] 3. NETs induce B7H3-CAR-T cell exhaustion

[0096] After NETs were co-incubated with B7H3-CAR-T cells in vitro, the memory phenotype of CAR-T cells was detected by flow cytometry. Fig.10 As shown in Figure 2, NETs can induce B7H3-CAR-T cells to transform from naive (T naive ) and central memory type (Tcm) to terminal type (TEMRA), further suggesting that NETs can inhibit the proliferation of CAR-T cells and may induce their exhaustion. Fig.11 As shown, B7H3-CAR-T cells were stimulated with NETs in vitro, and CD4 + and CD8 +The results showed that after NETs stimulated B7H3-CAR-T cells for 6h, 12h and 24h, the expression of Tim3 on the surface of B7H3-CAR-T cells was significantly upregulated; after NETs stimulation for 24h, the expression of Lag3 on the surface of B7H3-CAR-T cells was significantly upregulated, but there was no significant difference in the expression of PD-1, TIGIT, CD244 and CTLA4, suggesting that NETs may induce B7H3-CAR-T cells to present Tim3. + / Lag3 + Upregulated depletion phenotype.

[0097] Example 5 Targeting NETs by DNase I can improve the efficacy of B7H3-CAR-T without obvious toxic side effects

[0098] 1. DNase I itself does not affect the function of B7H3-CAR-T

[0099] To explore the possibility of DNase I combined with B7H3-CAR-T in the treatment of lung cancer, we first evaluated the effect of DNase I on B7H3-CAR-T cell function in terms of cell proliferation, apoptosis, migration, cytokine secretion and targeted killing. The results of CFSE, apoptosis detection and Transwell migration assay showed that DNase I treatment did not affect the proliferation, survival and migration of B7H3-CAR-T cells ( Fig.12 AC). At the same time, DNase I treatment did not affect the secretion of IFN-γ by B7H3-CAR-T cells, nor did it induce changes in the memory phenotype of B7H3-CAR-T cells ( Fig.13 AB). Further studies found that at effector-target ratios of 1:1, 1:2, and 1:4, DNase I did not affect the targeted killing of lung cancer A549 and H1299 cells by B7H3-CAR-T cells ( Fig.14 ). These research results suggest that DNase I has no significant effect on the survival and anti-tumor function of B7H3-CAR-T cells.

[0100] 2. Targeting NETs can improve the efficacy of B7H3-CAR-T in vivo

[0101] To determine whether targeting NETs can improve the efficacy of B7H3-CAR-T in lung cancer, Fig.15As shown in AD, a subcutaneous transplant tumor model of human lung cancer A549 was constructed based on severely immunodeficient NCG mice. With PBS as the control group, the mice were treated with DNase I, B7H3-CAR-T, and DNase I combined with B7H3-CAR-T cells. Mice were randomly divided into groups before treatment: the tumor volume of mice was monitored, and the average tumor volume was 50 mm 3 At the same time, the mice were divided into PBS group; DNase I group: each mouse was intraperitoneally injected with 200 μL working solution (1000 U / mL) every day; B7H3-CAR-T group: each mouse was injected with 3×10 6 B7H3-CAR-T cells; combined treatment group: 3×10 6 B7H3-CAR-T cells + daily intraperitoneal injection of 200μL DNase I working solution (1000U / mL). The results showed that compared with the PBS group, the mean tumor volume and tumor weight of the DNase I treatment group alone were reduced, but the difference was not statistically significant, indicating that DNase I had no obvious cytotoxicity; the tumor volume and tumor weight of the B7H3-CAR-T treatment group alone were significantly lower than those of the PBS group, confirming that targeted B7H3-CAR-T can significantly inhibit the progression of lung cancer. In addition, the tumor volume and tumor weight of mice in the DNase I combined with B7H3-CAR-T group were significantly lower than those in the B7H3-CAR-T group, indicating that clearing NETs can significantly improve the efficacy of B7H3-CAR-T in lung cancer.

[0102] At the same time, in order to clarify the targeting effect of DNase I on NETs, ​​the NETs content in the tumor was detected by immunofluorescence. Compared with the B7H3-CAR-T treatment group alone, the NETs content in the tumor of mice treated with the combination of DNase I and B7H3-CAR-T was significantly reduced ( Fig.16 ), suggesting that DNase I can effectively degrade intratumoral NETs. In addition, we further detected the number of T lymphocytes infiltrating in tumor tissues by flow cytometry and immunohistochemistry. Fig.17 As shown in AB, compared with the B7H3-CAR-T treatment group alone, the CD3 + The number of cells increased significantly, suggesting that targeting NETs promoted the infiltration of T lymphocytes in tumor tissues. To further clarify the role of NETs, ​​we confirmed in vitro through proliferation, invasion and migration experiments that DNase I had no obvious anti-tumor activity, confirming that DNase I mainly enhanced the efficacy of B7H3-CAR-T cells in vivo by clearing NETs ( Fig.18 AC).

[0103] 3. DNase I and B7H3-CAR-T treatment have no obvious toxic side effects

[0104] DNase I has been approved by the US FDA for the treatment of pulmonary cystic fibrosis, which confirms the safety of DNase I drugs to a certain extent. However, the approval of this indication is limited to the inhalation of DNase I, and the safety of systemic medication has not yet been clarified. In order to clarify whether DNase I and B7H3-CAR-T cells have systemic toxicity and side effects, we took the main organs of mice, such as heart, liver, spleen, lung and kidney. The results of HE staining showed that the cell nuclear morphology in the heart, liver, spleen, lung and kidney sections of mice in the DNase I treatment group, B7H3-CAR-T treatment group and DNase I combined with B7H3-CAR-T group was normal, the tissue structure was intact, and there was no obvious inflammatory infiltration; compared with the PBS group, there was no significant difference ( Fig.19 ). These results suggest that systemic administration of DNase I and B7H3-CAR-T therapy have no obvious toxic side effects.

[0105] Example 6 Carrying DNase I or DNase IL3 can significantly improve the efficacy of B7H3-CAR-T in lung cancer

[0106] Previous research results have shown that targeting NETs by DNase I can improve the efficacy of B7H3-CAR-T in lung cancer. Literature research found that DNase IL3, similar to DNase I, also plays a role in degrading DNA. In order to more effectively remove NETs and increase the concentration of DNase I around CAR-T cells, we intend to construct B7H3-CAR-T cells that carry and secrete DNase I or DNase IL3, hoping to promote NETs degradation locally through the secretion of CAR-T cells themselves, promote the infiltration of B7H3-CAR-T, and reduce the effect of NETs on the function of B7H3-CAR-T cells.

[0107] 1. Successfully constructed B7H3-CAR-T cells carrying DNase I or DNase IL3

[0108] like Fig. 20 As shown in A, based on the B7H3-CAR-T cell sequence, two sequences, DNase I and DNaseIL3, were connected in series after the CAR structure to obtain CAR-T cells carrying DNase I or DNase IL3 targeting B7H3 (B7H3-CAR-T DNase I and B7H3-CAR-T DNase IL3 ). Based on PCR and WB experiments, DNase I and DNase IL3 were respectively expressed in B7H3-CAR-T cells compared with B7H3-CAR-T cells. DNase I and B7H3-CAR-T DNase IL3Highly expressed in cells ( Fig. 20 BC), indicating that B7H3-CAR-T cells successfully carried DNase I or DNase IL3 protein.

[0109] Among them, the B7H3-CAR-T DNase I The CAR in the antibody is obtained by sequentially connecting the signal peptide, the heavy chain variable region of antibody AHF23050, the linker, the light chain variable region of antibody AHF23050, the CD8 hinge region and transmembrane domain, the CD28 costimulatory signal domain, the CD3ζ intracellular signal transduction domain, the T2A self-cleaving peptide, and DNAse I, and the corresponding sequences are shown in Table 2;

[0110] The B7H3-CAR-T DNase IL3 The CAR in the sequence is obtained by sequentially connecting a signal peptide, a heavy chain variable region of antibody AHF23050, a linker, a light chain variable region of antibody AHF23050, a CD8 hinge region and a transmembrane domain, a CD28 co-stimulatory signal domain, a CD3ζ intracellular signal transduction domain, a T2A self-cleaving peptide, and a DNAse IL3, and the corresponding sequences are shown in Table 2.

[0111] Table 2 Coding sequences of enhanced chimeric antigen receptors targeting B7H3 and carrying DNase I or DNase IL3 (B7H3.DNaseI.CAR or B7H3.DNase IL3.CAR)

[0112]

[0113]

[0114] To clarify whether the overexpressed DNase I and DNase IL3 can be secreted into the extracellular space and exert their effects, we co-incubated the cell culture supernatant with plasmid DNA and analyzed the DNA degradation by gel electrophoresis. Fig.21 As shown in A, B7H3-CAR-T DNase I and B7H3-CAR-T DNase IL3 The cell culture supernatant can significantly promote DNA degradation, and its degradation effect is similar to that of the positive control DNase I, which confirms that overexpressed DNase I and DNase IL3 can be secreted from B7H3-CAR-T cells to the extracellular space to degrade DNA. In addition, SytoxGreen staining also showed that the NETs mesh structure was destroyed, indicating that B7H3-CAR-T DNase I and B7H3-CAR-T DNase IL3 Cell culture supernatant can effectively degrade NETs of human and mouse neutrophils ( Fig.21B). To evaluate whether carrying DNase I and DNase IL3 affects the expression of CAR molecules on the membrane surface, we used flow cytometry to analyze the expression level of CAR molecules on the membrane surface of B7H3-CAR-T cells after overexpressing DNase I and DNase IL3. The results showed that B7H3-CAR-T cells carrying DNase I or DNase IL3 could express CAR structure normally, and there was no significant difference in CAR positive rate among the groups, indicating that carrying DNase I or DNase IL3 did not affect the expression of CAR molecules on the membrane surface of B7H3-CAR-T cells ( Fig. 22 ).

[0115] 2. Carrying DNase I or DNase IL3 does not affect the function of B7H3-CAR-T cells

[0116] To clarify whether carrying DNase I or DNase IL3 affects the targeted killing function of B7H3-CAR-T cells, we used RTCA to record the real-time activity of B7H3-CAR-T cells. DNase I and B7H3-CAR-T DNase IL3 The results showed that at the effector-target ratios of 1:1, 1:2 and 1:4, B7H3-CAR-T cells carrying two DNA enzymes could effectively kill A549 and H1299 cells, with no significant difference in the anti-tumor ability of B7H3-CAR-T cells themselves ( Fig.23 ). The above research results show that carrying DNase I or DNase IL3 did not weaken the killing function of B7H3-CAR-T cells in in vitro experiments. The results of CFSE, apoptosis detection and Transwell migration experiments showed that carrying DNase I or DNase IL3 did not affect the proliferation, survival and migration of B7H3-CAR-T cells ( Fig.24 AC). At the same time, carrying DNase I or DNase IL3 promoted the secretion of IFN-γ by B7H3-CAR-T cells, but did not induce changes in the memory phenotype of B7H3-CAR-T cells ( Fig.25 AB). These research results suggest that carrying DNA enzyme has no inhibitory effect on the survival and anti-tumor function of B7H3-CAR-T cells.

[0117] 3. Carrying DNase I or DNase IL3 in vivo can improve the efficacy of B7H3-CAR-T cells

[0118] To investigate whether carrying DNase I or DNase IL3 can improve the efficacy of B7H3-CAR-T in lung cancer, a subcutaneous transplant tumor model of human lung cancer A549 was constructed based on severely immunodeficient NCG mice. The mice were given B7H3-CAR-T, B7H3-CAR-T and DNase I / IL3, respectively, with PBS as the control group. DNase I and B7H3-CAR-T DNase IL3 Cell therapy ( Fig.26 A). Before treatment, mice were randomly divided into groups: the tumor volume of mice was monitored and the average tumor volume reached 50 mm. 3 At the same time, the mice were divided into PBS group; B7H3-CAR-T treatment group: each mouse was injected with 3×10 6 B7H3-CAR-T cells; B7H3-CAR-T DNase I Treatment group: Each mouse was injected intravenously with 3×10 6 B7H3-CAR-T DNase I Cells; B7H3-CAR-T DNase IL3 Treatment group: Each mouse was injected intravenously with 3×10 6 B7H3-CAR-T DNase IL3 The results showed that compared with the PBS group, B7H3-CAR-T, B7H3-CAR-T DNase I and B7H3-CAR-T DNase IL3 The tumor volume and weight in the cell therapy group were significantly lower than those in the PBS group, confirming that the three CAR-T cells can effectively inhibit the progression of lung cancer. DNase I and B7H3-CAR-T DNase IL3 The tumor volume and weight of the cell therapy group were significantly reduced ( Fig.26 BD), indicating that carrying DNase I or DNase IL3 can improve the efficacy of B7H3-CAR-T cells in vivo.

[0119] 4. DNase I and DNase IL3 clear NETs and promote B7H3-CAR-T tumor infiltration

[0120] We speculate that DNase I and DNase IL3 can clear intratumoral NETs and promote the infiltration and survival of B7H3-CAR-T cells. The results of tumor tissue immunofluorescence showed that compared with the PBS and B7H3-CAR-T treatment groups, the B7H3-CAR-T DNase I and B7H3-CAR-T DNase IL3 The content of NETs in the tumor of the cell therapy group was significantly reduced ( Fig. 27), suggesting that DNase I or DNaseIL3 can effectively degrade intratumoral NETs. Flow cytometry and IHC results showed that B7H3-CAR-T DNase I and B7H3-CAR-T DNase IL3 The number of infiltrating T cells in the tumor increased significantly in the cell therapy group ( Fig.28 AB), indicating that the elimination of NETs could promote the intratumoral infiltration of B7H3-CAR-T or promote its survival.

[0121] 5. B7H3-CAR-T cell therapy carrying DNase I or DNase IL3 has no obvious toxic side effects

[0122] To determine whether B7H3-CAR-T cells carrying DNase I or DNase IL3 have systemic toxicity, we collected the heart, liver, spleen, lung, and kidney of mice. HE staining results showed that compared with the PBS and B7H3-CAR-T treatment groups, B7H3-CAR-T DNase I and B7H3-CAR-T DNase IL3 The cell nuclei in the heart, liver, spleen, lung and kidney sections of the mice in the cell therapy group were normal, the tissue structure was intact, and there was no obvious inflammatory infiltration ( Fig.29 ). These results suggest that B7H3-CAR-T cells carrying DNase I or DNaseIL3 have no obvious toxic side effects in vivo.

Claims

1. A chimeric antigen receptor carrying DNase I or DNase IL3 targeting B7H3, characterized in that: The chimeric antigen receptor comprises an antibody targeting B7H3, and DNase I and / or DNase IL3; The antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1-3, and the light chain variable region comprises LCDR1-3; The HCDR1-3 are the HCDR1-3 in the heavy chain variable region as shown in SEQ ID NO:4; The LCDR1-3 is LCDR1-3 in the light chain variable region as shown in SEQ ID NO:9; The amino acid sequence of the DNase I is as shown in SEQ ID NO:27 or an amino acid sequence having at least 80% homology to SEQ ID NO:27; The amino acid sequence of the DNase IL3 is shown in SEQ ID NO: 29 or an amino acid sequence having at least 80% homology with SEQ ID NO:

29.

2. The chimeric antigen receptor according to claim 1, characterized in that The amino acid sequences of HCDR1-3 are shown in SEQ ID NOs: 1-3 respectively; Optionally, the amino acid sequences of LCDR1-3 are shown in SEQ ID NOs: 6-8, respectively; Optionally, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:4 or an amino acid sequence having at least 80% homology to SEQ ID NO:4; Optionally, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:9 or an amino acid sequence having at least 80% homology to SEQ ID NO:9; Optionally, the chimeric antigen receptor further comprises a transmembrane domain, an intracellular signaling domain, a hinge region, and a co-stimulatory signaling domain; Optionally, the chimeric antigen receptor further comprises any one or more of a signal peptide, a self-cleaving peptide, a safety switch, an immunomodulatory molecule or a cytokine; Optionally, the transmembrane domain is selected from the transmembrane domain of any one of the following molecules: CD8, 4-1BB, CD28, CD34, CD3ε, PD-1, IgG1, IgG4, OX40, IL-2 receptor, IL-11 receptor; Optionally, the intracellular signaling domain is selected from the intracellular signaling domain of any one of the following molecules: CD3ζ, FcRγ, FcRβ, TCRζ, CD3γ, CD3δ, CD3ε, CD4, CD5, CD8, CD21, CD22, CD79a, CD79b, CD278, FcεRI, DAP10, DAP12; Optionally, the hinge region is selected from the hinge region of any one of the following molecules: CD8, 4-1BB, CD28, CD34, OX40, CD3ε, IgG1, IgG4, PD-1, IL-2 receptor, IL-7 receptor, IL-11 receptor; Optionally, the costimulatory signaling domain is selected from the costimulatory signaling domains of any one of the following molecules: CD28, 4-1BB, CD19, CD4, CD27, ICOS, CD8α, CD8β, HVEM, LIGHT, NKp30, NKp46, CD40, CD30, TIM1, CD2, CD7, CD226; Optionally, the signal peptide is selected from the signal peptides of any one of the following molecules: α chain and β chain of T cell receptor, CD3ζ, CD33, CD3ε, CD16, CD22, CD28, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, GITR; Optionally, the self-cleaving peptide is selected from any one of the following self-cleaving peptides: T2A, P2A, E2A, F2A; Optionally, the safety switch is selected from any one of the following safety switches: tEGFR, iCaspase-9, RQR8; Optionally, the immunomodulatory molecule or cytokine is selected from any one of the following immunomodulatory molecules or cytokines: B7.1, CCL19, CCL21, CD40L, CD137L, GITRL, GM-CSF, IL-2, IL-15, IL-18, IL-12, IL-21, LEC, OX40L; Optionally, the chimeric antigen receptor is selected from any one of the following groups: (1) A chimeric antigen receptor obtained by sequentially connecting a signal peptide, a heavy chain variable region of the antibody, a linker, a light chain variable region of the antibody, a CD8 hinge region and a transmembrane domain, a CD28 co-stimulatory signal domain, a CD3ζ intracellular signal transduction domain, a T2A self-cleaving peptide, and DNAse I; (2) a chimeric antigen receptor obtained by sequentially connecting a signal peptide, a heavy chain variable region of the antibody, a linker, a light chain variable region of the antibody, a CD8 hinge region and a transmembrane domain, a CD28 co-stimulatory signal domain, a CD3ζ intracellular signal transduction domain, a T2A self-cleaving peptide, and a DNAse IL3; (3) a derivative chimeric antigen receptor formed by replacing, deleting or adding one or more amino acids based on the amino acid sequence of the chimeric antigen receptor described in (1) or (2); Optionally, the Linker is (G4S)3; Optionally, the amino acid sequence of the signal peptide is shown in SEQ ID NO: 11; Optionally, the amino acid sequence of the Linker is shown in SEQ ID NO: 15; Optionally, the amino acid sequence of the CD8 hinge region and transmembrane domain is as shown in SEQ ID NO: 19; Optionally, the amino acid sequence of the CD28 costimulatory signal domain is as shown in SEQ ID NO: 21; Optionally, the amino acid sequence of the CD3ζ intracellular signaling domain is as shown in SEQ ID NO: 23; Optionally, the amino acid sequence of the T2A self-cleaving peptide is as shown in SEQ ID NO: 25; Optionally, the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO:31 or SEQ ID NO:

33.

3. A nucleic acid molecule encoding the chimeric antigen receptor according to claim 1 or 2; Optionally, the nucleotide sequence encoding the heavy chain variable region of the antibody in the chimeric antigen receptor of claim 1 or 2 is as shown in SEQ ID NO: 14; Optionally, the nucleotide sequence encoding the light chain variable region of the antibody in the chimeric antigen receptor of claim 1 or 2 is as shown in SEQ ID NO: 18; Optionally, the nucleotide sequence encoding the signal peptide in the chimeric antigen receptor according to claim 1 or 2 is shown in SEQ ID NO: 12; Optionally, the nucleotide sequence encoding the Linker in the chimeric antigen receptor according to claim 1 or 2 is shown in SEQ ID NO: 16; Optionally, the nucleotide sequence encoding the CD8 hinge region and transmembrane domain in the chimeric antigen receptor of claim 1 or 2 is as shown in SEQ ID NO: 20; Optionally, the nucleotide sequence encoding the CD28 co-stimulatory signaling domain in the chimeric antigen receptor of claim 1 or 2 is as shown in SEQ ID NO: 22; Optionally, the nucleotide sequence encoding the CD3ζ intracellular signaling domain in the chimeric antigen receptor of claim 1 or 2 is shown in SEQ ID NO: 24; Optionally, the nucleotide sequence encoding the T2A self-cleaving peptide in the chimeric antigen receptor of claim 1 or 2 is shown as SEQ ID NO: 26; Optionally, the nucleotide sequence encoding DNAse I in the chimeric antigen receptor according to claim 1 or 2 is shown in SEQ ID NO: 28; Optionally, the nucleotide sequence encoding DNAse IL3 in the chimeric antigen receptor of claim 1 or 2 is shown in SEQ ID NO: 30; Optionally, the nucleotide sequence encoding the chimeric antigen receptor of claim 1 or 2 is shown as SEQ ID NO:32 or SEQ ID NO:

34.

4. An expression vector comprising the nucleic acid molecule of claim 3; Optionally, the vector is a DNA vector, an RNA vector, a plasmid, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, a retroviral vector or a transposon system.

5. A genetically engineered immune cell comprising the nucleic acid molecule of claim 3 or the expression vector of claim 4; Optionally, the genetically engineered immune cell expresses the chimeric antigen receptor of claim 1 or 2; Optionally, the immune cells are T cells, B cells, NK cells, iNKT cells, CTL cells, dendritic cells, myeloid cells, monocytes, macrophages or any combination thereof.

6. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the chimeric antigen receptor according to claim 1 or 2, the nucleic acid molecule according to claim 3, the expression vector according to claim 4 and / or the genetically engineered immune cell according to claim 5; Optionally, the pharmaceutical composition comprises DNase I and / or DNase IL3 as described in claim 1, and chimeric antigen receptor immune cells targeting B7H3; Optionally, the chimeric antigen receptor in the chimeric antigen receptor immune cell targeting B7H3 is obtained by sequentially connecting the signal peptide described in claim 1 or 2, the heavy chain variable region of the antibody, the linker, the light chain variable region of the antibody, the CD8 hinge region and the transmembrane domain, the CD28 costimulatory signal domain, and the CD3ζ intracellular signal transduction domain; Optionally, the immune cells are T cells, B cells, NK cells, iNKT cells, CTL cells, dendritic cells, myeloid cells, monocytes, macrophages or any combination thereof.

7. A biological agent, characterized in that: The biological preparation comprises the pharmaceutical composition according to claim 6.

8. A kit, characterized in that: The kit comprises the nucleic acid molecule of claim 3 and / or the expression vector of claim 4; Optionally, the kit further comprises reagents for introducing the nucleic acid molecules and expression vectors into immune cells.

9. A method for preparing the genetically engineered immune cell according to claim 5, characterized in that: The method comprises the following steps: introducing the nucleic acid molecule of claim 3 or the expression vector of claim 4 into immune cells; Optionally, the introduction method is calcium phosphate transfection, DEAE-dextran-mediated transfection, microinjection, electroporation, TALEN method, ZFN method, liposome-mediated transfection, lentiviral infection, retroviral infection, adenoviral infection, transposon technology or CRISPR-Cas9 technology.

10. Any of the following applications, characterized in that: The applications include: (1) Use of the chimeric antigen receptor according to claim 1 or 2, the nucleic acid molecule according to claim 3, the expression vector according to claim 4, the genetically engineered immune cell according to claim 5, the pharmaceutical composition according to claim 6, the biological preparation according to claim 7 and / or the kit according to claim 8 in the preparation of a drug for treating and / or preventing a B7H3-positive related disease; (2) Use of the chimeric antigen receptor according to claim 1 or 2, the nucleic acid molecule according to claim 3, the expression vector according to claim 4, the genetically engineered immune cell according to claim 5, the pharmaceutical composition according to claim 6 and / or the kit according to claim 8 in the preparation of a biological agent for treating and / or preventing a B7H3-positive related disease; (3) Use of the chimeric antigen receptor according to claim 1 or 2, the nucleic acid molecule according to claim 3 and / or the expression vector according to claim 4 in the preparation of a kit for preparing genetically engineered immune cells for treating and / or preventing B7H3-positive related diseases; (4) Use of the chimeric antigen receptor according to claim 1 or 2, the nucleic acid molecule according to claim 3, the expression vector according to claim 4 and / or the kit according to claim 8 in the preparation of genetically engineered immune cells for treating and / or preventing B7H3-positive related diseases; (5) Use of the kit according to claim 8 in the preparation of genetically engineered immune cells for treating and / or preventing B7H3-positive related diseases; (6) Use of NETs degraders in combination with chimeric antigen receptor immune cells targeting B7H3 in the preparation of drugs for treating and / or preventing B7H3-positive related diseases; (7) Use of NETs degraders in the preparation of drugs for enhancing the anti-tumor efficacy of chimeric antigen receptor immune cells; (8) Use of NETs degraders in the preparation of drugs for increasing the number of intratumoral infiltrating chimeric antigen receptor immune cells; Optionally, the NETs degrading agent is DNase I, DNase IL3, DNase IL1 and / or DNase IL2; Optionally, the NETs degrading agent is DNase I and / or DNase IL3; Optionally, the chimeric antigen receptor immune cell is the chimeric antigen receptor immune cell targeting B7H3 as described in claim 6; Optionally, the B7H3-positive related disease is lung cancer, ovarian cancer, prostate cancer, liver cancer, kidney cancer, breast cancer, colorectal cancer, esophageal cancer, oral cancer, gastric cancer, pancreatic cancer, endometrial cancer, bladder cancer, osteosarcoma, glioma or myeloid leukemia.

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