WO2024065297A1 - 嵌合抗原受体及其衍生物和应用 - Google Patents

嵌合抗原受体及其衍生物和应用 Download PDF

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WO2024065297A1
WO2024065297A1 PCT/CN2022/122211 CN2022122211W WO2024065297A1 WO 2024065297 A1 WO2024065297 A1 WO 2024065297A1 CN 2022122211 W CN2022122211 W CN 2022122211W WO 2024065297 A1 WO2024065297 A1 WO 2024065297A1
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chimeric antigen
antigen receptor
sequence
cells
axl
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朱棣
余科
吴重恩
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复旦大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C12N15/867—Retroviral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the invention belongs to the technical field of genetic engineering, and in particular relates to a chimeric antigen receptor and its derivatives and applications.
  • Axl is a receptor tyrosine kinase that is highly expressed in a variety of cancers, such as myeloid leukemia, pyrethroid leukemia, megakaryocytic leukemia, endometrial cancer, gastric cancer, colon cancer, prostate cancer, thyroid cancer, lung cancer, breast cancer, ovarian cancer, liver cancer, renal cell carcinoma, glioblastoma, melanoma, and osteosarcoma, and is a potential anti-cancer drug target.
  • cancers such as myeloid leukemia, pyrethroid leukemia, megakaryocytic leukemia, endometrial cancer, gastric cancer, colon cancer, prostate cancer, thyroid cancer, lung cancer, breast cancer, ovarian cancer, liver cancer, renal cell carcinoma, glioblastoma, melanoma, and osteosarcoma, and is a potential anti-cancer drug target.
  • the present invention provides a chimeric antigen receptor, which can kill tumor cells, especially tumor cells expressing Axl.
  • the chimeric antigen receptor of the present invention includes Axl-specific antibodies, which can be single-chain antibodies (scFV), monoclonal antibodies, domain antibodies, Fab fragments, Fd fragments, Fv fragments, F(ab')2 fragments and their derivatives, or other forms of antibodies.
  • Axl-specific antibodies which can be single-chain antibodies (scFV), monoclonal antibodies, domain antibodies, Fab fragments, Fd fragments, Fv fragments, F(ab')2 fragments and their derivatives, or other forms of antibodies.
  • the Axl-specific antibody is a humanized antibody.
  • the humanized antibody is a full-length antibody, and the subtype is any one of hIgG1, hIgG2, hIgG3 and hIgG4.
  • sequence of the light chain variable region is shown in SEQ ID NO: 1; the sequence of the heavy chain variable region is shown in SEQ ID NO: 2.
  • the Axl-specific antibody also includes a connecting region located between the light chain variable region and the heavy chain variable region, and the connecting region sequence is shown in SEQ ID NO: 3.
  • the Axl-specific antibody is formed by sequentially connecting the light chain variable region, the connecting region and the heavy chain variable region.
  • the chimeric antigen receptor further comprises a hinge region connected to the C-terminus of the heavy chain variable region of the Axl-specific antibody, and a transmembrane domain connected to the hinge region.
  • the sequence of the hinge region is derived from at least one of CD8 ⁇ , CD28, 4-1BB, ICOS, OX40, CD40, CD80 and IgG.
  • the sequence of the transmembrane domain is derived from at least one of CD2, CD27, LFA-1, CD8 ⁇ , CD28, 4-1BB, ICOS, OX40, CD40, CD80, CD3 ⁇ and CD3 ⁇ .
  • sequence of the hinge region is shown in SEQ ID NO: 4, and the sequence of the transmembrane domain is shown in SEQ ID NO: 5.
  • the chimeric antigen receptor further comprises an expression driver region and a signal peptide connecting the expression driver region and the light chain variable region of the AXL-specific antibody.
  • the expression driver region of the present invention can mark and encode the promoter for the amplification and suicide of CAR cells.
  • the sequence of the expression driver region is derived from at least one of EF1 ⁇ , CMV, PGK, MPSV, MMLV and SFFV.
  • sequence of the expression driver region is as shown in SEQ ID NO: 6.
  • sequence of the signal peptide is as shown in SEQ ID NO: 7.
  • sequence of the chimeric antigen receptor is as shown in SEQ ID NO: 8.
  • the present invention also provides a nucleic acid encoding at least a partial fragment of the chimeric antigen receptor, and a vector comprising the nucleic acid.
  • the vector may be an adenoviral vector, a lentiviral vector or a retroviral vector.
  • the present invention also provides a vector comprising the nucleic acid, and a host cell comprising the vector.
  • the host cell may be a recombinant microbial host cell or a mammalian cell.
  • the host cell is capable of expressing the Axl-specific antibody.
  • the present invention also provides the application of the chimeric antigen receptor in the preparation of tumor-related drugs and tumor treatment.
  • the tumor is an Axl-high-expressing tumor.
  • FIG1 is a plasmid map of the shuttle plasmid of an embodiment of the present invention.
  • FIG2 is a flow cytometry analysis result comparison diagram of the transfection control group and the transfection experimental group of the embodiment of the present invention.
  • FIG. 5 is a comparison chart of the MDA-MB-231 blank group, the control group corresponding to different E:T, and the experimental group corresponding to different E:T at different co-culture times according to the embodiment of the present invention
  • FIG6 is a comparison chart of the proliferation rate trends of the corresponding control group and the experimental group of LCLC-103H under different E:Ts, and the proliferation rate trends of the corresponding control group and the experimental group of H460 under different E:Ts;
  • FIG7 is a comparison chart of the proliferation rate trends of the corresponding control group and the experimental group of MDA-MB-231 under different E:Ts, and the proliferation rate trends of the corresponding control group and the experimental group of MDA-MB-453 under different E:Ts;
  • FIG8 is a comparison chart of the relative expression levels of INF- ⁇ in the blank group, the control group and the experimental group corresponding to MV4-11 in the embodiment of the present invention.
  • FIG9 is a comparison chart of the relative expression levels of INF- ⁇ in the blank group, the control group and the experimental group corresponding to HL60 in an embodiment of the present invention.
  • FIG10 is a comparison chart of the relative expression levels of INF- ⁇ in the blank group, control group and experimental group corresponding to MDA-MB-231 in an embodiment of the present invention.
  • FIG11 is a comparison photo of tumor metastasis of each lung tissue sample of the PBS group, the animal control group and the animal treatment group according to an embodiment of the present invention.
  • FIG12 is a comparison chart of the lung metastasis area ratios of lung tissue samples in each group calculated based on the photos shown in FIG11 ;
  • FIG13 is a comparison of immunostaining photographs of in situ tumor tissue samples of the PBS group, the animal control group, and the animal treatment group according to an embodiment of the present invention.
  • FIG14 is a comparison of the percentage of positive areas of CD3 and CD8 in the in situ tumor tissue samples of the PBS group, the animal control group and the animal treatment group according to an embodiment of the present invention.
  • 15 is a graph comparing the expression levels of CD3, CD8A, CD4, IFNG, GZMB and TNFA in the PBS group, the animal control group and the animal treatment group according to an embodiment of the present invention.
  • Antibodies in the embodiments of the present invention refer to antigen-binding proteins of the immune system, including complete full-length antibodies with antigen-binding regions and any fragments thereof in which the "antigen-binding portion” or “antigen-binding region” is retained, or single chains thereof such as single-chain variable fragments (scFv).
  • Antibodies also include all recombinant forms of antibodies (particularly the antibodies described herein), such as antibodies expressed in prokaryotic cells, non-glycosylated antibodies, and antibody fragments and derivatives that bind to antigens.
  • CD3 ⁇ is defined as the protein provided by GenBank Accession No. BAG36664.1, or equivalent residues from non-human species such as mice, rodents, monkeys, apes, etc.
  • CD3 ⁇ domain is defined as the amino acid residues from the cytoplasmic domain of the ⁇ chain that are sufficient to functionally transmit the initial signal required for T cell activation.
  • the cytoplasmic domain of ⁇ comprises residues 52 to 163 of GenBank Accession No. BAG36664.1, which are functionally directed to homologs - equivalent residues from non-human species such as mice, rodents, monkeys, apes, etc.
  • CD28 is defined as the protein provided by GenBank Accession No. NP_006130.1, or equivalent residues from non-human species such as mice, rodents, monkeys, apes, etc.
  • CD28 signaling region is defined as amino acid residues from the cytoplasmic domain of CD28, which are capable of transmitting the co-stimulatory signal required for T cell activation; its sequence comprises residues 180 to 220 of GenBank Accession No. NP_006130.1, its functional homologs-equivalent residues from non-human species such as mice, rodents, monkeys, apes, etc.
  • CD28 hinge region comprises residues 114 to 152 of GenBank Accession No.
  • NP_006130.1 its functional homologs-equivalent residues from non-human species such as mice, rodents, monkeys, apes, etc.
  • the "CD28 hinge transmembrane region” comprises residues 153 to 179 of GenBank Accession No. NP_006130.1, which functionally refers to homologs - equivalent residues from non-human species such as mouse, rodent, monkey, ape, etc.
  • CD8 is defined as a molecule that is a leukocyte differentiation antigen, a glycoprotein on the surface of some T cells, which is used to assist the T cell receptor (TCR) in recognizing antigens and participating in the transduction of T cell activation signals, also known as the co-receptor of TCR;
  • the "CD8 hinge region” is defined as the presence of a variable region (V-like region) in the extracellular part of the ⁇ chain and ⁇ chain of the CD8 molecule, and between the V-like region and the cell membrane part is a hinge region (connecting peptide) rich in proline, threonine and serine residues.
  • EF1 ⁇ is defined as a strong mammalian expression promoter of the elongation factor 1 alpha (EF1A) gene, which can stably drive the constitutive expression of its downstream genes in a variety of cells.
  • the EF1 ⁇ promoter is suitable for the expression of stem cells, primary cells, hematopoietic cells, etc., and is weaker than CMV in commonly used cells such as HEK293, tumor cell lines, etc.
  • Axl is also known as Ark, Ufo or Tyro-7, and belongs to the TAM family of receptor tyrosine kinases. Its extracellular segment is similar to the structure of neural cell adhesion molecule (NCAM), containing two immunoglobulin-like regions (Ig) and two fibronectin III (FNIII, FN3)-like regions.
  • NCAM neural cell adhesion molecule
  • Ig immunoglobulin-like regions
  • FNIII, FN3 fibronectin III
  • the intracellular region of Axl protein is a tyrosine kinase-like region with kinase activity.
  • the reagents and consumables involved in the embodiments of the present invention can all be purchased through commercial channels, and unless otherwise specified, they are all from Hanheng Biotechnology (Shanghai) Co., Ltd.
  • the cell culture described in the embodiments of the present invention is carried out in a carbon dioxide cell culture incubator, and the temperature is controlled at 37 ⁇ 0.5 degrees Celsius and the volume concentration of carbon dioxide is 5 ⁇ 0.5%.
  • the confidence interval of qPCR in the embodiment of the present invention is 95%.
  • each group of experiments was repeated at least 3 times, and the experimental results were statistically analyzed using GraphPad Prism 8.0 software.
  • the two-tailed unpaired t-test was used to calculate the statistical difference between the two groups of data, and the ANOVA analysis of variance was used to calculate the statistical difference between the differences between multiple groups of data.
  • p ⁇ 0.05 is considered to be statistically significant.
  • * represents P ⁇ 0.05
  • *** represents P ⁇ 0.001.
  • Example 1 Axl-specific chimeric antigen receptor
  • This embodiment provides an Axl-specific chimeric antigen receptor (abbreviated as Axl-CAR) having a sequence as shown in SEQ ID NO: 8.
  • Axl-CAR Axl-specific chimeric antigen receptor
  • the nucleotide sequence of Axl-CAR in Example 1 was amplified by PCR and then digested with restriction enzymes, and the recovered digested fragments were inserted into the multiple cloning site of the shuttle plasmid to obtain a recombinant shuttle plasmid.
  • Figure 1 for the plasmid map of the shuttle plasmid; the specific operation process is a conventional technical means of those skilled in the art.
  • the DH5 ⁇ strain in the E. coli DH5 ⁇ competent cell suspension (product number MCC0010, Frdbio) was used as the recipient cell. 10 ng of the above-obtained recombinant shuttle plasmid was added to every 100 ⁇ l of the E. coli DH5 ⁇ competent cell suspension, mixed well, placed in an ice bath for 20-30 minutes, and then heat-shocked at 42 degrees Celsius for 90 seconds to allow the recombinant shuttle plasmid to enter the cells for transformation.
  • the specific operation process please refer to the product manual of the E. coli DH5 ⁇ competent cell suspension, which will not be described here.
  • the reaction system consists of 2 ⁇ l bacterial liquid, 5 ⁇ l 2xHieff PCR Master MIX, 2 ⁇ l ddH 2 O, 0.5 ⁇ l upstream primer and 0.5 ⁇ l downstream primer.
  • the sequencing results confirm that the sequencing results are consistent with the target sequence.
  • the upstream primer sequence was 5’-GTGTGACCCTGACTGTGGAC-3’, and the downstream primer sequence was 5’-AGTAGTCGAAGAAGCCGGTG-3’.
  • the recombinant shuttle plasmid bacterial solution was amplified and extracted and purified using a plasmid extraction kit (Tiangen, DP117), and the purified recombinant shuttle plasmid was used to transfect cells.
  • the purified recombinant shuttle plasmid of Example 2 is packaged into lentivirus, and the specific packaging method is as follows:
  • 293T cells were cultured to a confluence of 70-80% using fresh complete medium DMEM (Thermo, Catalog No. 11965118) containing 10% fetal bovine serum and then resuspended to obtain a cell suspension (cell number 5 ⁇ 10 6 , volume 20 ml).
  • 10 ⁇ g of pSPAX2 plasmid, 5 ⁇ g of pMD2G plasmid, 10 ⁇ g of purified recombinant shuttle plasmid package and 75 ⁇ l of transfection reagent (Hanbio Biotechnology, Catalog No. HB-TRCF-1000) were mixed and incubated at room temperature for 15 minutes, and then slowly added dropwise to the above cell suspension for transfection. The culture medium was replaced 16 hours after transfection.
  • the culture was centrifuged at 2000g for 10 minutes at 4 degrees Celsius, the viral supernatant was collected and filtered using a sterile filter to remove cell debris and impurities, and then the supernatant was centrifuged at 82700g for 120 minutes at 4 degrees Celsius to obtain a lentiviral precipitate; the lentiviral precipitate was resuspended in 200 ⁇ l of the aforementioned DMEM complete medium to obtain a lentiviral resuspension.
  • the titer of the lentiviral resuspension was tested. The specific steps are as follows:
  • the virus titer was calculated to be 1 ⁇ 10 8 TU/ml.
  • the lentivirus resuspension obtained in Example 3 was used to infect human peripheral blood mononuclear cells (PBMC) to construct Axl-CAR-T cells.
  • PBMC peripheral blood mononuclear cells
  • the culture product containing the T cells to be transfected was collected and centrifuged at 1500 rpm for 5 min at room temperature, and the supernatant was discarded.
  • the obtained precipitate was resuspended in EasySep Buffer and transferred to a 5 mL round-bottom test tube, and placed in a magnet for 5 min. The magnet and the test tube were tilted together, and the cell fluid was poured out and collected, and then centrifuged at 1500 rpm for 5 min at room temperature, and the supernatant was discarded.
  • the cells were resuspended in RPMI medium to 1 ⁇ 10 6 /mL to form a cell suspension to be transfected.
  • the amount of lentivirus used was calculated based on the virus titer of 1 ⁇ 10 8 TU/ml and the selected MOI (multiplicity of infection). 250 ⁇ l of the cell suspension to be transfected was added to the transfection system to form the following transfection control group and transfection experimental group:
  • the difference between the added control virus suspension and the lentivirus suspension of Example 3 is that the control shuttle plasmid is packaged as a control lentivirus according to the method described in Example 3, and the control shuttle plasmid is obtained by referring to the steps for preparing the recombinant shuttle plasmid in Example 2, except that the GFP protein sequence is used instead of the Axl-CAR sequence.
  • Transfection experimental group The difference from the transfection control group is that the added virus suspension is the lentivirus suspension obtained in Example 3.
  • the transfection control group and the transfection experimental group were centrifuged at 200 g for 1 hour at room temperature and cultured for 6 hours. Then, RPMI complete medium containing 200 U/mL rhIL-2 was added to each group to replenish the sample volume to 500 ⁇ L per group, and the culture was continued for 48 hours.
  • the cells obtained in each group were cultured for 2 days and then stained with biotinylated protein L, streptavidin-PE, and anti-CD3-APC, and then detected using flow cytometry to obtain a flow analysis result comparison chart of the transfection control group and the transfection experimental group shown in Figure 2.
  • the staining steps and flow cytometry detection steps are conventional technical means used by those skilled in the art. Referring to Figure 2, it can be seen from the comparison of the transfection control group and the transfection experimental group that after transfection of the lentivirus of Example 3, the T cells successfully expressed Axl-CAR.
  • the cells obtained in each group were cultured for 2 days and then lysed, and total RNA was extracted and reverse transcribed.
  • the obtained cDNA was used as a template, and qPCR was performed with GAPDH as the internal reference gene to obtain the relative expression level comparison chart of anti-Axl-scFv mRNA in the transfection control group and the transfection experimental group shown in Figure 3.
  • the steps of total RNA extraction, reverse transcription and qPCR are conventional technical means used by those skilled in the art. Referring to Figure 3, compared with the transfection control group, the relative expression level of anti-Axl-scFv mRNA in the transfection experimental group was significantly increased by about 3 times.
  • the primer pair sequences of the target gene are as follows:
  • AXL-humab1-F 5′-GGTGGAGGAAGCCAAGTTCA-3′ and AXL-humab1-R: 5′-CTCTAAACCTTGGCCGGGAG-3′;
  • CD3-F 5′-TGCCTCTTATCAGTTGGCGT-3′ and CD3-R: 5′-TTCCTCTGGGGTAGCAGACA-3′;
  • CD8-F 5′-TTACTGCAACCACAGGAACCG-3′ and CD8-R: 5′-AGTAATCTTTCCCACCCCGC-3′;
  • CD4-F 5′-CCAGAGGCCCTGCCATTTC-3′ and CD4-R: 5′-TTCTTTCCCTGAGTGGCTGC-3′;
  • IFNG-F 5′-CGTTTTGGGTTCTCTTGGCT-3′ and IFNG-R: 5′-TTTCTGTCACTCTCCTCTTTCC-3′;
  • Gzmb-F 5′-GATCATCGGGGGACATGAGG-3′ and Gzmb-R: 5′-TGACATTTATGGAGCTTCCCCA-3′;
  • TNFA-F 5’-GTAGCCCATGTTGTAGCAAACC-3’ and TNFA-R: 5’-TATCTCTCAGCTCCACGCCA-3’.
  • Example 5 In vitro killing experiment of tumor cells by Axl-CAR-T cells
  • LCLC-103H, HL60, MDA-MB-231, MDA-MB-453, and MV4-11, which highly express Axl, and H460, which lowly express Axl were co-cultured with the Axl-CAR-T cells obtained in Example 4 to examine the killing ability of Axl-CAR-T cells against various tumor cells.
  • LCLC-103H and H460 are both human large cell lung cancer lines
  • HL60 is a human leukemia tumor cell
  • MV4-11 is a human acute monocytic leukemia cell, all of which are from Nanjing Kebai Biotechnology Co., Ltd.
  • MDA-MB-231 and MDA-MB-453 are human breast cancer cells obtained from ATCC.
  • Each of the above tumor cells was cultured in RPMI complete medium and passaged at a ratio of 1:3 until the corresponding logarithmic phase cells with good growth were obtained. Each tumor cell was then resuspended in RPMI complete medium and the concentration was adjusted to obtain a tumor cell suspension.
  • the T cells to be transfected in Example 4 were resuspended in RPMI complete medium and the concentration was adjusted to obtain a T cell suspension.
  • the Axl-CAR-T cells in Example 4 were resuspended in RPMI complete medium and the concentration was adjusted to obtain an Axl-CAR-T cell suspension.
  • the blank group, control group and experimental group corresponding to each tumor cell were monitored by real-time label-free dynamic cell analysis technology (RTCA).
  • RTCA real-time label-free dynamic cell analysis technology
  • the cell index was detected every 15 minutes, and the proliferation rate was calculated as (real-time cell index-cell index when T cells were added)/cell index when T cells were added.
  • the number of tumor cells in each well of the blank group, control group and experimental group corresponding to each tumor cell was adjusted to 3 ⁇ 10 4 .
  • the relative expression level of INF- ⁇ in each control group and experimental group relative to the blank group was detected using Human IFN ⁇ ELISPOT Kit (abcam).
  • Axl-CAR-T cells have a selective killing effect on tumor cells, as follows:
  • E:T is 2:1 and 4:1
  • Axl-CAR-T cells show a significant killing effect on MDA-MB-453 relative to the corresponding MDA-MB-453 control group
  • E:T is 2:1
  • Axl-CAR-T cells have no significant killing effect on MDA-MB-453 relative to the corresponding MDA-MB-453 control group.
  • E:T ratio Axl-CAR-T cells showed stronger killing ability against MDA-MB-453.
  • IFN- ⁇ plays an important immunomodulatory role in inducing antiviral immunity as a cytokine secreted by immune active cells
  • detecting the level of IFN- ⁇ is to indirectly detect the activity of effector cells.
  • the comparison of the relative expression levels of INF- ⁇ in the blank group, control group and experimental group corresponding to MV4-11, HL60 and MDA-MB-231 shown in Figures 8 to 10 is shown as follows: Compared with the blank group and control group corresponding to each tumor cell, the relative expression level of INF- ⁇ in each experimental group has a significant upward trend. It shows that Axl-CAR-T cells have a good killing effect on MV4-11, HL60 and MDA-MB-231.
  • Example 6 In vivo killing experiment of Axl-CAR-T cells on tumor cells
  • mice Ten female, 6-8-week-old NSG mice (Shanghai Model Organisms Science Co., Ltd.) were used in each group for the in vivo killing experiment. Specifically, 1 ⁇ 10 7 MDA-MB-231 cells were implanted into the fourth mammary pad of each mouse, and tumors were formed after 18 days. Then, intervention was performed to form the following PBS group, animal control group, and animal treatment group:
  • PBS group 100 ⁇ l of PBS solution was injected into the tail vein each time;
  • Animal control group resuspend the T cells to be transfected in Example 4 with 100 ⁇ l of PBS solution and adjust the cell number to 8 ⁇ 10 6 , and inject into the tail vein of mice each time;
  • Animal treatment group The Axl-CAR-T cells of Example 4 were resuspended in 100 ⁇ l of PBS solution and the cell number was adjusted to 8 ⁇ 10 6 cells, and the mice were injected via the tail vein each time.
  • the lung tissue samples of mice were immersed in 4% paraformaldehyde for fixation, then paraffin-embedded, sliced, and then microscopically examined after hematoxylin-eosin staining (HE).
  • ImageJ was used to process the HE staining pictures, circle the tumor part in the lung tissue and calculate the area, and obtain the tumor metastasis comparison photos of each lung tissue sample of the PBS group, animal control group and animal treatment group shown in Figure 11, as well as the lung metastasis area ratio comparison diagram of each group of lung tissue samples according to the photos shown in Figure 11.
  • Specific staining, microscopic examination and image acquisition and analysis methods are conventional technical means for those skilled in the art. Referring to Figures 11 and 12, compared with the PBS group and the animal control group, the proportion of lung tumor area in the animal experimental group decreased significantly or even approached 0, which shows that Axl-CAR-T cells can inhibit the lung metastasis of breast cancer.
  • the average tumor volume of the PBS control group is 2800 cubic centimeters
  • the average tumor volume of the animal control group is 2430 cubic centimeters
  • the average tumor volume of the animal experimental group is 1650 cubic centimeters.
  • P relative to the control group is 0.0005.
  • the in situ tumor tissue samples of each mouse were fixed by immersion in 4% paraformaldehyde, then embedded in paraffin, sliced and subjected to immunohistochemical staining (IHC).
  • the antibodies and proportions used included: anti-CD3 1:100 (Servicebio, GB13014); anti-CD8 1:100 (Servicebio, GB13068); secondary antibody HRP-goat anti-rabbit antibody 1:200 (Servicebio, GB23303). Then, microscopic examination was performed, and immunohistochemical images were processed using ImageJ.
  • IHCTool was used to extract the immunohistochemical positive area and calculate the area, and the immunostaining photo comparison of the in situ tumor tissue samples of each group shown in Figure 13 and the positive area ratio comparison of CD3 and CD8 in the in situ tumor tissue samples of each group were obtained as shown in Figure 14. Referring to Figures 13 and 14, compared with the animal control group, the protein levels of CD3 and CD8 in the tumor samples of the animal experimental group were significantly upregulated.
  • mRNA of each in situ tumor tissue sample was extracted for qPCR analysis, and the expression levels of CD3, CD8A, CD4, IFNG, GZMB and TNFA were detected, and the expression level comparison chart of the above factors in each group was obtained as shown in FIG15.
  • PBS represents the PBS group
  • Mock T cell represents the animal control group
  • AXL-CAR T cell represents the animal experimental group.
  • the three T cell marker genes CD3, CD8A, and CD4 in the tumor samples of the animal experimental group were significantly upregulated, and the cytokine genes IFNG, GZMB and TNFA secreted by T cells were also significantly upregulated.
  • the significantly upregulated protein levels of CD3 and CD8 in the tumor samples of the animal experimental group indicated in FIG13 and FIG14 it is proved that compared with the animal control group, Axl-CAR-T cells have a stronger ability to infiltrate tumors.

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Abstract

本发明实施例提供了轻链可变区的序列如SEQIDNO：1所示，重链可变区的序列如SEQIDNO：2所示的嵌合抗原受体，能够对肿瘤细胞，特别是表达Axl的肿瘤细胞具有杀伤作用。本发明实施例还提供了所述嵌合抗原受体的衍生物及其应用。

Description

嵌合抗原受体及其衍生物和应用 技术领域
本发明属于基因工程技术领域，尤其涉及嵌合抗原受体及其衍生物和应用。
技术背景
Axl是一种受体酪氨酸激酶，在多种癌症，诸如粒细胞性白血病、拟除虫菊酯白血病、巨核细胞白血病、子宫内膜癌、胃癌、结肠癌、前列腺癌、甲状腺癌、肺癌、乳腺癌、卵巢癌、肝癌、肾细胞癌、胶质母细胞瘤、黑素瘤、骨肉瘤中高表达，是具有潜力的抗癌药物靶点。
因此有必要提供新型的Axl特异性嵌合抗原受体。
发明概要
本发明提供了一种嵌合抗原受体，能够对肿瘤细胞，特别是表达Axl的肿瘤细胞具有杀伤作用。
本发明的所述嵌合抗原受体包括Axl特异性抗体，所述Axl特异性抗体可以是单链抗体(scFV)，单克隆抗体，结构域抗体，Fab片段，Fd片段，Fv片段，F(ab’)2片段和其衍生物，或其它形式的抗体。
优选的，所述Axl特异性抗体为人源化抗体。所述人源化抗体为全长抗体，亚型为hIgG1、hIgG2、hIgG3和hIgG4的任意一种。
优选的，所述轻链可变区的序列如SEQ ID NO：1所示；所述重链可变区的序列如SEQ ID NO：2所示。
优选的，所述Axl特异性抗体还包括位于所述轻链可变区和所述重链可变区之间的连接区，所述连接区序列如SEQ ID NO：3所示。
优选的，所述Axl特异性抗体由所述轻链可变区、所述连接区和所述重 链可变区顺次连接而成。
优选的，所述嵌合抗原受体还包括连接所述Axl特异性抗体的重链可变区C端的铰链区，以及连接所述铰链区的跨膜结构域。
优选的，所述铰链区的序列来源于CD8α、CD28、4-1BB、ICOS、OX40、CD40、CD80和IgG的至少一种。
优选的，所述跨膜结构域的序列来源于CD2、CD27、LFA-1、CD8α、CD28、4-1BB、ICOS、OX40、CD40、CD80、CD3ζ和CD3ε的至少一种。
优选的，所述铰链区的序列如SEQ ID NO：4所示，所述跨膜结构域的序列如SEQ ID NO：5所示。
优选的，所述嵌合抗原受体还包括表达驱动区以及连接所述表达驱动区和所述AXL特异性抗体的轻链可变区的信号肽。
本发明的所述表达驱动区能够标记及编码CAR细胞的扩增、自杀的启动子。
优选的，所述表达驱动区的序列来源于EF1α、CMV、PGK、MPSV、MMLV和SFFV的至少一种。
优选的，所述表达驱动区的序列如SEQ ID NO：6所示。
优选的，所述信号肽的序列如SEQ ID NO：7所示。
优选的，所述嵌合抗原受体的序列如SEQ ID NO：8所示。
本发明还提供了编码所述嵌合抗原受体至少部分片段的核酸，以及包含所述核酸的载体。
优选的，所述载体可以是腺病毒载体、慢病毒载体或逆转录病毒载体。
本发明还提供了包含所述核酸的载体，以及包含所述载体的宿主细胞。
优选的，所述宿主细胞可以是重组微生物宿主细胞或哺乳动物细胞。
优选的，所述宿主细胞能够表达所述Axl特异性抗体。
本发明还提供了所述嵌合抗原受体在肿瘤相关药物制备以及肿瘤治疗方面的应用。
优选的，所述肿瘤为Axl高表达肿瘤。
附图说明
图1为本发明实施例穿梭质粒的质粒图谱；
图2为本发明实施例的转染对照组和转染实验组的流式分析结果对照图；
图3为本发明实施例的转染对照组和转染实验组的抗Axl-scFv的mRNA相对表达水平对比图；
图4为本发明实施例的LCLC-103H空白组以及在E：T＝4：1情况下对应的对照组和实验组在不同共培养时间下的LCLC-103H增殖率的变化趋势对比图；
图5为本发明实施例的MDA-MB-231空白组、不同E:T下对应的对照组以及不同E:T下对应的实验组在不同共培养时间下MDA-MB-231增殖率的变化趋势对比图；
图6为本发明实施例的LCLC-103H的对应对照组、实验组在不同E:T下的增殖率趋势，以及H460的对应对照组和实验组在不同E:T下的增殖率趋势对比图；
图7为本发明实施例的MDA-MB-231的对应对照组、实验组在不同E:T下的增殖率趋势，以及MDA-MB-453的对应对照组、实验组在不同E:T下的增殖率趋势对比图；
图8为本发明实施例的MV4-11对应的各空白组、对照组和实验组的INF-γ相对表达水平对比图；
图9为本发明实施例的HL60对应的各空白组、对照组和实验组的INF-γ相对表达水平对比图；
图10为本发明实施例的MDA-MB-231对应的各空白组、对照组和实验组的INF-γ相对表达水平对比图；
图11为本发明实施例的PBS组、动物对照组和动物治疗组的各肺组织样本的肿瘤转移情况对比照片；
图12为根据图11所示照片统计的各组肺组织样本的肺转移区域占比对比图；
图13为本发明实施例PBS组、动物对照组和动物治疗组原位肿瘤组织样本的免疫染色照片对比图；
图14为本发明实施例PBS组、动物对照组和动物治疗组的原位肿瘤组织样本中CD3和CD8的阳性区域占比对比图；
图15为本发明实施例PBS组、动物对照组和动物治疗组的CD3、CD8A、CD4、IFNG、GZMB及TNFA的表达水平对比图。
发明内容
为使本发明实施例的目的、技术方案和优点更加清楚，下面将对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。除非另外定义，此处使用的技术术语或者科学术语应当为本发明所属领域内具有一般技能的人士所理解的通常意义。本文中使用的“包括”等类似的词语意指出现该词前面的元件或者物件涵盖出现在该词后面列举的元件或者物件及其等同，而不排除其他元件或者物件。
本发明实施例采用的科技术语具有与本领域技术人员常规理解的相同 或相似的含义。为便于理解本发明，一些术语定义如下：
本发明实施例中的“抗体”指免疫系统的抗原结合蛋白，包括具有抗原结合区域的完整全长抗体及其中“抗原结合部分”或“抗原结合区域”保留的其任何片段、或其单链例如单链可变片段(scFv)。“抗体”还包括抗体(特别是本文所述抗体)的所有重组形式，例如在原核细胞中表达的抗体，未糖基化的抗体以及与抗原结合的抗体片段和衍生物。
“CD3ζ”定义为GenBan登录号BAG36664.1提供的蛋白质、或来自非人类物种例如小鼠、啮齿类动物、猴、猿等的等价残基。“CD3ζ结构域”定义为来自ζ链的胞质结构域的氨基酸残基，其足以功能性地传递T细胞活化所需的初始信号。一方面，ζ的胞质结构域包含GenBank登录号BAG36664.1的残基52至163、其功能性指向同源物—来自非人物种例如小鼠、啮齿类动物、猴、猿等的等价残基。
“CD28”定义为GenBan登录号NP_006130.1提供的蛋白质、或来自非人类物种例如小鼠、啮齿类动物、猴、猿等的等价残基。“CD28信号区”定义为来自CD28的胞质结构域的氨基酸残基，其能够传递T细胞活化所需的共刺激信号；其序列包含GenBank登录号NP_006130.1的残基180至220、其功能性指向同源物—来自非人物种例如小鼠、啮齿类动物、猴、猿等的等价残基。“CD28铰链区”包含GenBank登录号NP_006130.1的残基114至152、其功能性指向同源物—来自非人物种例如小鼠、啮齿类动物、猴、猿等的等价残基。“CD28铰跨膜区”包含GenBank登录号NP_006130.1的残基153至179、其功能性指向同源物—来自非人物种例如小鼠、啮齿类动物、猴、猿等的等价残基。
“CD8”定义为分子是一种白细胞分化抗原，为部分T细胞表面所具有的一种糖蛋白，用以辅助T细胞受体(TCR)识别抗原并参与T细胞活化信号的转导，又称为TCR的共受体；
“CD8铰链区”定义为CD8分子α链和β链的胞外部分均存在一个类可变区(V样区)，V样区和胞膜部分之间是富含脯氨酸、苏氨酸和丝氨酸残基的铰链区(连接肽)。
“EF1α”定义为延伸因子1α(elongation factor 1 alpha,EF1A)基因的强哺乳动物表达启动子，可在多种细胞中稳定驱动其下游基因的组成型表达，EF1α启动子适合干细胞、原代细胞、造血细胞等的表达，在常用细胞如HEK293、肿瘤等细胞系中弱于CMV。
Axl为又名Ark，Ufo或Tyro-7，属于受体酪氨酸激酶TAM家族。其胞外段与神经细胞粘附分子(neural cell adhesion molecule，NCAM)的结构相似，包含2个免疫球蛋白样区(Ig)和2个纤连蛋白Ⅲ(FNⅢ，FN3)样区。其中Ig样区是与配体结合的区域，而FNⅢ区在Axl蛋白与其配体结合的过程中起调节作用。Axl蛋白的胞内区为酪氨酸激酶样区，具有激酶活性。
本发明实施例所涉及的试剂和耗材均可以通过商业途径购买，除特别说明的外，均来自汉恒生物科技(上海)有限公司。
本发明实施例所述的细胞培养，如无特别说明，均在二氧化碳细胞培养箱中进行，并控制温度为37±0.5摄氏度，二氧化碳体积浓度为5±0.5％。
本发明实施例中qPCR置信区间为95％。涉及统计学分析的数据，每组实验至少重复3次，实验结果数据利用GraphPad Prism 8.0软件进行统计学分析。两组数据间比较使用双尾非配对t检验来计算统计学差异，多组数据 间差异的比较使用ANOVA方差分析计算统计学差异。p<0.05被认为具有统计学差异，说明书附图中：*代表P<0.05；***代表P<0.001。
实施例1：Axl特异性嵌合抗原受体
本实施例提供了序列如SEQ ID NO：8所示的Axl特异性嵌合抗原受体(简记为Axl-CAR)。
实施例2：构建穿梭质粒
本实施例将实施例1的Axl-CAR的核苷酸序列经PCR扩增后用限制酶酶切，将回收得到的酶切片段插入穿梭质粒的多克隆位点，得到重组穿梭质粒。穿梭质粒的质粒图谱请参见图1；具体操作过程为本领域技术人员的常规技术手段。
本实施例以E.coli DH5α感受态细胞悬液(产品编号MCC0010，Frdbio)中的DH5α菌株为受体细胞，在每100微升E.coli DH5α感受态细胞悬液中加入10纳克前述得到的重组穿梭质粒混匀后在冰浴中放置20-30分钟后，在42摄氏度下热击90秒，使所述重组穿梭质粒进入细胞进行转化。具体的操作过程请参见E.coli DH5α感受态细胞悬液的产品说明书，在此不做赘述。
取转化后得到的细胞悬液100微升加入到300微升新鲜无抗LB液体培养基混匀后在37摄氏度、230rpm下震荡培养45-60分钟，然后在预先包被有20毫升新鲜无抗LB固体培养基的10厘米培养皿中涂布均匀。涂布均匀后的培养皿倒置并在37摄氏度下恒温培养12-16小时直至出现明显且未重叠的单菌落，显色后挑选白色菌落并使用新鲜无抗LB液体培养配置为菌液后进行PCR测序鉴定，反应体系由2微升菌液、5微升2xHieff PCR Master MIX、2微升ddH 2O、0.5微升上游引物和0.5微升下游引物组成，测序结果 确认测序结果与目的序列一致。
上游引物序列为5’-GTGTGACCCTGACTGTGGAC-3’，下游引物序列为5’-AGTAGTCGAAGAAGCCGGTG-3’。
使用质粒抽提试剂盒(Tiangen，DP117)将重组穿梭质粒菌液进行扩增和抽提纯化，得到的纯化重组穿梭质粒用于转染细胞。
实施例3：慢病毒包装
本实施例将实施例2的纯化重组穿梭质粒包装为慢病毒，具体包装方法如下：
使用含10％胎牛血清的新鲜完全培养基DMEM(Thermo，货号11965118)将293T细胞培养至汇合度在70-80％后再重悬得到细胞悬液(细胞数量5×10 6，体积为20毫升)，将由10微克pSPAX2质粒、5微克pMD2G质粒、10微克纯化重组穿梭质粒包和75微升转染试剂(Hanbio Biotechnology，货号HB-TRCF-1000)混匀后室温孵育15分钟，然后缓慢滴加至上述细胞悬液进行转染。转染后16小时置换培养基，转染后48小时将培养物在2000g离心力、4摄氏度下离心10分钟后收集病毒上清并使用无菌过滤器过滤去除细胞碎片和杂质，然后对得到的上清在82700g离心力，4摄氏度下离心120分钟得到慢病毒沉淀；使用200微升前述DMEM完全培养基重悬慢病毒沉淀，得到慢病毒重悬液。
取10微升慢病毒重悬液加入预先接种Hela细胞的96孔板培养24小时后进行显微镜镜检，观察到培养基澄清透明，细胞间隙无明显颗粒，无任何细菌及真菌污染。
取10微升慢病毒重悬液在96℃水浴孵育15min后进行PCR反应，观察 到PCR胶图无明显条带，说明慢病毒没有支原体污染情况。
对慢病毒重悬液进行滴度检测，具体步骤如下：
第一天：将良好增殖状态的293T细胞消化计数后稀释至1×10 5/mL,加入96孔板进行48小时的细胞培养。
第二天：准备6个1.5mL EP管，第一个EP管中加入10μL慢病毒重悬液，然后做3倍梯度稀释，得到6个稀释度的慢病毒重悬液。
第三天，有需要加puromycin筛选的孔，先吸去100mL含病毒培养基，加入100μL含1.5μg/mL puromycin的10％FBS完全培养基。
第五天，在荧光显微镜下观察结果，在观察结果前6h需更换新鲜10％FBS完全培养基，从孔中吸出80μL培养基，然后加入80μL新鲜10％FBS完全培养基，放入37℃，5％CO2培养箱中培养。6h后荧光显微镜下观察结果，荧光百分比在10～50％的孔计算病毒滴度。
滴度(TU/mL)＝细胞数×阳性克隆百分比×MOI(1)×病毒稀释倍数×10 3TU/mL
计算得到病毒滴度为1×10 8TU/ml。
实施例4：Axl-CAR-T细胞的构建
本实施例以实施例3得到的慢病毒重悬液感染人外周血单个核细胞(PBMC)以构建Axl-CAR-T细胞。具体构建过程如下：
取健康志愿者血样10毫升加入20毫升PBS稀释后缓慢加入10ml Ficoll-Paque PLUS试剂(GE healthcare)中，800g离心力下室温离心30min后去除血清层，吸取中间白色絮状细胞层至离心管中并加入30mL PBS稀释细胞液，然后在400g离心力下室温离心15min弃去上清，用1mL PBS重悬 得到的沉淀后，用70um滤膜过滤以及1500rmp 5min室温离心。弃上清，用RPMI完全培养基加200U/mL rhIL-2(Biolegend)重悬细胞至1×10 6个/mL。培养2小时后，使用人源T细胞激活/增殖试剂盒(Miltenyi Biotec GmbH)进行刺激，随后培养4-5天，每两天进行一次传代，得到待转染T细胞。
收集前述得到的包含待转染T细胞的培养产物在1500rmp下室温离心5min后弃上清。得到的沉淀使用EasySep Buffer重悬后转入5mL圆底试管，放入磁体5min。磁体与试管一同倾斜，倒出并收集细胞液，然后在1500rmp下室温离心5min后弃上清，用RPMI培养基重悬细胞至1×10 6个/mL，形成待转染细胞悬液。
根据病毒滴度1×10 8TU/ml以及选择的MOI(multiplicity of infection)计算慢病毒的使用量，分别取250微升待转染细胞悬液加入转染体系形成以下转染对照组和转染实验组：
转染对照组：加入0.5微升聚凝胺(终浓度5微克/毫升)和0.5微升IL-2(终浓度5微克/毫升)，控制MOI＝5，加入的对照病毒悬液与实施例3的慢病毒悬液的区别在于，将对照穿梭质粒按照实施例3所述方法包装为对照慢病毒，且对照穿梭质粒参照实施例2的制备重组穿梭质粒的步骤得到，区别为使用GFP蛋白序列替代Axl-CAR序列。
转染实验组：与转染对照组的区别在于，加入的病毒悬液为实施例3得到的慢病毒悬液。
将转染对照组和转染实验组分别在200g离心力和室温下离心1小时后培养6h。然后向每组加入包含200U/mL rhIL-2的RPMI完全培养基补液至每组500微升样品量，继续培养48小时。
本实施例将每组得到的细胞培养2天后使用生物素化的蛋白L、链酶亲和素-PE、抗CD3-APC进行染色，然后使用流式细胞技术检测，得到图2所示的转染对照组和转染实验组的流式分析结果对照图。染色步骤和流式细胞术检测步骤为本领域技术人员使用的常规技术手段。参照图2，由转染对照组和转染实验组的对比可以看到，转染实施例3的慢病毒后，T细胞成功表达了Axl-CAR。
本实施例将每组得到的细胞培养2天后裂解细胞，提取总RNA后进行逆转录，将获得的cDNA作为模板，以GAPDH为内参基因进行qPCR，得到图3所示的转染对照组和转染实验组的抗Axl-scFv的mRNA相对表达水平对比图。总RNA提取、逆转录以及qPCR的步骤为本领域技术人员使用的常规技术手段。参照图3，与转染对照组相比，转染实验组的抗Axl-scFv的mRNA相对表达水平显著升高了3倍左右。
进行qPCR使用Takara
Figure PCTCN2022122211-appb-000001
RT Master Mix试剂配制反应体系，目的基因的引物对序列如下：
AXL-humab1-F：5’-GGTGGAGGAAGCCAAGTTCA-3’与AXL-humab1-R：5’-CTCTAAACCTTGGCCGGGAG-3’；
CD3-F：5’-TGCCTCTTATCAGTTGGCGT-3’与CD3-R：5’-TTCCTCTGGGGTAGCAGACA-3’；
CD8-F：5’-TTACTGCAACCACAGGAACCG-3’与CD8-R：5’-AGTAATCTTTCCCACCCCGC-3’；
CD4-F：5’-CCAGAGGCCCTGCCATTTC-3’与CD4-R：5’-TTCTTTCCCTGAGTGGCTGC-3’；
IFNG-F：5’-CGTTTTGGGTTCTCTTGGCT-3’与IFNG-R：5’-TTTCTGTCACTCTCCTCTTTCC-3’；
GZMB-F：5’-GATCATCGGGGGACATGAGG-3’与GZMB-R：5’-TGACATTTATGGAGCTTCCCCA-3’；
TNFA-F：5’-GTAGCCCATGTTGTAGCAAACC-3’与TNFA-R：5’-TATCTCTCAGCTCCACGCCA-3’。
实施例5：Axl-CAR-T细胞对肿瘤细胞体外杀伤实验
本实施例将分别使用高表达Axl的LCLC-103H、HL60、MDA-MB-231、MDA-MB-453、MV4-11，以及低表达Axl的H460与实施例4得到的Axl-CAR-T细胞共培养，考察Axl-CAR-T细胞对各肿瘤细胞的杀伤能力。
LCLC-103H和H460均为人大细胞肺癌株，HL60为人白血病肿瘤细胞，MV4-11为人急性单核细胞白血病细胞，均来源于南京科佰生物科技有限公司。
MDA-MB-231和MDA-MB-453均为人乳腺癌细胞，来源于ATCC。
分别将上述各肿瘤细胞在RPMI完全培养基中培养，并以1：3比例传代直至得到相应的生长情况良好的对数期细胞。然后将各肿瘤细胞使用RPMI完全培养基重悬并调整浓度，得到各肿瘤细胞悬液。取实施例4的待转染T细胞经RPMI完全培养基重悬并调整浓度，得到T细胞悬液。取实施例4的Axl-CAR-T细胞经RPMI完全培养基重悬并调整浓度，得到Axl-CAR-T细胞悬液。
取预先每孔包被50微升RPMI完全培养基的16孔板，各孔加入肿瘤细胞悬液并调整浓度为5000个细胞/100微升培养基后培养至肿瘤细胞贴壁。 然后：加入T细胞悬液得到对照组，并调节T细胞与肿瘤细胞数量比(E：T)；加入Axl-CAR-T细胞悬液得到实验组并调节E：T；加入RPMI完全培养基并调整为与对照组和实验组等量得到空白组。
将各肿瘤细胞所对应的空白组、对照组和实验组采用实时无标记动态细胞分析技术(Real Time Cellular Analysis，RTCA)进行监测。每15min检测一次细胞指数，并计算增殖率＝(实时细胞指数-加入T细胞时的细胞指数)/加入T细胞时的细胞指数。
各肿瘤细胞所对应的空白组、对照组和实验组调整各孔肿瘤细胞数目为3×10 4个，经5天共培养后，使用Human IFNγ ELISPOT Kit(abcam)对各对照组和实验组相对空白组的INF-γ相对表达水平进行检测。
从图4所示LCLC-103H空白组以及在E：T＝4：1情况下对应的对照组和实验组在不同共培养时间下的LCLC-103H增殖率的变化趋势对比图可以看出，Axl-CAR-T细胞对LCLC-103H的杀伤效果要强于对照组的T细胞，使得LCLC-103H的增殖率显著下降。可见Axl-CAR-T细胞对LCLC-103H具有良好的杀伤效果。
从图5所示的MDA-MB-231空白组、不同E:T下对应的对照组以及不同E:T下对应的实验组在不同共培养时间下MDA-MB-231增殖率的变化趋势对比图可以看出：在E:T为1:2和1:1下，Axl-CAR-T细胞对MDA-MB-231的杀伤效果显著优于对应的对照组。可见Axl-CAR-T细胞对MDA-MB-231具有良好的杀伤效果。
Axl-CAR-T细胞对肿瘤细胞的杀伤效果具有选择性，具体如下：
从图6所示的LCLC-103H的对应对照组、实验组在不同E:T下的增殖 率趋势，以及H460的对应对照组和实验组在不同E:T下的增殖率趋势对比可以看到：不同E:T下相较于对应的LCLC-103H对照组，Axl-CAR-T细胞对LCLC-103H均具有良好的杀伤效果；E:T为2:1和4:1下相较于对应的H460对照组，Axl-CAR-T细胞对H460的杀伤效果显著，当E:T为8:1时相较于对应的H460对照组，Axl-CAR-T细胞对H460的杀伤效果不显著，相较LCLC-103H各组杀伤情况显示了较强的剂量依赖性。E:T为2:1和4:1下，Axl-CAR-T细胞对LCLC-103H的杀伤效果相较于对H460的杀伤效果更显著，而在E:T为8:1下，Axl-CAR-T细胞对LCLC-103H的杀伤效果相较于对H460的杀伤效果而言并不显著。
从图7所示的MDA-MB-231的对应对照组、实验组在不同E:T下的增殖率趋势，以及MDA-MB-453的对应对照组、实验组在不同E:T下的增殖率趋势对比可以看到：E:T为4:1和8:1时，Axl-CAR-T细胞相对于对应的MDA-MB-231对照组而言显示出了对MDA-MB-231显著的杀伤效果，E:T为2:1时；Axl-CAR-T细胞相对于对应的对照组而言对MDA-MB-231的杀伤效果并不显著。E:T为2:1和4:1时，Axl-CAR-T细胞相对于对应的MDA-MB-453对照组而言显示出了对MDA-MB-453显著的杀伤效果，E:T为2:1时；Axl-CAR-T细胞相对于对应的MDA-MB-453对照组而言对MDA-MB-453的杀伤效果并不显著。在同等E:T下，Axl-CAR-T细胞显示出了对MDA-MB-453更强的杀伤性能。
由于IFN-γ作为免疫活性细胞分泌的细胞因子在诱导抗病毒免疫中起着重要的免疫调理作用，业内公认基体受免疫刺激后的IFN-γ的水平实际上反映了效应细胞的活动。因此，检测IFN-γ的水平就是间接地检测效应细胞 的活性。图8至图10所示的MV4-11、HL60和MDA-MB-231对应的各空白组、对照组和实验组的INF-γ相对表达水平对比图所示：相较于各肿瘤细胞对应的空白组和对照组，各实验组的INF-γ相对表达水平都有显著的上升趋势。说明Axl-CAR-T细胞对于MV4-11、HL60和MDA-MB-231均具有良好的杀伤效果。
实施例6：Axl-CAR-T细胞对肿瘤细胞体内杀伤实验
每组10只雌性、6-8周的NSG小鼠(上海南方模式生物科技股份有限公司)进行体内杀伤实验。具体的，向每只小鼠的第四乳垫种植1×10 7个MDA-MB-231细胞，18天后成瘤，然后进行干预形成以下PBS组、动物对照组和动物治疗组：
PBS组：每次尾静脉注射100微升PBS溶液；
动物对照组：用100微升PBS溶液重悬实施例4的待转染T细胞并调整细胞数为8×10 6个，每次对小鼠实施尾静脉注射；
动物治疗组：用100微升PBS溶液重悬实施例4的Axl-CAR-T细胞并调整细胞数为8×10 6个，每次对小鼠实施尾静脉注射。
以上各组每两天尾静脉注射一次，共注射4次。开始给药后的第34天对小鼠安乐死并取组织进行分析。
本实施例取小鼠的肺组织样本浸泡于4％多聚甲醛中固定，随后石蜡包埋、切片后进行苏木精-伊红染色(HE)后显微镜镜检，使用ImageJ进行HE染色图片处理，圈出肺组织中的肿瘤部分并计算面积，得到图11所示的PBS组、动物对照组和动物治疗组的各肺组织样本的肿瘤转移情况对比照片以及根据图11所示照片统计的各组肺组织样本的肺转移区域占比对比图。 具体的染色、显微镜镜检和图像采集分析方法为本领域技术人员的常规技术手段。参照图11和图12，相比于PBS组和动物对照组，动物实验组的肺部肿瘤区域占比显著下降甚至接近于0，可见Axl-CAR-T细胞能够抑制乳腺癌的肺转移。
本实施例取各小鼠的原位肿瘤组织样本计算肿瘤体积＝0.5×L×W 2；其中L为长度，W为宽度。通过计算可知，PBS对照组的平均肿瘤体积为2800立方厘米，动物对照组的平均肿瘤体积为2430立方厘米，动物实验组的平均肿瘤体积为1650立方厘米。相对对照组的p＝0.0005。
各小鼠的原位肿瘤组织样本浸泡于4％多聚甲醛中固定，随后石蜡包埋、切片后进行免疫组织化学染色(IHC)，使用的抗体及比例包括：anti-CD3 1:100(Servicebio,GB13014)；anti-CD8 1:100(Servicebio,GB13068)；二抗HRP-山羊抗兔抗体1:200(Servicebio,GB23303)。然后进行显微镜镜检，使用ImageJ进行免疫组化图片处理，使用IHCTool提取出免疫组化阳性区域并计算面积，得到图13所示的各组原位肿瘤组织样本的免疫染色照片对比图，以及图14统计的各组原位肿瘤组织样本中CD3和CD8的阳性区域占比对比图。参照图13和图14，相比于动物对照组，动物实验组的肿瘤样本中CD3和CD8的蛋白水平显著上调。
进一步的，本实施例提取了各原位肿瘤组织样本的mRNA进行qPCR分析，检测CD3、CD8A、CD4、IFNG、GZMB及TNFA的表达水平，得到图15所示的各组上述因子表达水平对比图。其中，PBS表示PBS组，Mock T cell表示动物对照组，AXL-CAR T cell表示动物实验组。具体分析步骤请参见前述。参见图15，相比于动物对照组，动物实验组的肿瘤样本中CD3、 CD8A、CD4这三个T细胞标志基因显著上调，T细胞分泌的细胞因子基因IFNG、GZMB及TNFA也显著上调，结合图13和图14所指示的动物实验组的肿瘤样本中CD3和CD8的蛋白水平显著上调，均证明了相较于动物对照组，Axl-CAR-T细胞对肿瘤的浸润能力更强。

Claims (11)

  1. 一种嵌合抗原受体，其特征在于，包括重链可变区和轻链可变区；
    所述轻链可变区的序列如SEQ ID NO：1所示；
    所述重链可变区的序列如SEQ ID NO：2所示。
  2. 根据权利要求1所述的嵌合抗原受体，其特征在于，还包括连接所述轻链可变区和所述重链可变区的连接区，所述连接区序列如SEQ ID NO：3所示。
  3. 根据权利要求1所述的嵌合抗原受体，其特征在于，还包括跨膜结构域，以及位于所述跨膜结构域和所述Axl特异性抗体的重链可变区之间的铰链区；
    所述铰链区的序列来源于CD8、CD8α、CD28、4-1BB、ICOS、OX40、CD40、CD80和IgG的至少一种；
    所述跨膜结构域的序列来源于CD2、CD27、LFA-1、CD8α、CD28、4-1BB、ICOS、OX40、CD40、CD80、CD3ζ和CD3ε的至少一种。
  4. 根据权利要求3所述的嵌合抗原受体，其特征在于，所述铰链区的序列如SEQ ID NO：4所示，所述跨膜结构域的序列如SEQ ID NO：5所示。
  5. 根据权利要求3所述的嵌合抗原受体，其特征在于，还包括表达驱动区以及位于所述表达驱动区和所述Axl特异性抗体的轻链可变区之间的信号肽；
    所述表达驱动区的序列来源于EF1α、CMV、PGK、MPSV、MMLV和SFFV的至少一种。
  6. 根据权利要求5所述的嵌合抗原受体，其特征在于，所述表达驱动区的序列如SEQ ID NO：6所示，所述信号肽的序列如SEQ ID NO：7所示。
  7. 根据权利要求1所述的嵌合抗原受体，其特征在于，所述嵌合抗原受体的序列如SEQ ID NO：8所示。
  8. 一种核酸，其特征在于，编码如权利要求1所述的嵌合抗原受体中至少部分片段。
  9. 一种载体，其特征在于，包含如权利要求8所述的核酸。
  10. 一种宿主细胞，其特征在于，包含如权利要求9所述的载体。
  11. 一种如权利要求1所述的嵌合抗原受体在肿瘤治疗药物制备或肿瘤治疗方面的应用。
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110483639A (zh) * 2018-05-15 2019-11-22 复旦大学 靶向axl的抗体及抗体-药物偶联物及其制备方法和用途
CN115125272A (zh) * 2021-03-29 2022-09-30 复旦大学 一种car-t治疗载体及其构建方法和应用

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110483639A (zh) * 2018-05-15 2019-11-22 复旦大学 靶向axl的抗体及抗体-药物偶联物及其制备方法和用途
CN115125272A (zh) * 2021-03-29 2022-09-30 复旦大学 一种car-t治疗载体及其构建方法和应用

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