WO2022126689A1 - 抗b7h3嵌合抗原受体及其应用 - Google Patents

抗b7h3嵌合抗原受体及其应用 Download PDF

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WO2022126689A1
WO2022126689A1 PCT/CN2020/138243 CN2020138243W WO2022126689A1 WO 2022126689 A1 WO2022126689 A1 WO 2022126689A1 CN 2020138243 W CN2020138243 W CN 2020138243W WO 2022126689 A1 WO2022126689 A1 WO 2022126689A1
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seq
chimeric antigen
antigen receptor
nucleic acid
car
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PCT/CN2020/138243
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French (fr)
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李光超
罗敏
丁雯
周兆
王学俊
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广州百暨基因科技有限公司
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Priority to US18/267,295 priority Critical patent/US20240059776A1/en
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Definitions

  • the present application belongs to the technical field of biomedicine, and relates to anti-B7H3 chimeric antigen receptors and applications thereof.
  • B7H3 is a type I transmembrane protein that belongs to the B7 immune co-stimulatory and co-suppressive family. It has immunosuppressive functions and can reduce type I interferon (IFN) released by T cells and reduce the cytotoxicity of NK cells.
  • IFN type I interferon
  • the B7H3 protein has limited expression in normal tissues (eg, prostate, breast, placenta, liver, colon, and lymphoid organs), but is abnormally highly expressed in most malignant tumors. The expression of B7H3 can be detected in non-small cell lung cancer cell lines and tumor tissues.
  • B7H3 High expression of B7H3 in tumor cells is often associated with decreased tumor-infiltrating lymphocytes, accelerated cancer progression, and malignancies (neurological, melanoma, lung, head and neck, colorectal, pancreatic, prostate, ovarian, lung, and clear cell renal carcinoma) are closely related to clinical outcomes. Due to its widespread expression in a variety of tumors, B7H3 has emerged as a potential target for cancer immunotherapy. However, there are few reports of immunotherapy targeting B7H3.
  • the present application provides an anti-B7H3 chimeric antigen receptor and its application.
  • the anti-B7H3 chimeric antigen receptor adopts an anti-B7H3 antibody capable of binding to human B7H3 as an antigen-binding domain, which can not only bind to free B7H3 protein, but also It can bind to the B7H3 protein on the cell surface and has important application prospects in the field of tumor therapy.
  • the application provides an anti-B7H3 chimeric antigen receptor comprising an antigen binding domain, a hinge region, a transmembrane domain and a signaling domain;
  • antigen binding domain is an anti-B7H3 antibody.
  • an anti-B7H3 antibody with binding ability to B7H3 is used as the antigen-binding domain of the chimeric antigen receptor, so that the chimeric antigen receptor can specifically bind to B7H3-positive tumor cells and achieve a specific target for B7H3-positive tumors. to the effect.
  • the antigen binding domain comprises the amino acid sequences set forth in SEQ ID NO: 1 and SEQ ID NO: 2, wherein SEQ ID NO: 1 and SEQ ID NO: 2 are linked by a linker peptide to form anti-B7H3 Antibody H26B6; wherein
  • the antigen binding domain comprises the amino acid sequences set forth in SEQ ID NO: 3 and SEQ ID NO: 4, wherein SEQ ID NO: 3 and SEQ ID NO: 4 are linked by a linker peptide to form anti-B7H3 Antibody H2B8; wherein
  • the antigen binding domain comprises the amino acid sequences set forth in SEQ ID NO:5 and SEQ ID NO:6, wherein SEQ ID NO:5 and SEQ ID NO:6 are linked by a linker peptide to form anti-B7H3 Antibody 26B6; wherein
  • the antigen binding domain comprises the amino acid sequences set forth in SEQ ID NO: 7 and SEQ ID NO: 8, wherein SEQ ID NO: 7 and SEQ ID NO: 8 are linked by a linker peptide to form anti-B7H3 Antibody 2B8; wherein
  • the antigen binding domain comprises the amino acid sequences set forth in SEQ ID NO: 9 and SEQ ID NO: 10, wherein SEQ ID NO: 9 and SEQ ID NO: 10 are linked by a linker peptide to form anti-B7H3 Antibody 23H1; wherein
  • the antigen binding domain comprises the amino acid sequences set forth in SEQ ID NO: 11 and SEQ ID NO: 12, wherein SEQ ID NO: 11 and SEQ ID NO: 12 are linked by a linker peptide to form anti-B7H3 Antibody 6F7; wherein
  • the antigen binding domain comprises the amino acid sequences set forth in SEQ ID NO: 13 and SEQ ID NO: 14, wherein SEQ ID NO: 13 and SEQ ID NO: 14 are linked by a linker peptide to form anti-B7H3 Antibody Enoblituzumab (Eno); of which
  • the antigen binding domain comprises the amino acid sequences set forth in SEQ ID NO: 15 and SEQ ID NO: 16, wherein SEQ ID NO: 15 and SEQ ID NO: 16 are linked by a linker peptide to form anti-B7H3 Antibody huM30; wherein
  • the hinge region comprises the CD8 ⁇ hinge region.
  • the transmembrane domain comprises a CD8 ⁇ transmembrane domain and/or a CD28 transmembrane domain.
  • the signaling domain comprises CD3 ⁇ .
  • the signaling domain further comprises any one or a combination of at least two of 4-1BB, CD28 intracellular domain, DAP10 or OX40.
  • the anti-B7H3 chimeric antigen receptor further comprises a signal peptide.
  • the signal peptide comprises IgG ⁇ light chain signal peptide, CD8 ⁇ signal peptide, GM-CSF signal peptide, HSA signal peptide, IgG heavy chain signal peptide, IgG light chain signal peptide, CD33 signal peptide, IL-2 signal peptide or Any of the insulin signal peptides.
  • the present application provides anti-B7H3 chimeric antigen receptors
  • the anti-B7H3 chimeric antigen receptors include signal peptide, anti-B7H3 antibody, CD8 ⁇ hinge region, CD8 ⁇ transmembrane region, 4-1BB and CD3 ⁇ .
  • the anti-B7H3 chimeric antigen receptor is H26B6-CAR, which is formed in tandem from IgG ⁇ light chain signal peptide, anti-B7H3 antibody H26B6, CD8 ⁇ hinge region, CD8 ⁇ transmembrane region, 4-1BB, and CD3 ⁇ .
  • H26B6-CAR includes the amino acid sequence shown in SEQ ID NO: 17; wherein
  • the anti-B7H3 chimeric antigen receptor is an H2B8-CAR formed in tandem from an IgG ⁇ light chain signal peptide, anti-B7H3 antibody H2B8, CD8 ⁇ hinge region, CD8 ⁇ transmembrane region, 4-1BB, and CD3 ⁇ .
  • H2B8-CAR includes the amino acid sequence shown in SEQ ID NO: 18; wherein
  • the anti-B7H3 chimeric antigen receptor is L26B6-CAR, which is formed in tandem from HuIgG ⁇ light chain signal peptide, anti-B7H3 antibody 26B6, CD8 ⁇ hinge region, CD8 ⁇ transmembrane region, 4-1BB, and CD3 ⁇ .
  • L26B6-CAR includes the amino acid sequence shown in SEQ ID NO: 19; wherein
  • the anti-B7H3 chimeric antigen receptor is 26B6-CAR, which is formed in tandem from CD8 ⁇ signal peptide, anti-B7H3 antibody 26B6, CD8 ⁇ hinge region, CD8 ⁇ transmembrane region, 4-1BB, and CD3 ⁇ .
  • 26B6-CAR includes the amino acid sequence shown in SEQ ID NO: 20; wherein
  • the anti-B7H3 chimeric antigen receptor is an L2B8-CAR formed in tandem from an IgG ⁇ light chain signal peptide, anti-B7H3 antibody 2B8, CD8 ⁇ hinge region, CD8 ⁇ transmembrane region, 4-1BB, and CD3 ⁇ .
  • L2B8-CAR includes the amino acid sequence shown in SEQ ID NO: 21; wherein
  • the anti-B7H3 chimeric antigen receptor is a 2B8-CAR formed in tandem from the CD8 ⁇ signal peptide, the anti-B7H3 antibody 2B8, the CD8 ⁇ hinge region, the CD8 ⁇ transmembrane region, 4-1BB, and CD3 ⁇ .
  • 2B8-CAR includes the amino acid sequence shown in SEQ ID NO: 22; wherein
  • the anti-B7H3 chimeric antigen receptor is an L23H1-CAR formed in tandem from an IgG ⁇ signal peptide, an anti-B7H3 antibody 23H1, a CD8 ⁇ hinge region, a CD8 ⁇ transmembrane region, 4-1BB, and CD3 ⁇ .
  • L23H1-CAR includes the amino acid sequence shown in SEQ ID NO: 23; wherein
  • the anti-B7H3 chimeric antigen receptor is a 23H1-CAR formed in tandem from the CD8 ⁇ signal peptide, the anti-B7H3 antibody 23H1, the CD8 ⁇ hinge region, the CD8 ⁇ transmembrane region, 4-1BB, and CD3 ⁇ .
  • 23H1-CAR includes the amino acid sequence shown in SEQ ID NO: 24; wherein
  • the anti-B7H3 chimeric antigen receptor is an L6F7-CAR formed in tandem from an IgG ⁇ signal peptide, anti-B7H3 antibody 6F7, CD8 ⁇ hinge region, CD8 ⁇ transmembrane region, 4-1BB, and CD3 ⁇ .
  • L6F7-CAR includes the amino acid sequence shown in SEQ ID NO: 25; wherein
  • the anti-B7H3 chimeric antigen receptor is a 6F7-CAR formed in tandem from the CD8 ⁇ signal peptide, the anti-B7H3 antibody 6F7, the CD8 ⁇ hinge region, the CD8 ⁇ transmembrane region, 4-1BB, and CD3 ⁇ .
  • 6F7-CAR includes the amino acid sequence shown in SEQ ID NO: 26; wherein
  • the anti-B7H3 chimeric antigen receptor is an Eno-CAR formed in tandem from the CD8 ⁇ signal peptide, the anti-B7H3 antibody Enoblituzumab, the CD8 ⁇ hinge region, the CD8 ⁇ transmembrane region, 4-1BB, and CD3 ⁇ .
  • Eno-CAR includes the amino acid sequence shown in SEQ ID NO: 27; wherein
  • the anti-B7H3 chimeric antigen receptor is a huM30-CAR formed in tandem from the CD8 ⁇ signal peptide, the anti-B7H3 antibody huM30, the CD8 ⁇ hinge region, the CD8 ⁇ transmembrane region, 4-1BB, and CD3 ⁇ .
  • huM30-CAR includes the amino acid sequence shown in SEQ ID NO: 28; wherein
  • the present application provides a nucleic acid molecule comprising the gene encoding the anti-B7H3 chimeric antigen receptor described in the first aspect.
  • the nucleic acid molecule comprises the nucleic acid sequence shown in SEQ ID NO: 29, which is the encoding gene of H26B8-CAR.
  • the nucleic acid molecule comprises the nucleic acid sequence shown in SEQ ID NO: 30, which is the encoding gene of H2B8-CAR.
  • the nucleic acid molecule comprises the nucleic acid sequence shown in SEQ ID NO: 31, which is the encoding gene of L26B6-CAR.
  • the nucleic acid molecule comprises the nucleic acid sequence shown in SEQ ID NO: 32, which is the encoding gene of 26B6-CAR.
  • the nucleic acid molecule comprises the nucleic acid sequence shown in SEQ ID NO: 33, which is the encoding gene of L2B8-CAR.
  • the nucleic acid molecule comprises the nucleic acid sequence shown in SEQ ID NO: 34, which is the encoding gene of 2B8-CAR.
  • the nucleic acid molecule comprises the nucleic acid sequence shown in SEQ ID NO: 35, which is the encoding gene of L23H1-CAR.
  • the nucleic acid molecule comprises the nucleic acid sequence shown in SEQ ID NO: 36, which is the encoding gene of 23H1-CAR.
  • the nucleic acid molecule comprises the nucleic acid sequence shown in SEQ ID NO: 37, which is the encoding gene of L6F7-CAR.
  • the nucleic acid molecule comprises the nucleic acid sequence shown in SEQ ID NO: 38, which is the encoding gene of 6F7-CAR.
  • the nucleic acid molecule comprises the nucleic acid sequence shown in SEQ ID NO: 39, which is the encoding gene of Eno-CAR.
  • the nucleic acid molecule comprises the nucleic acid sequence shown in SEQ ID NO: 40, which is the encoding gene of huM30-CAR.
  • the present application provides an expression vector, the expression vector comprising the nucleic acid molecule described in the second aspect.
  • the expression vector is any one of a lentiviral vector, a retroviral vector or an adeno-associated virus vector containing the nucleic acid molecule described in the second aspect, preferably a lentiviral vector.
  • the present application provides a recombinant lentivirus prepared from mammalian cells transfected with the expression vector and the helper plasmid described in the third aspect.
  • the present application provides a chimeric antigen receptor T cell, the chimeric antigen receptor T cell expressing the anti-B7H3 chimeric antigen receptor of the first aspect.
  • T cells expressing anti-B7H3 chimeric antigen receptor use the antigen binding domain of chimeric antigen receptor to target B7H3 positive tumor cells, exert the killing function of T cells, and realize the killing effect on B7H3 positive tumors.
  • the nucleic acid molecule of the second aspect is integrated into the genome of the chimeric antigen receptor T cell.
  • the chimeric antigen receptor T cells comprise the expression vector described in the third aspect and/or the recombinant lentivirus described in the fourth aspect.
  • the present application provides a pharmaceutical composition comprising the chimeric antigen receptor T cell according to the fifth aspect.
  • the pharmaceutical composition further comprises any one or a combination of at least two pharmaceutically acceptable carriers, excipients or diluents.
  • the present application provides the anti-B7H3 chimeric antigen receptor described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, the recombinant lentivirus described in the fourth aspect, Application of the chimeric antigen receptor T cell described in the fifth aspect or the pharmaceutical composition described in the sixth aspect in the preparation of a medicine for treating malignant tumors.
  • the malignancy comprises acute lymphoblastic leukemia, myeloid leukemia, melanoma, neuroblastoma, non-small cell lung cancer, nasopharyngeal cancer, breast cancer, colorectal cancer, liver cancer, pancreatic cancer or cervical cancer Any one or a combination of at least two.
  • This application uses anti-human B7H3 antibody as the antigen binding domain to construct a CAR molecule.
  • T cells expressing anti-B7H3 CAR have significant killing effects on B7H3-positive tumor cells under different effector-target ratios.
  • H26B6-CAR- T has the optimal killing function;
  • the anti-B7H3 CAR-T of this application has significant in vivo efficacy. After administration to tumor model mice, it can significantly inhibit the growth of tumor cells, promote tumor cell apoptosis, and secrete cells at the same time.
  • the factor IFN- ⁇ can effectively remove tumor cells.
  • Fig. 1 is the structural representation of anti-B7H3CAR molecule
  • Figure 2 is the map of the recombinant lentiviral vector pCDH-EF1-anti-B7H3-CAR;
  • Figure 3A shows the flow cytometry of H26B6-CAR-T, H2B8-CAR-T, L2B8-CAR-T, and L26B6-CAR-T
  • Figure 3B shows the flow cytometry of Eno-CAR-T and huM30-CAR-T
  • Figure 3C is the flow cytometry of huM30-CAR-T, 26B6-CAR-T in another experiment
  • Figure 3D is the flow cytometry of huM30-CAR-T, L2B8-CAR-T, 2B8-CAR-T in another experiment cell map;
  • Figure 4A shows the killing efficiency of H26B6-CAR-T, H2B8-CAR-T, L2B8-CAR-T, and L26B6-CAR-T on human hepatoma HepG2 cells under different effector-target ratios
  • Figure 4B shows H26B6-CAR-T , H2B8-CAR-T, L2B8-CAR-T, L26B6-CAR-T killing human pancreatic cancer cell PL45 under different effector-target ratios
  • Figure 4C shows H26B6-CAR-T, H2B8-CAR-T, The killing efficiency of L2B8-CAR-T and L26B6-CAR-T on human cervical cancer cell SiHa under different effector-target ratios.
  • Figure 4D shows the different effector targets of huM30-CAR-T, 2B8-CAR-T and T cells. The killing efficiency of target cells was compared.
  • Figure 4E shows the killing efficiency of huM30-CAR-T, 2B8-CAR-T and T cells on target cells under different effector-target ratios.
  • Figure 4F shows the killing efficiency of huM30-CAR-T, 26B6 -The killing efficiency of CAR-T and T cells on PL45 under different effector-target ratios
  • Figure 4G shows the killing efficiency of huM30-CAR-T, 26B6-CAR-T and T cells on PC9 under different effector-target ratios
  • Figure 4H shows the killing efficiency of huM30-CAR-T, 26B6-CAR-T and T cells on SiHa under different effector-target ratios
  • Figure 4I shows the killing efficiency of huM30-CAR-T, 26B6-CAR-T and T cells at different The killing efficiency of HepG2.0 under the effector-target ratio
  • Figure 4J shows the killing efficiency of huM30-CAR-T, 2B8-CAR-T, L2B8-CAR-T and T cells under different effector-target ratios to PL45
  • Figure 4K The killing efficiency of huM30-CAR
  • Figure 5A shows the secretion of IFN- ⁇ by Eno-CAR-T after co-incubating with target cells according to different effector-target ratios
  • Figure 5B shows the secretion of IFN- ⁇ after huM30-CAR-T co-incubated with target cells according to different effector-target ratios the case of ⁇ ;
  • Figure 6A is the body weight change of the A375 tumor model during administration
  • Figure 6B is the body weight change of the Hep3B tumor model during the administration
  • Figure 6C is the SiHa tumor model The body weight change of the mice during administration
  • Fig. 7A shows the tumor volume change of the A375 tumor model during administration
  • Fig. 7B shows the tumor volume change of the Hep3B tumor model during administration
  • Fig. 7C shows the tumor volume change of the SiHa tumor model during administration
  • Figure 8A is the in vivo imaging fluorescence data of the A375 tumor model during administration
  • Figure 8B is the in vivo imaging fluorescence data of the Hep3B tumor model during the administration
  • Figure 8C is the SiHa tumor model in vivo in the mice during the administration imaging fluorescence data;
  • Figure 9A shows the serum IFN- ⁇ secretion level of the mice in the A375 tumor model during the administration period
  • Figure 9B shows the serum IFN- ⁇ secretion level of the mice in the Hep3B tumor model during the administration period
  • Figure 9C shows the SiHa tumor model during the administration period. Serum IFN- ⁇ secretion levels in mice.
  • anti-B7H3 antibodies H26B6, H2B8, 26B6, 2B8, 23H1, 6F7, Enoblituzumab (Eno) and huM30 were selected as the antigen-binding domains to construct CAR molecules.
  • 26B6 and its humanized H26B6, 2B8 and its humanized H2B8, 23H1, and 6F7 have significant binding ability to B7H3, not only can bind to free B7H3 protein, but also can bind to cell surface B7H3 protein;
  • huM30 is a humanized B7H3 antibody (CN103687945B) from Daiichi Sankyo (Japan).
  • Enoblituzumab (MGA271) is a new monoclonal antibody that has been optimized by immune molecules against the B7H3 target. MacroGenics uses exclusive Fc-optimized technology Developed, with unique antibody advantages and therapeutic potential, no such drug has been approved in the world, Enoblituzumab represents the world's leading B7H3 antibody drug.
  • the above-mentioned anti-B7H3 antibody is used as the antigen-binding domain of the CAR molecule, combined with the hinge region, transmembrane domain and signaling domain, to construct the anti-B7H3 CAR molecule shown in Figure 1.
  • the CAR molecule is:
  • IgG ⁇ light chain signal peptide H26B6, CD8 ⁇ hinge region, CD8 ⁇ transmembrane region, 4-1BB and CD3 ⁇ (SEQ ID NO: 17);
  • IgG ⁇ light chain signal peptide H2B8, CD8 ⁇ hinge region, CD8 ⁇ transmembrane region, 4-1BB and CD3 ⁇ (SEQ ID NO: 18);
  • HuIgG ⁇ light chain signal peptide L26B6, CD8 ⁇ hinge region, CD8 ⁇ transmembrane region, 4-1BB and CD3 ⁇ (SEQ ID NO: 19);
  • CD8 ⁇ signal peptide 26B6, CD8 ⁇ hinge region, CD8 ⁇ transmembrane region, 4-1BB and CD3 ⁇ (SEQ ID NO: 20);
  • CD8 ⁇ signal peptide 2B8, CD8 ⁇ hinge region, CD8 ⁇ transmembrane region, 4-1BB and CD3 ⁇ (SEQ ID NO: 22);
  • CD8 ⁇ signal peptide 23H1, CD8 ⁇ hinge region, CD8 ⁇ transmembrane region, 4-1BB and CD3 ⁇ (SEQ ID NO: 24);
  • IgG ⁇ light chain signal peptide L6F7, CD8 ⁇ hinge region, CD8 ⁇ transmembrane region, 4-1BB and CD3 ⁇ (SEQ ID NO:25);
  • CD8 ⁇ signal peptide 6F7, CD8 ⁇ hinge region, CD8 ⁇ transmembrane region, 4-1BB and CD3 ⁇ (SEQ ID NO: 26);
  • CD8 ⁇ signal peptide Eno, CD8 ⁇ hinge region, CD8 ⁇ transmembrane region, 4-1BB and CD3 ⁇ (SEQ ID NO: 27);
  • CD8 ⁇ signal peptide huM30, CD8 ⁇ hinge region, CD8 ⁇ transmembrane region, 4-1BB and CD3 ⁇ (SEQ ID NO:28).
  • the encoding gene of the above CAR molecule was synthesized by the whole gene, and the synthesized CAR molecule encoding gene was cloned into the lentiviral vector pCDH through PCR, enzyme digestion, recombination and other steps to obtain the recombinant lentiviral vector pCDH-EF1-anti as shown in Figure 2. -B7H3-CAR.
  • the recombinant lentiviral plasmid vector was packaged into recombinant lentiviral particles using 293T cells and helper plasmids, and the activated T cells were infected to obtain CAR-T cells expressing different CARs H26B6-CAR-T, H2B8-CAR-T, L26B6-CAR-T T, 26B6-CAR-T, L2B8-CAR-T, 2B8-CAR-T, L23H1-CAR-T, 23H1-CAR-T, L6F7-CAR-T, 6F7-CAR-T, Eno-CAR-T, huM30-CAR-T.
  • the expression rate of CAR in CAR-T cells was detected by flow cytometry.
  • the expression rate of CAR in H26B6-CAR-T cells was 65.72%, and the expression rates of CAR in H2B8-CAR-T, L2B8-CAR-T, L26B6-CAR-T cells were 31.73%, 38.15%, and 38.15%, respectively. 44.14%.
  • the expression rate of CAR in Eno-CAR-T cells was 27.3%, and the expression rate of CAR in huM30-CAR-T cells was 45.2%.
  • H26B6-CAR-T, H2B8-CAR-T, L2B8-CAR-T, L26B6-CAR-T were combined with human liver cancer cell HepG2, human pancreatic cancer cell PL45, and human cervical cancer cell SiHa in a ratio of 2:1, 1:1 , 1:4 effector-target ratio was incubated for 16h, and the killing efficiency of CAR-T was detected by RTCA technology.
  • Figures 4A, 4B, and 4C show that the four CAR-T cells have killing effects on the three tumor cells under different effector-target ratios, and the larger the effector-target ratio, the stronger the killing ability; the effective-target ratio is 2 : 1, the killing efficiency of H26B6-CAR-T on three tumor cells was better than that of H2B8-CAR-T, L2B8-CAR-T and L26B6-CAR-T.
  • CAR-T cells huM30-CAR-T and 2B8-CAR-T were prepared from PBMCs of healthy donors (donor 1 and donor 2), and the target cells were 3:1, 1:1, 1
  • the effector-target ratio of :3 was incubated for 16h, and the killing efficiency of CAR-T was detected by RTCA technology, and a T cell control group was set at the same time.
  • 26B6-CAR-T, huM30-CAR-T, L2B8-CAR-T, 2B8-CAR-T were combined with human pancreatic cancer cell PL45, human lung cancer cell PC9, human cervical cancer cell SiHa, human liver cancer cell Cells HepG2 were co-incubated for 16 h at the effector-target ratio of 1:2, 1:1, and 2:1, and the killing efficiency of CAR-T was detected by RTCA technology.
  • Figure 4F Figure 4G, Figure 4H, Figure 4I, 26B6-CAR-T is superior to huM30-CAR-T in killing ability
  • Figure 4J Figure 4K, Figure 4L, Figure 4M show that L2B8-CAR-T, 2B8-CAR-T was comparable to huM30-CAR-T in killing PL45 and PC9 cells, and L2B8-CAR-T was better than huM30-CAR-T and 2B8-CAR-T in killing SiHa and HepG2.
  • Example 4 The ability of CAR-T cells co-cultured with tumor cells to secrete IFN- ⁇
  • Eno-CAR-T cells were diluted with RPMI-1640 serum-free medium containing 2 mM GlutaMAX, 10 mM HEPES, 100 U/mL penicillin and 100 ⁇ g/mL streptomycin, and were compared with 1 ⁇ 104 targets according to different effector-target ratios.
  • Target cells Jurkat, A375, A549, HCT116, K562, SK-N-BE(2), HONE1 or HTB20
  • Target cells were co-cultured in a 96-well round bottom plate with 3 replicate wells for each experiment, 37°C, 5% CO2 Incubate in incubator for 16h; take 50 ⁇ L of supernatant from each well to detect the secretion of cytokine IFN- ⁇ .
  • Human IFN- ⁇ enzyme-linked immunosorbent assay kit (Shenzhen Xinbosheng Biotechnology Co., Ltd.) was used to detect the IFN- ⁇ content in the supernatant: dilute the supernatant 20-30 times with the sample diluent in the kit, absorb Add 100 ⁇ L to the pre-coated ELISA plate, incubate at 37°C for 1.5 hours after sealing; wash the incubated ELISA plate with PBST, spin dry, add 100 ⁇ L biotinylated antibody to each well, incubate at 37°C for 1 hour, wash , spin dry; add 100 ⁇ L of HRP-labeled streptavidin to each well, wrap with platinum paper, incubate at 37 °C for 30 min, wash, and spin dry; add 100 ⁇ L of TMB substrate chromogenic solution to each well, and react at 37 °C in the dark After 15 min, 100 ⁇ L/well of stop solution was added to stop the reaction; the OD value at
  • Eno-CAR-T cells were associated with B7H3-positive melanoma cells A375, lung cancer cells A549, PC9, colon cancer cells HCT116, neuroblastoma SH-SY5Y, SK-N-SH, SK-N- Co-incubation with MC cells can release a large amount of IFN- ⁇ ; but co-incubation with B7H3-negative target cells Daudi, H929, and jurkat cannot release significant IFN- ⁇ .
  • both huM30-CAR-T and Eno-CAR-T cells can kill A375, A549, HTC116, K562, HONE1 and HTB20, but not Jurkat, SK-N-BE(2).
  • H26B6-CAR-T Human skin melanoma cells A375, human liver cancer cells Hep 3B2.1-7 or human cervical cancer cells SiHa were used to subcutaneously inoculate NOD-Prkdcscid Il2rgtm1/Bcgen mice ( B-NDG mice), established a solid tumor model, and observed the inhibitory effect of H26B6-CAR-T on tumor growth in mice.
  • 10 female B-NDG mice were selected subcutaneously inoculated with A375-luc (luciferase-labeled human A375 cells; 5E+06 per mouse; 5 days after tumor formation), and 10 female B-NDG mice were subcutaneously inoculated with Hep3B-luc ( Luciferase-labeled human Hep 3B2.1-7 cells; 5E+06 per mouse; 9 days after tumor formation), 25 female B-NDG mice were subcutaneously inoculated with SiHa-luc (luciferase-labeled human SiHa cells; 5E +06/only; 9 days after tumor formation);
  • 1Human skin melanoma A375 group 5 mice in each group, a total of two groups were administered Mock T 5 ⁇ 106cell/piece and H26B6-CAR-T 3 ⁇ 106cell/piece respectively;
  • mice in each group 5 mice in each group, a total of two groups were administered Mock T 5 ⁇ 106cell/piece and H26B6-CAR-T 3 ⁇ 106cell/piece respectively;
  • 3Human cervical cancer SiHa group 5 animals in each group, a total of 5 groups, respectively, intravenously administered vehicle (DMSO) 200 ⁇ L/a, Mock T 5 ⁇ 106cell/a, H26B6-CAR-T high-dose administration group 5 ⁇ 106cell/ 1 ⁇ 106 cells / only in the middle-dose group and 0.2 ⁇ 106 cells / in the low-dose group;
  • DMSO intravenously administered vehicle
  • Clinical observation was carried out twice a day.
  • the patients were weighed once before grouping and twice a week after administration.
  • the tumor size was measured with vernier calipers.
  • the day of CAR-T administration was recorded as D0 day, and the fluorescence signal was captured with a small animal in vivo imager. , the blood was collected to detect the content of IFN- ⁇ by ELISA.
  • the body weight changes of each group of animals during the experiment are shown in Figure 6A, Figure 6B and Figure 6C, respectively.
  • the body weight was stable in the first 15 days after Mock T administration, and the body weight decreased slightly after 15 days.
  • H26B6-CAR-T administration The body weight increased steadily after the drug;
  • Hep3B group The average body weight of Mock T and H26B6-CAR-T before and after administration was stable;
  • SiHa group The body weight of the mice was stable before and after Mock T and H26B6-CAR-T administration, and there was no statistically significant difference. Variety.
  • H26B6-CAR-T completely inhibited tumor growth in A375 and Hep3B tumor models, and in SiHa tumor model, high-dose H26B6-CAR-T inhibited tumor growth , the tumor shrank significantly, and the tumor volume treated with low-dose H26B6-CAR-T partially shrank.
  • the tumor inhibitory effect of H26B6-CAR-T relative to Mock T was obvious on the 16th day of administration; in the Hep3B tumor model, the administration of the first day The tumor inhibitory effect of H26B6-CAR-T relative to Mock T was obvious at 19 days; in the SiHa tumor model, the tumor inhibition effect of high dose H26B6-CAR-T was obvious relative to Mock T on the 10th day of administration, and the low dose H26B6-CAR -T has a partial suppression effect.
  • the secretion of IFN- ⁇ was detected on the first day, and the secretion of IFN- ⁇ was detected at D19 in the Mock T group; in the SiHa tumor model, blood was collected from D1, D5, D12, and D19 to detect IFN- ⁇ .
  • the secretion of ⁇ peaked on the 5th day, and the secretion of IFN- ⁇ in the low and medium dose H26B6-CAR-T group and the Mock T group peaked on the 12th day.
  • H26B6-CAR-T can effectively remove tumor cells in three mouse solid tumor models, and the tumor in mice was significantly reduced, and no drug-related abnormalities were found.
  • the anti-B7H3 CAR-T cells of this application have a significant killing effect on B7H3-positive tumor cells under different effector-target ratios. After co-culture with tumor cells, they secrete a large amount of cytokine IFN- ⁇ , and the in vivo efficacy is remarkable. , can effectively remove B7H3 positive tumor cells.
  • the present application illustrates the detailed method of the present application through the above-mentioned embodiments, but the present application is not limited to the above-mentioned detailed method, which does not mean that the present application must rely on the above-mentioned detailed method for implementation.
  • Those skilled in the art should understand that any improvement to the application, the equivalent replacement of each raw material of the product of the application, the addition of auxiliary components, the selection of specific methods, etc., all fall within the scope of protection and disclosure of the application.

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Abstract

提供一种抗B7H3嵌合抗原受体及其应用，所述抗B7H3嵌合抗原受体包括抗原结合结构域、铰链区、跨膜结构域和信号传导结构域；所述抗原结合结构域为抗人B7H3抗体。该抗B7H3嵌合抗原受体对B7H3阳性肿瘤细胞具有特异性靶向作用，表达抗B7H3嵌合抗原受体的T细胞体内外杀伤作用显著，能够有效清除B7H3阳性肿瘤细胞，在肿瘤治疗领域具有重要意义。

Description

抗B7H3嵌合抗原受体及其应用 技术领域
本申请属于生物医药技术领域，涉及抗B7H3嵌合抗原受体及其应用。
背景技术
B7H3是I型跨膜蛋白，属于B7免疫共刺激和共抑制家族，具有免疫抑制功能，可以减少T细胞释放的I型干扰素(IFN)、降低NK细胞的细胞毒性。B7H3蛋白在正常组织(例如前列腺、乳腺、胎盘、肝脏、结肠和淋巴器官)中表达有限，但是在大部分恶性肿瘤中异常高表达。在非小细胞肺癌细胞株和肿瘤组织中可以检测到B7H3的表达。在表达B7H3的肿瘤组织中，浸润性淋巴样细胞的数目显著降低，与淋巴结转移呈正相关(Sun Y,Wang Y,Zhao J,et al.B7-H3 and B7-H4 expression in non-small-cell lung cancer[J].Lung Cancer,2006,53(2):143-151.；赵文建，陈春燕，眭文妍等.B7-H3及其与肿瘤关系的研究进展[J].医学综述,2009,15(22):3430-3433.)。
B7H3在肿瘤细胞中的高表达通常与肿瘤浸润淋巴细胞减少、癌症进展加快以及恶性肿瘤(神经系统肿瘤、黑色素瘤、肺癌、头颈癌、结肠直肠癌、胰腺癌、前列腺癌、卵巢癌、肺癌和透明细胞肾癌)的临床结果密切相关。由于其在多种肿瘤中广泛表达，B7H3已成为癌症免疫疗法的潜在靶标。但目前鲜有靶向B7H3的免疫疗法报道。
发明内容
本申请提供了抗B7H3嵌合抗原受体及其应用，所述抗B7H3嵌合抗原受体采用对人B7H3具有结合能力的抗B7H3抗体为抗原结合结构域，不仅可以结合游离的B7H3蛋白，还可以结合细胞表面的B7H3蛋白，在肿瘤治疗领域具有重要应用前景。
第一方面，本申请提供了抗B7H3嵌合抗原受体，所述抗B7H3嵌合抗原受体包括抗原结合结构域、铰链区、跨膜结构域和信号传导结构域；
其中所述抗原结合结构域为抗B7H3抗体。
本申请中，采用对B7H3具有结合能力的抗B7H3抗体作为嵌合抗原受体的抗原结合结构域，使得嵌合抗原受体可以特异性结合B7H3阳性肿瘤细胞，实现对B7H3阳性肿瘤的特异性靶向作用。
在一些具体实施方案中，所述抗原结合结构域包括SEQ ID NO:1和SEQ ID NO:2所示的氨基酸序列，其中SEQ ID NO:1和SEQ ID NO:2通过连接肽连接形成抗B7H3抗体H26B6；其中
Figure PCTCN2020138243-appb-000001
在一些具体实施方案中，所述抗原结合结构域包括SEQ ID NO:3和SEQ ID NO:4所示 的氨基酸序列，其中SEQ ID NO:3和SEQ ID NO:4通过连接肽连接形成抗B7H3抗体H2B8；其中
Figure PCTCN2020138243-appb-000002
在一些具体实施方案中，所述抗原结合结构域包括SEQ ID NO:5和SEQ ID NO:6所示的氨基酸序列，其中SEQ ID NO:5和SEQ ID NO:6通过连接肽连接形成抗B7H3抗体26B6；其中
Figure PCTCN2020138243-appb-000003
在一些具体实施方案中，所述抗原结合结构域包括SEQ ID NO:7和SEQ ID NO:8所示的氨基酸序列，其中SEQ ID NO:7和SEQ ID NO:8通过连接肽连接形成抗B7H3抗体2B8；其中
Figure PCTCN2020138243-appb-000004
在一些具体实施方案中，所述抗原结合结构域包括SEQ ID NO:9和SEQ ID NO:10所示的氨基酸序列，其中SEQ ID NO:9和SEQ ID NO:10通过连接肽连接形成抗B7H3抗体23H1；其中
Figure PCTCN2020138243-appb-000005
在一些具体实施方案中，所述抗原结合结构域包括SEQ ID NO:11和SEQ ID NO:12所示的氨基酸序列，其中SEQ ID NO:11和SEQ ID NO:12通过连接肽连接形成抗B7H3抗体6F7；其中
Figure PCTCN2020138243-appb-000006
在一些具体实施方案中，所述抗原结合结构域包括SEQ ID NO:13和SEQ ID NO:14所示的氨基酸序列，其中SEQ ID NO:13和SEQ ID NO:14通过连接肽连接形成抗B7H3抗体Enoblituzumab(Eno)；其中
Figure PCTCN2020138243-appb-000007
在一些具体实施方案中，所述抗原结合结构域包括SEQ ID NO:15和SEQ ID NO:16所示的氨基酸序列，其中SEQ ID NO:15和SEQ ID NO:16通过连接肽连接形成抗B7H3抗体huM30；其中
Figure PCTCN2020138243-appb-000008
优选地，所述铰链区包括CD8α铰链区。
优选地，所述跨膜结构域包括CD8α跨膜区和/或CD28跨膜区。
优选地，所述信号传导结构域包括CD3ζ。
优选地，所述信号传导结构域还包括4-1BB、CD28胞内区、DAP10或OX40中的任意一种或至少两种的组合。
优选地，所述抗B7H3嵌合抗原受体还包括信号肽。
优选地，所述信号肽包括IgGκ轻链信号肽、CD8α信号肽、GM-CSF信号肽、HSA信号肽、IgG重链信号肽、IgG轻链信号肽、CD33信号肽、IL-2信号肽或胰岛素信号肽中的任意一种。
作为优选技术方案，本申请提供了抗B7H3嵌合抗原受体，所述抗B7H3嵌合抗原受体包括信号肽、抗B7H3抗体、CD8α铰链区、CD8α跨膜区、4-1BB和CD3ζ。
在一些具体实施方案中，所述抗B7H3嵌合抗原受体是H26B6-CAR，其由IgGκ轻链信号肽、抗B7H3抗体H26B6、CD8α铰链区、CD8α跨膜区、4-1BB和CD3ζ串联形成。H26B6-CAR包括SEQ ID NO:17所示的氨基酸序列；其中
Figure PCTCN2020138243-appb-000009
在一些具体实施方案中，所述抗B7H3嵌合抗原受体是H2B8-CAR，其由IgGκ轻链信号肽、抗B7H3抗体H2B8、CD8α铰链区、CD8α跨膜区、4-1BB和CD3ζ串联形成。H2B8-CAR包括SEQ ID NO:18所示的氨基酸序列；其中
Figure PCTCN2020138243-appb-000010
在一些具体实施方案中，所述抗B7H3嵌合抗原受体是L26B6-CAR，其由HuIgGκ轻链信号肽、抗B7H3抗体26B6、CD8α铰链区、CD8α跨膜区、4-1BB和CD3ζ串联形成。L26B6-CAR包括SEQ ID NO:19所示的氨基酸序列；其中
Figure PCTCN2020138243-appb-000011
在一些具体实施方案中，所述抗B7H3嵌合抗原受体是26B6-CAR，其由CD8α信号肽、抗B7H3抗体26B6、CD8α铰链区、CD8α跨膜区、4-1BB和CD3ζ串联形成。26B6-CAR包括SEQ ID NO:20所示的氨基酸序列；其中
Figure PCTCN2020138243-appb-000012
在一些具体实施方案中，所述抗B7H3嵌合抗原受体是L2B8-CAR，其由IgGκ轻链信号肽、抗B7H3抗体2B8、CD8α铰链区、CD8α跨膜区、4-1BB和CD3ζ串联形成。L2B8-CAR包括SEQ ID NO:21所示的氨基酸序列；其中
Figure PCTCN2020138243-appb-000013
在一些具体实施方案中，所述抗B7H3嵌合抗原受体是2B8-CAR，其由CD8α信号肽、抗B7H3抗体2B8、CD8α铰链区、CD8α跨膜区、4-1BB和CD3ζ串联形成。2B8-CAR包括SEQ ID NO:22所示的氨基酸序列；其中
Figure PCTCN2020138243-appb-000014
在一些具体实施方案中，所述抗B7H3嵌合抗原受体是L23H1-CAR，其由IgGκ信号肽、抗B7H3抗体23H1、CD8α铰链区、CD8α跨膜区、4-1BB和CD3ζ串联形成。L23H1-CAR 包括SEQ ID NO:23所示的氨基酸序列；其中
Figure PCTCN2020138243-appb-000015
在一些具体实施方案中，所述抗B7H3嵌合抗原受体是23H1-CAR，其由CD8α信号肽、抗B7H3抗体23H1、CD8α铰链区、CD8α跨膜区、4-1BB和CD3ζ串联形成。23H1-CAR包括SEQ ID NO:24所示的氨基酸序列；其中
Figure PCTCN2020138243-appb-000016
在一些具体实施方案中，所述抗B7H3嵌合抗原受体是L6F7-CAR，其由IgGκ信号肽、抗B7H3抗体6F7、CD8α铰链区、CD8α跨膜区、4-1BB和CD3ζ串联形成。L6F7-CAR包括SEQ ID NO:25所示的氨基酸序列；其中
Figure PCTCN2020138243-appb-000017
在一些具体实施方案中，所述抗B7H3嵌合抗原受体是6F7-CAR，其由CD8α信号肽、抗B7H3抗体6F7、CD8α铰链区、CD8α跨膜区、4-1BB和CD3ζ串联形成。6F7-CAR包括SEQ ID NO:26所示的氨基酸序列；其中
Figure PCTCN2020138243-appb-000018
Figure PCTCN2020138243-appb-000019
在一些具体实施方案中，所述抗B7H3嵌合抗原受体是Eno-CAR，其由CD8α信号肽、抗B7H3抗体Enoblituzumab、CD8α铰链区、CD8α跨膜区、4-1BB和CD3ζ串联形成。Eno-CAR包括SEQ ID NO:27所示的氨基酸序列；其中
Figure PCTCN2020138243-appb-000020
在一些具体实施方案中，所述抗B7H3嵌合抗原受体是huM30-CAR，其由CD8α信号肽、抗B7H3抗体huM30、CD8α铰链区、CD8α跨膜区、4-1BB和CD3ζ串联形成。huM30-CAR包括SEQ ID NO:28所示的氨基酸序列；其中
Figure PCTCN2020138243-appb-000021
第二方面，本申请提供了核酸分子，所述核酸分子包括第一方面所述的抗B7H3嵌合抗原受体的编码基因。
在一些具体实施方案中，所述核酸分子包括SEQ ID NO:29所示的核酸序列，为H26B8-CAR的编码基因。
在一些具体实施方案中，所述核酸分子包括SEQ ID NO:30所示的核酸序列，为H2B8-CAR的编码基因。
在一些具体实施方案中，所述核酸分子包括SEQ ID NO:31所示的核酸序列，为L26B6-CAR的编码基因。
在一些具体实施方案中，所述核酸分子包括SEQ ID NO:32所示的核酸序列，为26B6-CAR的编码基因。
在一些具体实施方案中，所述核酸分子包括SEQ ID NO:33所示的核酸序列，为L2B8-CAR的编码基因。
在一些具体实施方案中，所述核酸分子包括SEQ ID NO:34所示的核酸序列，为2B8-CAR的编码基因。
在一些具体实施方案中，所述核酸分子包括SEQ ID NO:35所示的核酸序列，为L23H1-CAR的编码基因。
在一些具体实施方案中，所述核酸分子包括SEQ ID NO:36所示的核酸序列，为23H1-CAR的编码基因。
在一些具体实施方案中，所述核酸分子包括SEQ ID NO:37所示的核酸序列，为L6F7-CAR的编码基因。
在一些具体实施方案中，所述核酸分子包括SEQ ID NO:38所示的核酸序列，为6F7-CAR的编码基因。
在一些具体实施方案中，所述核酸分子包括SEQ ID NO:39所示的核酸序列，为Eno-CAR的编码基因。
在一些具体实施方案中，所述核酸分子包括SEQ ID NO:40所示的核酸序列，为huM30-CAR的编码基因。
第三方面，本申请提供了一种表达载体，所述表达载体包括第二方面所述的核酸分子。
优选地，所述表达载体为含有第二方面所述的核酸分子的慢病毒载体、逆转录病毒载体或腺相关病毒载体中的任意一种，优选为慢病毒载体。
第四方面，本申请提供了一种重组慢病毒，所述重组慢病毒由转染有第三方面所述的表达载体和辅助质粒的哺乳动物细胞制备得到。
第五方面，本申请提供了一种嵌合抗原受体T细胞，所述嵌合抗原受体T细胞表达第一方面所述的抗B7H3嵌合抗原受体。
本申请中，表达抗B7H3嵌合抗原受体的T细胞利用嵌合抗原受体的抗原结合结构域靶向B7H3阳性肿瘤细胞，发挥T细胞的杀伤功能，实现了对B7H3阳性肿瘤的杀伤作用。
优选地，所述嵌合抗原受体T细胞的基因组中整合有第二方面所述的核酸分子。
优选地，所述嵌合抗原受体T细胞包括第三方面所述的表达载体和/或第四方面所述的重组慢病毒。
第六方面，本申请提供了一种药物组合物，所述药物组合物包括第五方面所述的嵌合抗原受体T细胞。
优选地，所述药物组合物还包括药学上可接受的载体、赋形剂或稀释剂中的任意一种或至少两种的组合。
第七方面，本申请提供了第一方面所述的抗B7H3嵌合抗原受体、第二方面所述的核酸分子、第三方面所述的表达载体、第四方面所述的重组慢病毒、第五方面所述的嵌合抗原受 体T细胞或第六方面所述的药物组合物在制备恶性肿瘤治疗药物中的应用。
优选地，所述恶性肿瘤包括急性淋巴细胞白血病、髓性白血病、黑色素瘤、神经母细胞瘤、非小细胞肺癌、鼻咽癌、乳腺癌、结直肠癌、肝癌、胰腺癌或宫颈癌中的任意一种或至少两种的组合。
与现有技术相比，本申请具有如下有益效果：
(1)本申请采用抗人B7H3抗体作为抗原结合结构域构建CAR分子，表达抗B7H3CAR的T细胞在不同的效靶比下对B7H3阳性肿瘤细胞均具有显著的杀伤作用，其中，H26B6-CAR-T具有最优的杀伤功能；
(2)本申请的抗B7H3CAR-T细胞与肿瘤细胞共培养后分泌大量的细胞因子IFN-γ，间接证明了CAR-T对肿瘤细胞的杀伤功效；
(3)本申请的抗B7H3CAR-T、尤其是H26B6-CAR-T具有显著的体内药效，向肿瘤模型小鼠给药后，可以明显抑制肿瘤细胞生长、促进肿瘤细胞凋亡、同时分泌细胞因子IFN-γ，有效清除肿瘤细胞。
附图说明
图1为抗B7H3CAR分子的结构示意图；
图2为重组慢病毒载体pCDH-EF1-anti-B7H3-CAR图谱；
图3A为H26B6-CAR-T、H2B8-CAR-T、L2B8-CAR-T、L26B6-CAR-T的流式细胞图，图3B为Eno-CAR-T、huM30-CAR-T流式细胞图，图3C为另一实验的huM30-CAR-T、26B6-CAR-T流式细胞图，图3D为另一实验的huM30-CAR-T、L2B8-CAR-T、2B8-CAR-T流式细胞图；
图4A为H26B6-CAR-T、H2B8-CAR-T、L2B8-CAR-T、L26B6-CAR-T在不同的效靶比下对人肝癌细胞HepG2的杀伤效率，图4B为H26B6-CAR-T、H2B8-CAR-T、L2B8-CAR-T、L26B6-CAR-T在不同的效靶比下对人胰腺癌细胞PL45的杀伤效率，图4C为H26B6-CAR-T、H2B8-CAR-T、L2B8-CAR-T、L26B6-CAR-T在不同的效靶比下对人宫颈癌细胞SiHa的杀伤效率，图4D为huM30-CAR-T、2B8-CAR-T和T细胞在不同的效靶比下对靶细胞的杀伤效率，图4E为huM30-CAR-T、2B8-CAR-T和T细胞在不同的效靶比下对靶细胞的杀伤效率，图4F为huM30-CAR-T、26B6-CAR-T和T细胞在不同的效靶比下对PL45的杀伤效率，图4G为huM30-CAR-T、26B6-CAR-T和T细胞在不同的效靶比下对PC9的杀伤效率，图4H为huM30-CAR-T、26B6-CAR-T和T细胞在不同的效靶比下对SiHa的杀伤效率，图4I为huM30-CAR-T、26B6-CAR-T和T细胞在不同的效靶比下对HepG2.0的杀伤效率，图4J为huM30-CAR-T、2B8-CAR-T、L2B8-CAR-T和T细胞在不同的效靶比下对PL45的杀伤效率，图4K为huM30-CAR-T、2B8-CAR-T、L2B8-CAR-T和T细胞在不同的效靶比下对PC9的杀伤效率，图4L为huM30-CAR-T、2B8-CAR-T、L2B8-CAR-T和T细胞在不同的效靶比下对SiHa的杀伤效率，图4M为huM30-CAR-T、2B8-CAR-T、L2B8-CAR-T和T细胞在不同的效靶比下对HepG2的杀伤效率；
图5A为Eno-CAR-T按照不同的效靶比与靶细胞共孵育后分泌IFN-γ的情况，图5B为huM30-CAR-T按照不同的效靶比与靶细胞共孵育后分泌IFN-γ的情况；
图6A为A375肿瘤模型在给药期间小鼠的体重变化，图6B为Hep3B肿瘤模型在给药期 间小鼠的体重变化，图6C为SiHa肿瘤模型在给药期间小鼠的体重变化；
图7A为A375肿瘤模型在给药期间的肿瘤体积变化，图7B为Hep3B肿瘤模型在给药期间的肿瘤体积变化，图7C为SiHa肿瘤模型在给药期间的肿瘤体积变化；
图8A为A375肿瘤模型在给药期间小鼠的活体成像荧光数据，图8B为Hep3B肿瘤模型在给药期间小鼠的活体成像荧光数据，图8C为SiHa肿瘤模型在给药期间小鼠的活体成像荧光数据；
图9A为A375肿瘤模型在给药期间小鼠的血清IFN-γ分泌水平，图9B为Hep3B肿瘤模型在给药期间小鼠的血清IFN-γ分泌水平，图9C为SiHa肿瘤模型在给药期间小鼠的血清IFN-γ分泌水平。
具体实施方式
为进一步阐述本申请所采取的技术手段及其效果，以下结合实施例和附图对本申请作进一步地说明。可以理解的是，此处所描述的具体实施方式仅仅用于解释本申请，而非对本申请的限定。
实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件，或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可通过正规渠道商购获得的常规产品。
实施例1 CAR-T细胞的制备
本实施例选择抗B7H3抗体H26B6、H2B8、26B6、2B8、23H1、6F7、Enoblituzumab(Eno)和huM30作为抗原结合结构域构建CAR分子，其中，26B6及其人源化H26B6、2B8及其人源化H2B8、23H1、6F7对B7H3具有显著的结合能力，不仅可以结合游离的B7H3蛋白，还可以结合细胞表面的B7H3蛋白；huM30是日本第一三共公司(Daiichi Sankyo)的人源化B7H3抗体(CN103687945B)，正在开展临床I期试验用于治疗B7H3阳性的实体肿瘤(NCT02192567)；Enoblituzumab(MGA271)是一款经过免疫分子优化的、针对B7H3靶点的全新单克隆抗体，由MacroGenics采用独家Fc优化技术开发，具有独特的抗体优势和治疗潜力，全球尚无此类药物获批，Enoblituzumab代表了全球领先的B7H3抗体药物。
本实施例以上述抗B7H3抗体为CAR分子的抗原结合结构域，结合铰链区、跨膜结构域和信号传导结构域，构建图1所示的抗B7H3CAR分子。
具体地，CAR分子为：
a IgGκ轻链信号肽、H26B6、CD8α铰链区、CD8α跨膜区、4-1BB和CD3ζ(SEQ ID NO:17)；
b IgGκ轻链信号肽、H2B8、CD8α铰链区、CD8α跨膜区、4-1BB和CD3ζ(SEQ ID NO:18)；
c HuIgGκ轻链信号肽、L26B6、CD8α铰链区、CD8α跨膜区、4-1BB和CD3ζ(SEQ ID NO:19)；
d CD8α信号肽、26B6、CD8α铰链区、CD8α跨膜区、4-1BB和CD3ζ(SEQ ID NO:20)；
e IgGκ轻链信号肽、L2B8、CD8α铰链区、CD8α跨膜区、4-1BB和CD3ζ(SEQ ID NO:21)；
f CD8α信号肽、2B8、CD8α铰链区、CD8α跨膜区、4-1BB和CD3ζ(SEQ ID NO:22)；
g IgGκ轻链信号肽、L23H1、CD8α铰链区、CD8α跨膜区、4-1BB和CD3ζ(SEQ ID NO:23)；
h CD8α信号肽、23H1、CD8α铰链区、CD8α跨膜区、4-1BB和CD3ζ(SEQ ID NO:24)；
i IgGκ轻链信号肽、L6F7、CD8α铰链区、CD8α跨膜区、4-1BB和CD3ζ(SEQ ID NO:25)；
j CD8α信号肽、6F7、CD8α铰链区、CD8α跨膜区、4-1BB和CD3ζ(SEQ ID NO:26)；
k CD8α信号肽、Eno、CD8α铰链区、CD8α跨膜区、4-1BB和CD3ζ(SEQ ID NO:27)；
l CD8α信号肽、huM30、CD8α铰链区、CD8α跨膜区、4-1BB和CD3ζ(SEQ ID NO:28)。
全基因合成以上CAR分子的编码基因，通过PCR、酶切、重组等步骤将合成的CAR分子编码基因克隆至慢病毒载体pCDH中，得到如图2所示的重组慢病毒载体pCDH-EF1-anti-B7H3-CAR。
利用293T细胞和辅助质粒将重组慢病毒质粒载体包装为重组慢病毒颗粒，感染激活的T细胞，得到表达不同CAR的CAR-T细胞H26B6-CAR-T、H2B8-CAR-T、L26B6-CAR-T、26B6-CAR-T、L2B8-CAR-T、2B8-CAR-T、L23H1-CAR-T、23H1-CAR-T、L6F7-CAR-T、6F7-CAR-T、Eno-CAR-T、huM30-CAR-T。
实施例2 CAR-T细胞对CAR的表达效率
采用流式细胞仪检测CAR-T细胞CAR的表达率。
如图3A所示，H26B6-CAR-T细胞CAR的表达率为65.72％，H2B8-CAR-T、L2B8-CAR-T、L26B6-CAR-T细胞CAR的表达率分别为31.73％、38.15％、44.14％。
如图3B所示，Eno-CAR-T细胞CAR的表达率为27.3％，huM30-CAR-T细胞CAR的表达率为45.2％。
在另一实验中，如图3C所示，huM30-CAR-T细胞CAR的表达率为23.09％，26B6-CAR-T细胞CAR的表达率为7.67％；
在另一实验中，如图3D所示，huM30-CAR-T细胞CAR的表达率为33.12％，L2B8-CAR-T细胞CAR的表达率为33.43％，2B8-CAR-T细胞CAR的表达率为12.55％。
实施例3 CAR-T细胞的杀伤功能
将H26B6-CAR-T、H2B8-CAR-T、L2B8-CAR-T、L26B6-CAR-T与人肝癌细胞HepG2、人胰腺癌细胞PL45、人宫颈癌细胞SiHa，按2:1、1:1、1:4的效靶比共孵育16h，利用RTCA技术检测CAR-T的杀伤效率。
图4A、图4B、图4C结果显示，四种CAR-T细胞在不同的效靶比下对三种肿瘤细胞均具有杀伤作用，效靶比越大杀伤能力越强；当效靶比为2:1时，H26B6-CAR-T对三种肿瘤细胞的杀伤效率优于H2B8-CAR-T、L2B8-CAR-T和L26B6-CAR-T。
利用健康供者(供者1和供者2)的PBMC分别制备不同的CAR-T细胞(huM30-CAR-T和2B8-CAR-T)，与靶细胞按3:1、1:1、1:3的效靶比共孵育16h，利用RTCA技术检测CAR-T的杀伤效率，同时设置T细胞对照组。
图4D和图4E结果显示，huM30-CAR-T、2B8-CAR-T和T细胞在不同的效靶比下对靶细胞均具有杀伤作用，其中，2B8-CAR-T的杀伤能力在不同的效靶比下均显著高于huM30-CAR-T和T细胞。
在另一个实验中，将26B6-CAR-T、huM30-CAR-T、L2B8-CAR-T、2B8-CAR-T与人胰腺癌细胞PL45、人肺癌细胞PC9、人宫颈癌细胞SiHa、人肝癌细胞HepG2按1:2、1:1、2:1的效靶比共孵育16h，利用RTCA技术检测CAR-T的杀伤效率。
如图4F、图4G、图4H、图4I显示，26B6-CAR-T在杀伤能力上优于huM30-CAR-T；图4J、图4K、图4L、图4M显示，L2B8-CAR-T、2B8-CAR-T在杀伤PL45和PC9细胞能力上与huM30-CAR-T相当，L2B8-CAR-T杀伤SiHa和HepG2的能力优于huM30-CAR-T和2B8-CAR-T。
实施例4 CAR-T细胞与肿瘤细胞共培养分泌IFN-γ的能力
将Eno-CAR-T细胞用含2mM GlutaMAX、10mM HEPES、100U/mL青霉素和100μg/mL链霉素的RPMI-1640无血清培养基稀释后，按照不同的效靶比分别与1×104个靶细胞(Daudi、H929、Jurkat、Tonly、A375、A549、PC9、HCT116、SY5Y、SH、MC或293T)共培养于96孔圆底板中，每个实验设置3个复孔，37℃、5％CO2培养箱孵育16h；每孔取50μL上清液检测细胞因子IFN-γ的分泌情况。
huM30-CAR-T细胞用含2mM GlutaMAX、10mM HEPES、100U/mL青霉素和100μg/mL链霉素的RPMI-1640无血清培养基稀释后，按照效靶比为10:1分别与1×104个靶细胞(Jurkat、A375、A549、HCT116、K562、SK-N-BE(2)、HONE1或HTB20)共培养于96孔圆底板中，每个实验设置3个复孔，37℃、5％CO2培养箱孵育16h；每孔取50μL上清液检测细胞因子IFN-γ的分泌情况。
采用人IFN-γ酶联免疫试剂盒(深圳欣博盛生物科技有限公司)检测上清液中的IFN-γ含量：用试剂盒内的样品稀释液将上清液稀释20～30倍，吸取100μL加入到预包被的酶标板中，密封后37℃孵育1.5小时；孵育后的酶标板用PBST洗涤、甩干后，每孔加入100μL生物素化抗体，37℃孵育1小时，洗涤、甩干；每孔加入100μL HRP标记链霉亲和素，用铂纸包裹，37℃培养箱温育30min，洗涤、甩干；每孔加入100μL TMB底物显色液，37℃避光反应15min，加入100μL/孔终止液终止反应；用Infinite F50酶标仪(TECAN)读取450nm波长的OD值。
如图5A所示，Eno-CAR-T细胞与B7H3阳性的黑色素瘤细胞A375、肺癌细胞A549、PC9、结肠癌细胞HCT116、神经母细胞瘤SH-SY5Y、SK-N-SH、SK-N-MC细胞等共孵育，都能释放大量的IFN-γ；而与B7H3表达阴性的靶细胞Daudi、H929、jurkat共孵育不能释放明显的IFN-γ。
如图5B所示，huM30-CAR-T和Eno-CAR-T细胞都能杀伤A375、A549、HTC116、K562、HONE1和HTB20，而对Jurkat、SK-N-BE(2)无杀伤作用。
实施例5 H26B6-CAR-T的体内药效
本实施例进一步评估H26B6-CAR-T的体内药效，采用人皮肤黑色素瘤细胞A375、人肝癌细胞Hep 3B2.1-7或人子宫颈癌细胞SiHa皮下接种NOD-Prkdcscid Il2rgtm1/Bcgen小鼠(B-NDG小鼠)，建立实体瘤模型，观察H26B6-CAR-T对小鼠体内肿瘤的生长抑制作用。
步骤如下：
选择10只雌性B-NDG小鼠皮下接种A375-luc(荧光素酶标记的人A375细胞；5E+06/只；成瘤5天)、10只雌性B-NDG小鼠皮下接种Hep3B-luc(荧光素酶标记的人Hep 3B2.1-7 细胞；5E+06/只；成瘤9天)、25只雌性B-NDG小鼠皮下接种SiHa-luc(荧光素酶标记的人SiHa细胞；5E+06/只；成瘤9天)；
待成瘤后，按照实验方案分组，分别注射溶媒(DMSO注射剂)、未经修饰的T细胞(Mock T)和H26B6-CAR-T，每组五只小鼠，其中SiHa-luc组设定H26B6-CAR-T高中低剂量给药组：
①人皮肤黑色素瘤A375组：每组5只共两组，分别给药Mock T 5×106cell/只、H26B6-CAR-T 3×106cell/只；
②人肝癌细胞Hep 3B2.1-7组：每组5只共两组，分别给药Mock T 5×106cell/只、H26B6-CAR-T 3×106cell/只；
③人子宫颈癌SiHa组：每组5只共5组，分别静脉给药溶媒(DMSO)200μL/只、Mock T 5×106cell/只、H26B6-CAR-T高剂量给药组5×106cell/只、中剂量给药组1×106cell/只、低剂量给药组0.2×106cell/只；
每日2次进行临床观察，分组前称重1次，给药后每周称重2次，游标卡尺测量肿瘤大小，CAR-T给药当天记为D0天，用小动物活体成像仪拍摄荧光信号，采血利用ELISA检测IFN-γ的含量。
一般临床观察未见给药相关异常。
各组动物实验期间的体重变化分别如图6A、图6B和图6C所示，在A375组中，Mock T给药后前15天体重平稳，15日后体重略有下降，H26B6-CAR-T给药后体重平稳略有上升；Hep3B组：Mock T和H26B6-CAR-T给药前后均体重平稳；SiHa组：Mock T和H26B6-CAR-T给药前后小鼠体重平稳未见统计学意义的变化。
根据图7A、图7B和图7C的游标卡尺测量肿瘤体积数据，在A375和Hep3B肿瘤模型中，H26B6-CAR-T完全抑制肿瘤生长，在SiHa肿瘤模型中，高中剂量H26B6-CAR-T抑制肿瘤生长，肿瘤缩小明显，低剂量H26B6-CAR-T处理的肿瘤体积部分缩小。
根据图8A、图8B和图8C的活体成像荧光数据，在A375肿瘤模型中，给药第16天H26B6-CAR-T相对于Mock T的肿瘤抑制作用明显；在Hep3B肿瘤模型中，给药第19天H26B6-CAR-T相对于Mock T的肿瘤抑制作用明显；在SiHa肿瘤模型中，给药第10天高中剂量H26B6-CAR-T相对于Mock T的肿瘤抑制作用明显，低剂量H26B6-CAR-T具有部分抑制效果。
ELISA检测血清中IFN-γ分泌结果如图9A、图9B和图9C所示，在A375肿瘤模型中，D2、D9、D16采血检测IFN-γ，在D2检测到IFN-γ的分泌，并逐步升高，Mock T组的IFN-γ分泌水平高于H26B6-CAR-T组；在Hep3B肿瘤模型中，D1、D5、D12、D19采血检测IFN-γ，H26B6-CAR-T组在D5、D12天检测到IFN-γ分泌，Mock T组在D19检测到IFN-γ分泌；在SiHa肿瘤模型中，D1、D5、D12、D19采血检测IFN-γ，高剂量H26B6-CAR-T组对IFN-γ的分泌在第5天出现峰值，低中剂量H26B6-CAR-T组以及Mock T组对IFN-γ的分泌在第12天出现峰值。
说明H26B6-CAR-T在三种小鼠实体瘤模型中均能有效清除肿瘤细胞，小鼠肿块显著缩小，且未见给药相关异常。
综上所述，本申请的抗B7H3CAR-T细胞在不同的效靶比下对B7H3阳性肿瘤细胞具有 显著的杀伤作用，与肿瘤细胞共培养后分泌大量的细胞因子IFN-γ，体内药效显著，能够有效清除B7H3阳性肿瘤细胞。
申请人声明，本申请通过上述实施例来说明本申请的详细方法，但本申请并不局限于上述详细方法，即不意味着本申请必须依赖上述详细方法才能实施。所属技术领域的技术人员应该明了，对本申请的任何改进，对本申请产品各原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本申请的保护范围和公开范围之内。

Claims (15)

  1. 抗B7H3嵌合抗原受体，其包括抗原结合结构域、铰链区、跨膜结构域和信号传导结构域；
    其中所述抗原结合结构域为抗B7H3抗体。
  2. 根据权利要求1所述的抗B7H3嵌合抗原受体，其中，
    所述抗原结合结构域包括SEQ ID NO:1和SEQ ID NO:2所示的氨基酸序列；或
    所述抗原结合结构域包括SEQ ID NO:3和SEQ ID NO:4所示的氨基酸序列；或
    所述抗原结合结构域包括SEQ ID NO:5和SEQ ID NO:6所示的氨基酸序列；或
    所述抗原结合结构域包括SEQ ID NO:7和SEQ ID NO:8所示的氨基酸序列；或
    所述抗原结合结构域包括SEQ ID NO:9和SEQ ID NO:10所示的氨基酸序列；或
    所述抗原结合结构域包括SEQ ID NO:11和SEQ ID NO:12所示的氨基酸序列；或
    所述抗原结合结构域包括SEQ ID NO:13和SEQ ID NO:14所示的氨基酸序列；或
    所述抗原结合结构域包括SEQ ID NO:15和SEQ ID NO:16所示的氨基酸序列。
  3. 根据权利要求1或2所述的抗B7H3嵌合抗原受体，其中，所述铰链区包括CD8α铰链区。
  4. 根据权利要求1-3任一项所述的抗B7H3嵌合抗原受体，其中，所述跨膜结构域包括CD8α跨膜区和/或CD28跨膜区。
  5. 根据权利要求1-4任一项所述的抗B7H3嵌合抗原受体，其中，所述信号传导结构域包括CD3ζ；
    优选地，所述信号传导结构域还包括4-1BB、CD28胞内区、DAP10或OX40中的任意一种或至少两种的组合。
  6. 根据权利要求1-5任一项所述的抗B7H3嵌合抗原受体，其中，所述抗B7H3嵌合抗原受体还包括信号肽；
    优选地，所述信号肽包括IgGκ轻链信号肽、CD8α信号肽、GM-CSF信号肽、HSA信号肽、IgG重链信号肽、IgG轻链信号肽、CD33信号肽、IL-2信号肽或胰岛素信号肽中的任意一种。
  7. 根据权利要求1-6任一项所述的抗B7H3嵌合抗原受体，其中，所述抗B7H3嵌合抗原受体包括信号肽、抗B7H3抗体、CD8α铰链区、CD8α跨膜区、4-1BB和CD3ζ。
  8. 根据权利要求1-7任一项所述的抗B7H3嵌合抗原受体，其中，
    所述抗B7H3嵌合抗原受体包括SEQ ID NO:17所示的氨基酸序列；或
    所述抗B7H3嵌合抗原受体包括SEQ ID NO:18所示的氨基酸序列；或
    所述抗B7H3嵌合抗原受体包括SEQ ID NO:19所示的氨基酸序列；或
    所述抗B7H3嵌合抗原受体包括SEQ ID NO:20所示的氨基酸序列；或
    所述抗B7H3嵌合抗原受体包括SEQ ID NO:21所示的氨基酸序列；或
    所述抗B7H3嵌合抗原受体包括SEQ ID NO:22所示的氨基酸序列；或
    所述抗B7H3嵌合抗原受体包括SEQ ID NO:23所示的氨基酸序列；或
    所述抗B7H3嵌合抗原受体包括SEQ ID NO:24所示的氨基酸序列；或
    所述抗B7H3嵌合抗原受体包括SEQ ID NO:25所示的氨基酸序列；或
    所述抗B7H3嵌合抗原受体包括SEQ ID NO:26所示的氨基酸序列；或
    所述抗B7H3嵌合抗原受体包括SEQ ID NO:27所示的氨基酸序列；或
    所述抗B7H3嵌合抗原受体包括SEQ ID NO:28所示的氨基酸序列。
  9. 核酸分子，其包括权利要求1-8任一项所述的抗B7H3嵌合抗原受体的编码基因。
  10. 根据权利要求9所述的核酸分子，其中，
    所述核酸分子包括SEQ ID NO:29所示的核酸序列；或
    所述核酸分子包括SEQ ID NO:30所示的核酸序列；或
    所述核酸分子包括SEQ ID NO:31所示的核酸序列；或
    所述核酸分子包括SEQ ID NO:32所示的核酸序列；或
    所述核酸分子包括SEQ ID NO:33所示的核酸序列；或
    所述核酸分子包括SEQ ID NO:34所示的核酸序列；或
    所述核酸分子包括SEQ ID NO:35所示的核酸序列；或
    所述核酸分子包括SEQ ID NO:36所示的核酸序列；或
    所述核酸分子包括SEQ ID NO:37所示的核酸序列；或
    所述核酸分子包括SEQ ID NO:38所示的核酸序列；或
    所述核酸分子包括SEQ ID NO:39所示的核酸序列；或
    所述核酸分子包括SEQ ID NO:40所示的核酸序列。
  11. 一种表达载体，其包括权利要求9或10所述的核酸分子；
    优选地，所述表达载体为含有权利要求9或10所述的核酸分子的慢病毒载体、逆转录病毒载体或腺相关病毒载体中的任意一种，优选为慢病毒载体。
  12. 一种重组慢病毒，其是由转染有权利要求11所述的表达载体和辅助质粒的哺乳动物细胞制备得到的。
  13. 一种嵌合抗原受体T细胞，其表达权利要求1-8任一项所述的抗B7H3嵌合抗原受体；
    优选地，所述嵌合抗原受体T细胞的基因组中整合有权利要求9或10所述的核酸分子；
    优选地，所述嵌合抗原受体T细胞包括权利要求11所述的表达载体和/或权利要求12所述的重组慢病毒。
  14. 一种药物组合物，其包括权利要求13所述的嵌合抗原受体T细胞；
    任选地，所述药物组合物还包括药学上可接受的载体、赋形剂或稀释剂中的任意一种或至少两种的组合。
  15. 权利要求1-8任一项所述的抗B7H3嵌合抗原受体、权利要求9或10所述的核酸分子、权利要求11所述的表达载体、权利要求12所述的重组慢病毒、权利要求13所述的嵌合抗原受体T细胞或权利要求14所述的药物组合物在制备恶性肿瘤治疗药物中的应用；
    优选地，所述恶性肿瘤包括急性淋巴细胞白血病、髓性白血病、黑色素瘤、神经母细胞瘤、非小细胞肺癌、鼻咽癌、乳腺癌、结直肠癌、肝癌、胰腺癌或宫颈癌中的任意一种或至少两种的组合。
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