A dual-target chimeric antigen receptor that simultaneously targets CD70 and B7H3, chimeric antigen receptor CAR-T cells, and their applications

Abstract

The present invention discloses a dual-target chimeric antigen receptor that simultaneously targets CD70 and B7H3, a chimeric antigen receptor CAR-T cell and its application, which relates to the technical field of biomedicine. The dual-target chimeric antigen receptor includes an antigen-binding domain, a hinge region, a transmembrane domain and a signal transduction domain; the antigen-binding domain contains the binding domains of CD70 and B7H3, forming CD8aL-VHH1-I-VHH2-H-CD28 TM CD28-CD3ζ fusion protein. The dual-target chimeric antigen receptor of the present invention can not only specifically recognize tumor cells that are single positive for B7H3 or CD70, but also recognize tumor cells that co-express B7H3 and CD70. The CAR-T cells with dual targets have stronger anti-tumor immune activity and a wider killing coverage rate, and can reduce the immune escape reaction of low-abundance positive tumor cells, reducing the recurrence probability of cancer.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and specifically relates to a dual-target chimeric antigen receptor that simultaneously targets CD70 and B7H3, a chimeric antigen receptor CAR-T cell, and their applications. Background Art

[0002] With the development of tumor immunology theory and clinical technology, chimeric antigen receptor T-cell immunotherapy (CAR-T) has become the most popular and valuable treatment method in current tumor immunotherapy. At present, CAR-T therapy has made good progress in hematological tumors. However, due to the complex microenvironment of solid tumors, the killing effect of CAR-T on solid tumors is limited. Therefore, new methods need to be explored to improve the activity of CAR-T in treating solid tumors.

[0003] The basic design of CAR includes a tumor-associated antigen binding region, an intracellular signaling region, a transmembrane region, an extracellular hinge region, etc. Nanobodies, also known as VHH antibodies or single-domain antibodies, are a special type of antibody produced by animals such as camels and mules, and have a structure and function similar to those of conventional antibodies. Nanobodies are currently the smallest functional antigen-specific binding natural fragments, consisting of about 120 amino acids, with a length of 4 nm and a diameter of 2.5 nm. Compared with traditional monoclonal antibodies and Fab fragments (55×103) or scFv (28×103), nanobodies have a smaller molecular weight, so they have stronger and faster tissue penetration ability and can reach dense tissues such as solid tumors to play a role. In addition, nanobodies have good stability, high affinity, weak immunogenicity, are easy to genetically modify, and do not have problems such as easy mispairing of traditional antibodies and the need to optimize the heavy-chain and light-chain connection sequences, showing broad application prospects in tumor immunotherapy and other aspects.

[0004] CD70 is one of the members of the tumor necrosis factor receptor (TNFR) superfamily, and has the ability to regulate the activation, proliferation, and differentiation of T cells and B cells, playing an important role in maintaining the body's immune response. At the same time, under physiological conditions, CD70 is only transiently expressed on activated lymphocytes, but is abnormally expressed in various cancers such as renal cell carcinoma, lung cancer, hematogenous tumors, and central nervous system gliomas, and is closely related to the occurrence and development of tumors and the prognosis of patients, and can be used as a new biomarker for early cancer diagnosis, a new target for clinical diagnosis and treatment, and for detecting disease prognosis.

[0005] B7-H3 is a member of the immunoglobulin superfamily and plays an important role in tumor immunology. It is usually highly expressed in various tumor types, including but not limited to lung cancer, breast cancer, prostate cancer, and kidney cancer. The high expression of B7-H3 is associated with tumor aggressiveness and poor prognosis.

[0006] Although current CAR-T cells targeting CD70 or B7-H3 have achieved certain therapeutic effects in the corresponding cancer types, they still face certain obstacles, such as the loss of target antigens or high tumor heterogeneity. Once the target antigens of tumor cells are lost, CAR-T cells cannot exert their due tumor-killing ability. Therefore, the dual-target CAR-T constructed in the present invention that targets two antigens simultaneously can reduce the tumor recurrence rate and thus improve the therapeutic effect of tumors. Summary of the Invention

[0007] The purpose of the present invention is to provide a dual-target chimeric antigen receptor that simultaneously targets CD70 and B7H3, chimeric antigen receptor CAR-T cells, and their applications to solve the problems raised in the above background technology.

[0008] To achieve the above purpose, the present invention provides the following technical solutions:

[0009] A dual-target chimeric antigen receptor that simultaneously targets CD70 and B7H3, the dual-target chimeric antigen receptor includes an antigen-binding domain, a hinge region, a transmembrane domain, and a signal transduction domain; the antigen-binding domain contains the binding domains of CD70 and B7H3.

[0010] As a further scheme of the present invention: the dual-target chimeric antigen receptor contains CD8Leader, a nanobody targeting CD70, a nanobody targeting B7H3, hinge region CD8a, transmembrane region CD28, intracellular co-stimulatory domain CD28, and intracellular signal transduction domain CD3ζ in series, forming CD8aL-VHH1-I-VHH2-H-CD28 TM CD28-CD3ζ fusion protein; wherein, VHH1 is the antigen-binding domain targeting CD70, and VHH2 is the antigen-antibody domain targeting B7H3.

[0011] As a further scheme of the present invention: the CD8a L-VHH1-I-VHH2-H-CD28 TM In the CD28-CD3ζ fusion protein, the amino acid sequence of CD8 Leader is as shown in SEQ ID NO: 1.

[0012] As a further scheme of the present invention: the CD8a L-VHH1-I-VHH2-H-CD28 TMIn the CD28-CD3ζ fusion protein, the amino acid sequence of anti-CD70 VHH is as shown in SEQ ID NO: 2.

[0013] As a further aspect of the present invention: The dual-target chimeric antigen receptor further includes a linker sequence, which is connected between the nanobody targeting CD70 and the nanobody targeting B7H3. The linker fragment is (G4S)n or (EAAAK)n, where n is 1, 2, or 3.

[0014] Preferably, the linker fragment is (G4S)n, where n is 3, and the nucleotide sequence of the linker sequence is as shown in SEQ ID NO: 3.

[0015] Preferably, the CD8a L-VHH1-I-VHH2-H-CD28 TM In the CD28-CD3ζ fusion protein, the amino acid sequence of anti-CD70 VHH is as shown in SEQ ID NO: 4.

[0016] As a further aspect of the present invention: The CD8a L-VHH1-I-VHH2-H-CD28 TM In the CD28-CD3ζ fusion protein, the amino acid sequence of CD8a hinge is as shown in SEQ ID NO: 5.

[0017] As a further aspect of the present invention: The CD8a L-VHH1-I-VHH2-H-CD28 TM In the CD28-CD3ζ fusion protein, the amino acid sequence of CD28TM is as shown in SEQ ID NO: 6.

[0018] As a further aspect of the present invention: The CD8a L-VHH1-I-VHH2-H-CD28 TM In the CD28-CD3ζ fusion protein, the amino acid sequence of CD28 is as shown in SEQ ID NO: 7, and the amino acid sequence of CD3ζ is as shown in SEQ ID NO: 8.

[0019] As a further aspect of the present invention: The CD8a

[0020] The amino acid sequence of the L-VHH1-I-VHH2-H-CD28TM CD28-CD3ζ fusion protein is as shown in SEQ ID NO: 9.

[0021] The B7H3 and CD70 antibody sequences are those described in this patent or amino acid sequences having 90-99% identity therewith.

[0022] A polynucleotide encoding the above dual-target chimeric antigen receptor.

[0023] A viral vector, wherein the vector comprises the corresponding coding gene of the dual-target chimeric antigen receptor that simultaneously targets CD70 and B7H3 as described above.

[0024] A dual-target chimeric antigen receptor CAR-T cell that simultaneously targets CD70 and B7H3, comprising the dual-target chimeric antigen receptor that simultaneously targets CD70 and B7H3 as described above, and the CAR-T cell expresses the chimeric antigen receptor as described above.

[0025] The CAR-T cells prepared by the present invention have good killing effects on CD70-positive or B7H3-positive tumor cells, have no killing effects on normal tissue cells with weak expression of CD70 or B7H3, and can maintain a high proliferation ability and a low exhaustion index.

[0026] An application of a dual-target chimeric antigen receptor CAR-T cell that simultaneously targets CD70 and B7H3 in the preparation of anti-tumor drugs, including solid tumors and hematological tumors. The tumor is preferably renal cancer, and the tumor is a tumor that singly expresses or co-expresses CD70 and B7H3, or a tumor that is ineffective due to antigen loss after treatment with B7H3 CAR-T or CD70 CAR-T.

[0027] Compared with the prior art, the beneficial effects of the present invention are as follows: The dual-target chimeric antigen receptor of the present invention can not only specifically recognize tumor cells with single positive of B7H3 or CD70, but also recognize tumor cells co-expressing B7H3 and CD70. The CAR-T cells with dual targets have stronger anti-tumor immune activity and a wider killing coverage rate, and can reduce the immune escape reaction of low-abundance positive tumor cells, reducing the recurrence probability of cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings required to be used in the embodiments. Obviously, the drawings described below are only some embodiments recorded in the present invention. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings.

[0029] Figure 1 It is a schematic diagram of the structures of the humanized single CAR and Tandem-CAR vectors provided in the embodiments of the present invention;

[0030] Figure 2 It is a flow cytometry detection result diagram of the transfection efficiency of the humanized bispecific Tandem CAR-T cells provided in the embodiments of the present invention;

[0031] Figure 3Flow cytometry detection results of CD70 expression in ovarian cancer cell line OVCAR3 and B7H3 in prostate cancer PC-3 provided by the embodiments of the present invention;

[0032] Figure 4 Cytotoxicity of humanized bispecific Tandem CAR-T cells provided by the embodiments of the present invention against ovarian cancer cells (A2780) that do not express B7H3 and CD70, ovarian cancer cells (OVCAR3) that naturally express CD70, and prostate cancer cells (PC-3) that naturally express B7H3. Among them, the ordinate is the cell killing efficiency, and the unit is%;

[0033] Figure 5 Comparison chart of cytokine IFNγ and IL-2 secretion levels after incubation of humanized bispecific Tandem CAR-T cells with OVCAR3 cells provided by the embodiments of the present invention. Among them, mock T is untransfected T cells. Detailed implementation manners

[0034] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention and the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0035] Please refer to Figures 1-5 , in the embodiments of the present invention,

[0036] Example 1

[0037] Preparation of lentiviral expression vector

[0038] Gene synthesis of CD8a L-VHH1-I-VHH2-H-CD28 TM CD28-CD3ζ fusion gene sequence, and the gene sequence is as shown in SEQ ID NO.9. It is ligated into the PLV vector through digestion and transformation, and the upstream of the gene is the EP-1a promoter.

[0039] Example 2

[0040] Preparation of lentivirus

[0041] 24 hours before transfection, inoculate 293T cells into a T75 culture flask at about 1×107 per flask, and ensure that when the cells reach about 80% confluence and are evenly distributed in the culture flask, lentivirus packaging is carried out.

[0042] Prepare plasmid and transfection reagent diluents

[0043] 1. Vortex and mix the PEI 40K transfection reagent thoroughly.

[0044] 2. Prepare 2 centrifuge tubes and prepare plasmid and transfection reagent dilutions respectively in the following order.

[0045]

[0046] 3. Mix well.

[0047] 4. Add the transfection reagent dilution (centrifuge tube 2) to the plasmid DNA solution (centrifuge tube 1), and immediately mix well. Note that the addition order is very important.

[0048] 5. Incubate the transfection mixture at room temperature for 15 - 20 minutes.

[0049] 6. Add 1 ml of each transfection mixture to a 293T cell culture flask that has been seeded, and gently pipette the medium to mix well.

[0050] 7. Incubate at 37°C for 6 hours.

[0051] 8. Remove the medium containing the transfection reagent, and replace it with 20 ml of virus medium.

[0052] 9. Collect the cell culture supernatant 48 hours after transfection, centrifuge at 500 g for 10 min to remove cell debris. This supernatant can be directly used for lentivirus infection, or can be used for virus titer determination or virus concentration. For long - term storage, it can be frozen at - 80°C.

[0053] Example 3

[0054] Preparation of CAR - T cells

[0055] 1. Isolate T cells using the EasySepTM Human T Cell Isolation Kit (STEMCELL) (specific steps according to the instruction manual). Add anti - CD3 / CD28 magnetic beads (Gibco) at a ratio of cell:magnetic bead = 1:3, and culture for 24 hours to obtain T cells before transfection.

[0056] 2. Take out the virus supernatant from - 80°C, thaw it at 4°C. Add 100 μl of virus supernatant, 4 μg / ml polybrene and 100 IU / mL IL - 2 per 1×106 T cells. After 24 h, add fresh medium and adjust the cell density to 5×105 / ml. Continue to culture at 37°C and 5% CO2, and perform half - volume medium change every 2 - 3 days to maintain the cell density at 0.5 - 1×106 / ml. The culture time is 8 - 14 days.

[0057] 3. After transfection is completed, aspirate the transfected CAR-T cells, centrifuge at 1500 rpm for 5 min to collect the precipitate, and wash the precipitate with normal saline.

[0058] Use the FITC channel of the flow cytometer to detect the proportion of fluorescent cells expressing the transfected CAR. The detection results are as Figure 2 shown. As can be seen from Figure 2 , B7H3 CAR-T, CD70 CAR-T, and Tandem CAR-T have all been constructed, and the positive rates are all above 50%.

[0059] Example 4

[0060] Detection of cell line surface antigens

[0061] OVCAR3 ovarian cancer cells and PC-3 prostate cancer cells were both purchased from ATCC in the United States. After separate culturing, the cells were digested, centrifuged, and collected. They were washed twice with PBS, the supernatant was discarded, and they were labeled with PE anti-human CD70 monoclonal antibody (Biolegend) and PE anti-human B7H3 monoclonal antibody (R&D), and incubated at room temperature for 30 minutes.

[0062] Use flow cytometry to detect the expression levels of B7H3 and CD70. The detection results are as Figure 3 shown. The results show that the OVCAR3 cell line used in this experimental example expresses CD70, the PC-3 cell line expresses B7H3, and the A2780 cell line does not express B7H3 and CD70, which can be used as a negative control.

[0063] Example 5

[0064] 1. Add OVCAR3-luc, PC-3-luc, and A2780-luc cells to a 96-well plate according to the required amount of the sample, with 2×104 cells per well and a volume of 100 μL.

[0065] 2. Adjust the suspension concentration of Tandem CAR-T, CD70 CAR-T, B7H3 CAR-T, and T cells according to different positive rates and effector-target ratios E:T. When adding, it should be noted that regardless of the effector-target ratio and positive rate, the added volume is always 100 μL.

[0066] 3. Return the co-incubated cells to the incubator and culture for 8 h.

[0067] 4. Turn on the multifunctional microplate reader and software, select the Luminescence mode, and perform the plate reading layout.

[0068] 5. Place the incubated 96-well plate in a centrifuge, centrifuge at 1000 g for 10 min, and discard the supernatant.

[0069] 6. Add 100 μL of Reagent E605A in the ONE-Glo Luciferase Assay System kit to each well of cells, pipette to mix evenly, place in the dark at room temperature for 10 min, and transfer the solution in the cell culture plate horizontally into a 96-well white flat-bottom plate.

[0070] 7. Place the 96-well white flat-bottom plate on the microplate reader to read the data, and export and save the data for calculating the cell killing rate. The cell killing rate = (background luminescence value - sample luminescence value) / background luminescence value * 100%.

[0071] The results are as Figure 4 shown. Tandem CAR-T has better tumor killing ability compared with CD70 CAR-T and B7H3 CAR-T.

[0072] Example 6

[0073] ELISA detection of the cytokine release levels of IFN-γ and IL-2 in the co-culture supernatant of cancer cell lines (OVCAR3, PC-3) and CAR-T cells

[0074] Inoculate the OVCAR3 cell line into a 96-well plate at 2×104 cells / well; add several types of CAR-T and mock-T cells at 2×104 cells per well respectively, and co-culture in a cell incubator for 6 - 8 hours; use a human IFN-γ ELISA detection kit (R&D company) and an IL-2 ELISA detection kit (Nanjing Senbeijia company) to detect the co-culture supernatant (see the ELISA detection kit instruction manual for specific steps).

[0075] The results are as Figure 5 shown. The results show that T cells basically do not produce or produce very low levels of IFN-γ cytokine after co-incubation with OVCAR3 cells, while Tandem CAR-T cells produce significant cytokine release after incubation with OVCAR3 cells. Compared with the T cell control group, although there is no significant increase in the release of IL-2, the IL-2 released by Tandem CAR-T cells is still increased compared with the T cell group. These results prove that Tandem CAR-T cells can be specifically activated by tumor cells naturally expressing B7H3 and CD70 antigens.

[0076] Only some exemplary embodiments of the present invention are described by way of illustration above. Without doubt, for those of ordinary skill in the art, the described embodiments can be modified in various different ways without departing from the spirit and scope of the present invention. Therefore, the above drawings and descriptions are illustrative in nature and should not be construed as limiting the protection scope of the claims of the present invention.

[0077] SEQ ID NO:1 (CD8 Leader sequence):

[0078] MALPVTALLLPLALLLHAARP

[0079] SEQ ID NO:2 (anti-CD70 VHH):

[0080] QVQLVESGGGLVQPGGSLRLSCAASGFTLDTFDYYNIGWFRQAPGKEREEVSCISSNDASTNYANSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAARKHYYCPMYGCCNEYDYWGQGTQVTVSS

[0081] SEQ ID NO:3 (linker):

[0082] GGGGSGGGGSGGGGS

[0083] SEQ ID NO:4 (anti-B7H3 VHH):

[0084] QVQLQESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVGWIYPGNGDTSYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARUYGCCGYYYAMDYWGQGTSVTVSS

[0085] SEQ ID NO:5 (CD8a hinge):

[0086] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIY

[0087] SEQ ID NO:6 (CD28TM):

[0088] FWVLVVVGGVLACYSLLVTVAFIIFWV

[0089] SEQ ID NO:7 (CD28):

[0090] RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS

[0091] SEQ ID NO:8 (CD3_zeta):

[0092] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0093] SEQ ID NO:9:

[0094] MALPVTALLLPLALLLHAARPQVQLVESGGGLVQPGGSLRLSCAASGFTLDTFDYYNIGWFRQAPGKEREEVSCISSNDASTNYANSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAARKHYYCPMYGCCNEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSQVQLQESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVGWIYPGNGDTSYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARUYGCCGYYYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

Claims

1. A dual-target chimeric antigen receptor that simultaneously targets CD70 and B7H3, characterized in that: The dual-target chimeric antigen receptor comprises an antigen-binding domain, a hinge region, a transmembrane domain and a signal transduction domain; the antigen-binding domain contains the binding domains of CD70 and B7H3, and the dual-target chimeric antigen receptor comprises, in series, CD8 Leader, a nanobody targeting CD70, a nanobody targeting B7H3, hinge region CD8a, transmembrane region CD28, intracellular co-stimulatory domain CD28 and intracellular signal transduction domain CD3ζ, forming a CD8a L-VHH1-I-VHH2-H-CD28 TM CD28-CD3ζ fusion protein; wherein, VHH1 is the antigen-binding domain targeting CD70, VHH2 is the antigen-antibody domain targeting B7H3. In the CD8a L-VHH1-I-VHH2-H-CD28 TM CD28-CD3ζ fusion protein, the amino acid sequence of CD8 Leader is as shown in SEQ ID NO:

1. In the CD8a L-VHH1-I-VHH2-H-CD28 TM CD28-CD3ζ fusion protein, the amino acid sequence of anti-CD70 VHH is as shown in SEQ ID NO:

2. In the CD8a L-VHH1-I-VHH2-H-CD28 TM CD28-CD3ζ fusion protein, the amino acid sequence of CD8a hinge is as shown in SEQ ID NO:

5. In the CD8a L-VHH1-I-VHH2-H-CD28 TM CD28-CD3ζ fusion protein, the amino acid sequence of CD28TM is as shown in SEQ ID NO:

6. In the CD8a L-VHH1-I-VHH2-H-CD28 TM CD28-CD3ζ fusion protein, the amino acid sequence of CD28 is as shown in SEQ ID NO: 7, and the amino acid sequence of CD3ζ is as shown in SEQ ID NO:

8. The amino acid sequence of the CD8a L-VHH1-I-VHH2-H-CD28 TM CD28-CD3ζ fusion protein is as shown in SEQ ID NO:

9.

2. A dual-target chimeric antigen receptor CAR-T cell that simultaneously targets CD70 and B7H3, characterized in that: A dual-target chimeric antigen receptor that simultaneously targets CD70 and B7H3 as claimed in claim 1.

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