CN117843809A - Double-target chimeric antigen receptor for simultaneously targeting CD70 and B7H3, chimeric antigen receptor CAR-T cell and application thereof - Google Patents
Double-target chimeric antigen receptor for simultaneously targeting CD70 and B7H3, chimeric antigen receptor CAR-T cell and application thereof Download PDFInfo
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Abstract
The invention discloses a double-target chimeric antigen receptor and chimeric antigen receptor CAR-T cell for simultaneously targeting CD70 and B7H3 and application thereof, and relates to the technical field of biological medicine, wherein the double-target chimeric antigen receptor comprises an antigen binding domain, a hinge region, a transmembrane domain and a signal transduction domain; the antigen binding domain comprises the binding domains of CD70 and B7H3, forming CD8aL-VHH1-I-VHH2-H-CD28 TM CD28-CD3 zeta fusion proteins. The double-target chimeric antigen receptor not only can specifically identify B7H3 or CD70 single-positive tumor cells, but also can identify B7H3 and CD70 co-expressed tumor cells, and the double-target CAR-T is fineThe cells have stronger anti-tumor immune activity and wider killing coverage rate, and can reduce immune escape reaction generated by low-abundance positive tumor cells, thereby reducing the recurrence probability of cancers.
Description
Technical Field
The invention relates to the technical field of biological medicine, in particular to a double-target chimeric antigen receptor and chimeric antigen receptor CAR-T cell for simultaneously targeting CD70 and B7H3 and application thereof.
Background
With the development of tumor immunology theory and clinical technology, chimeric antigen receptor T cell therapy (Chimeric antigen receptor T-cell immunotherapy, CAR-T) has become the most popular treatment of research value in current tumor immunotherapy. At present, CAR-T treatment has been well developed in blood tumors, but because of the complex microenvironment of solid tumors, CAR-T has limited killing effect on solid tumors, and thus new methods need to be explored to improve the activity of CAR-T in treating solid tumors.
The basic design of the CAR includes tumor-associated antigen binding regions, intracellular signaling regions, transmembrane regions, extracellular hinge regions, and the like. Nanobodies, also known as VHH antibodies or single domain antibodies, are a special type of antibody produced by animals such as camels, mules, etc., having a structure and function similar to conventional antibodies. Nanobodies are currently the smallest functional antigen-specific binding natural fragments consisting of about 120 amino acids, 4nm in length and 2.5nm in diameter. Compared with the traditional monoclonal antibody and Fab fragment (55X 103) or scFv (28X 103), the molecular weight of the nano antibody is smaller, so that the nano antibody has stronger and faster tissue penetration capability and can reach compact tissues such as solid tumors to play a role. In addition, the nano antibody has the advantages of good stability, higher affinity, weak immunogenicity, easiness in gene modification, no occurrence of the problems that the traditional antibody is easy to produce wrong pairing and the connecting sequence of the heavy chain and the light chain needs to be optimized, and the like, and has wide application prospect in the aspects of tumor immunotherapy and the like.
CD70 is one of the members of the tumor necrosis factor receptor (tumor necrosis factor receptor, TNFR) superfamily, has the ability to regulate T cell and B cell activation, proliferation and differentiation, and plays an important role in maintaining the immune response of the organism. Meanwhile, under physiological conditions, CD70 is only transiently expressed in activated lymphocytes, but is abnormally expressed in various cancers such as renal cell carcinoma, lung cancer, hematogenous tumors, central nervous system glioma and the like, is closely related to the occurrence and development of tumors and prognosis of patients, and can be used as a novel biomarker for early diagnosis of cancers, clinical diagnosis and treatment and a novel target for detecting disease prognosis.
B7-H3 is a member of the immunoglobulin superfamily and plays an important role in tumor immunology. It is often highly expressed in a variety of tumor types including, but not limited to, lung, breast, prostate and kidney cancers. High expression of B7-H3 is associated with tumor invasiveness and poor prognosis.
Although CAR-T cells targeting CD70 or B7-H3 have achieved a certain therapeutic effect in the corresponding cancer species at present, there is still a certain impediment, such as loss of target antigen or high tumor heterogeneity, etc., and once the target antigen of tumor cells is lost, CAR-T cells cannot exert the due tumor killing ability. Therefore, the double-target CAR-T constructed by the invention and targeting two antigens can reduce the recurrence rate of the tumor, thereby improving the treatment effect of the tumor.
Disclosure of Invention
The invention aims to provide a double-target chimeric antigen receptor, a chimeric antigen receptor CAR-T cell and application thereof for simultaneously targeting CD70 and B7H3, so as to solve the problems in the background art.
In order to achieve the above purpose, the present invention provides the following technical solutions:
a dual-target chimeric antigen receptor that targets CD70 and B7H3 simultaneously, the dual-target chimeric antigen receptor comprising an antigen binding domain, a hinge region, a transmembrane domain, and a signaling domain; the antigen binding domain comprises the binding domains of CD70 and B7H 3.
As a further scheme of the invention: the double-target chimeric antigen receptor comprises a CD8Leader, a CD 70-targeting nanobody, a B7H 3-targeting nanobody, a hinge region CD8a, a transmembrane region CD28, an intracellular co-stimulatory domain CD28 and an intracellular signal transduction domain CD3 zeta which are sequentially connected in series to form a CD8aL-VHH1-I-VHH2-H-CD28 TM CD28-CD3 zeta fusion protein; wherein VHH1 is an antigen binding domain targeting CD70 and VHH2 is an antigen antibody domain targeting B7H 3.
As still further aspects of the invention: the CD8a L-VHH1-I-VHH2-H-CD28 TM In the CD28-CD3 zeta fusion protein, the amino acid sequence of the CD8Leader is shown in SEQ ID NO: 1.
As still further aspects of the invention: the CD8a L-VHH1-I-VHH2-H-CD28 TM In the CD28-CD3 zeta fusion protein, the amino acid sequence of the anti-CD70 VHH is shown in SEQ ID NO: 2.
As still further aspects of the invention: the double-target chimeric antigen receptor further comprises a connecting sequence, wherein the connecting sequence is connected between the CD 70-targeting nano-antibody and the B7H 3-targeting nano-antibody, the connecting fragment is (G4S) n or (EAAAK) n, and n is 1, 2 or 3.
Preferably, the connecting fragment is (G4S) n, n is 3, and the nucleotide sequence of the connecting sequence is shown in SEQ ID NO: 3.
Preferably, the CD8a L-VHH1-I-VHH2-H-CD28 TM In the CD28-CD3 zeta fusion protein, the amino acid sequence of the anti-CD70 VHH is shown in SEQ ID NO: 4.
As still further aspects of the invention: the CD8a L-VHH1-I-VHH2-H-CD28 TM In the CD28-CD3 zeta fusion protein, the amino acid sequence of CD8a range is shown as SEQ ID NO. 5.
As still further aspects of the invention: the CD8a L-VHH1-I-VHH2-H-CD28 TM In the CD28-CD3 zeta fusion protein, the amino acid sequence of CD28TM is shown as SEQ ID NO. 6.
As still further aspects of the invention: the CD8a L-VHH1-I-VHH2-H-CD28 TM In the CD28-CD3 zeta fusion protein, the amino acid sequence of CD28 is shown as SEQ ID NO. 7, and the amino acid sequence of CD3 zeta is shown as SEQ ID NO. 8.
As still further aspects of the invention: the CD8a
The amino acid sequence of the L-VHH1-I-VHH2-H-CD28TMCD28-CD3 zeta fusion protein is shown as SEQ ID NO. 9.
The B7H3 and CD70 antibody sequences are the amino acid sequences described in the patent or have 90-99% identity with the same.
Polynucleotides encoding the above-described dual-target chimeric antigen receptors.
A viral vector comprising the corresponding coding gene of the dual-target chimeric antigen receptor described above that targets both CD70 and B7H 3.
A dual-target chimeric antigen receptor CAR-T cell that targets CD70 and B7H3 simultaneously, comprising a dual-target chimeric antigen receptor that targets CD70 and B7H3 simultaneously as described above, the CAR-T cell expressing the chimeric antigen receptor as described above.
The CAR-T cells prepared by the invention have good killing effect on CD70 positive or B7H3 positive tumor cells, have no killing effect on CD70 or B7H3 weakly expressed normal tissue cells, and can keep higher proliferation capacity and lower depletion index.
Use of a dual-target chimeric antigen receptor CAR-T cell that targets CD70 and B7H3 simultaneously, in the manufacture of an anti-tumor medicament, including solid tumors and hematological tumors, preferably renal cancers, that are tumors that express CD70 and B7H3 either singly or simultaneously, or that are ineffective due to antigen loss following B7H3 CAR-T or CD70CAR-T treatment.
Compared with the prior art, the invention has the beneficial effects that: the double-target chimeric antigen receptor not only can specifically identify B7H3 or CD70 single-positive tumor cells, but also can identify B7H3 and CD70 co-expressed tumor cells, and the double-target CAR-T cells have stronger anti-tumor immune activity and wider killing coverage rate, can reduce immune escape reaction generated by low-abundance positive tumor cells, and reduce the recurrence probability of cancers.
Drawings
In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments described in the present invention, and other drawings may be obtained according to these drawings for a person having ordinary skill in the art.
FIG. 1 is a schematic diagram of the humanized single CAR and the Tanmem-CAR vector structures provided by the embodiment of the invention;
FIG. 2 is a graph of the results of the flow assay of transfection efficiency of humanized bispecific Tanmem CAR-T cells provided by the examples of the present invention;
FIG. 3 is a graph showing the results of flow detection of B7H3 of prostate cancer PC-3 and CD70 expression of OVCAR3, an ovarian cancer cell line according to an embodiment of the present invention;
FIG. 4 is a graph showing cytotoxicity of humanized bispecific Tanmem CAR-T cells provided in the examples of the present invention against ovarian cancer cells (A2780) that do not express B7H3 and CD70, ovarian cancer cells (OVCAR 3) that naturally express CD70, and prostate cancer cells (PC-3) that naturally express B7H3, wherein the ordinate represents cell killing efficiency in units of;
fig. 5 is a graph showing comparison of secretion amounts of cytokines ifnγ and IL-2 after incubation of humanized bispecific Tandem CAR-T cells and OVCAR3 cells, wherein mock T is an untransfected T cell, provided in the examples of the present invention.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the embodiments of the present invention and the accompanying drawings, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Referring to fig. 1-5, in an embodiment of the present invention,
example 1
Lentiviral expression vector preparation
Gene synthesis of CD8a L-VHH1-I-VHH2-H-CD28 TM The CD28-CD3 zeta fusion gene sequence is shown as SEQ ID NO.9, and is connected to PLV carrier through enzyme digestion and conversion, and the upstream of the gene is EP-1a promoter.
Example 2
Lentivirus preparation
24 hours prior to transfection, 293T cells were seeded into T75 flasks at about 1X 107 cells per flask, ensuring that the cells were lentivirally packaged at about 80% confluency and evenly distributed in the flask.
Preparation of plasmid and transfection reagent dilutions
1. Vortex shaking and mixing PEI 40K transfection reagent.
2. 2 centrifuge tubes were prepared and dilutions of plasmid and transfection reagent were prepared separately in the following order.
3. Fully and uniformly mixing.
4. The transfection reagent diluent (centrifuge tube 2) was added to the plasmid DNA solution (centrifuge tube 1) and immediately mixed well, and it was important to note the order of addition.
5. The transfection mixture was incubated at room temperature for 15-20 min.
6. Each 1ml of the transfection mixture was added to a well-spread 293T cell culture flask, and the medium was gently aspirated and mixed.
7. Culturing at 37deg.C for 6 hr.
8. The medium containing the transfection reagent was removed and replaced with 20ml of virus medium.
9. Cell culture supernatant is collected 48 hours after transfection, and cell fragments are removed by centrifugation at 500g for 10min, and the supernatant can be directly used for lentivirus infection, and can also be used for virus titer measurement or virus concentration, and can be frozen at-80 ℃ if long-term storage is needed.
Example 3
CAR-T cell preparation
1. T cells were isolated using EasySepTM Human T Cell Isolation Kit (stemcel) kit (specific procedure according to the instructions), following cells: magnetic beads = 1:3 proportion of anti-CD 3/CD28 magnetic beads (Gibco company) are added, and the T cells before transfection are obtained after culturing for 24 hours.
2. The virus supernatant was removed from-80℃and thawed at 4℃and 100. Mu.l of virus supernatant, 4ug/mL polybrene and 100IU/mL IL-2 were added per 1X 106T cell. After 24h, fresh medium was added and the cell density was adjusted to 5X 105/ml, cultivation was continued at 37℃with 5% CO2, half-volume liquid changes were performed every 2-3 days, and the cell density was maintained at 0.5-1X 106/ml for 8-14 days.
3. After the transfection was completed, the transfected CAR-T cells were aspirated, and the pellet was collected by centrifugation at 1500rpm for 5min and washed with physiological saline.
The proportion of transfected CAR expressed fluorescent cells is detected by using a flow cytometer FITC channel, the detection result is shown in figure 2, and as can be seen from figure 2, the construction of B7H3 CAR-T, CD70CAR-T and Tandem CAR-T is completed, and the positive rate is above 50%.
Example 4
Detection of cell line surface antigens
OVCAR3 ovarian cancer cells, PC-3 prostate cancer cells were purchased from ATCC in the united states, and after incubation, the cells were digested, centrifuged, collected, washed 2 times with PBS, the supernatant was discarded, and PE anti-human CD70 mab (Biolegend company), PE anti-human B7H3 mab (R & D company) were labeled and incubated at room temperature for 30 minutes.
The expression levels of B7H3 and CD70 were measured by flow cytometry, and the measurement results are shown in FIG. 3, which shows that the OVCAR3 cell line used in the experimental example expresses CD70, the PC-3 cell line expresses B7H3, and the A2780 cell line does not express B7H3 and CD70, and can be used as a negative control.
Example 5
1. OVCAR3-luc, PC-3-luc and A2780-luc cells were added in the required amounts to the samples in 96-well plates, 2X 104 cells per well, in a volume of 100. Mu.L.
2. T-regulatory cell suspension concentrations of T-regulatory cells were based on different positive and negative ratios of T-regulatory cells, with respect to both negative and positive ratios, the volume of addition was always 100. Mu.L.
3. The co-incubated cells were returned to the incubator for 8h.
4. And opening the multifunctional microplate reader and software, selecting a luminencemode, and performing plate reading layout.
5. The 96-well plate after incubation was placed in a centrifuge, centrifuged at 1000g for 10min and the supernatant was discarded.
6. And adding an E605A reagent in a 100 mu LONE-Glo Luciferase Assay System kit into each cell, blowing and uniformly mixing, standing at room temperature in a dark place for 10min, and transferring the solution sucked into a cell culture plate into a 96-well white flat bottom plate in a translation way.
7. The 96-well white flat bottom plate is placed with an enzyme label instrument to read data, and the data are derived and stored for calculating the cell killing rate, wherein the cell killing rate is = (background luminescence value-sample luminescence value)/background luminescence value is 100%.
The results are shown in FIG. 4, where Tandem CAR-T has better tumor killing ability compared to CD70CAR-T and B7H3 CAR-T.
Example 6
ELISA detection of cytokine IFN-gamma, IL-2 cytokine release levels in cancer cell lines (OVCAR 3, PC-3) and CAR-T cell co-culture supernatants
OVCAR3 cell lines were seeded in 96-well plates at 2 x 104 cells/well; adding several CAR-T, mock-T cells into 2X 104 cells per well, and co-culturing in a cell incubator for 6-8 hours; the co-culture supernatants were tested using human IFN-. Gamma.ELISA test kit (R & D Co.) and IL-2ELISA test kit (Nanjing Sen Bei Ga Co.), for specific steps as described in ELISA test kit instructions.
The results are shown in fig. 5, which demonstrate that T cells produce substantially no or very low IFN- γ cytokines after co-incubation with OVCAR3 cells, while Tandem CAR-T cells produce significant cytokine release after incubation with OVCAR3 cells, and that the release of IL-2 from the Tandem CAR-T cells is still elevated compared to the T cell control group, although there is no significant increase in IL-2 release, demonstrating that the Tandem CAR-T cells can be specifically activated by tumor cells naturally expressing B7H3 and CD70 antigens.
While certain exemplary embodiments of the present invention have been described above by way of illustration only, it will be apparent to those of ordinary skill in the art that modifications may be made to the described embodiments in various different ways without departing from the spirit and scope of the invention. Accordingly, the drawings and description are to be regarded as illustrative in nature and not as restrictive of the scope of the invention, which is defined by the appended claims.
SEQ ID NO:1(CD8 Leader sequence)：
MALPVTALLLPLALLLHAARP
SEQ ID NO:2(anti-CD70 VHH)：
QVQLVESGGGLVQPGGSLRLSCAASGFTLDTFDYYNIGWFRQAPGKEREEVSCISSNDASTNYANSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAARKHYYCPMYGCCNEYDYWGQGTQVTVSS
SEQ ID NO:3(linker)：
GGGGSGGGGSGGGGS
SEQ ID NO:4(anti-B7H3 VHH)：
QVQLQESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVGWIYPGNGDTSYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARUYGCCGYYYAMDYWGQGTSVTVSS
SEQ ID NO:5(CD8a hinge)：
TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIY
SEQ ID NO:6(CD28TM)：
FWVLVVVGGVLACYSLLVTVAFIIFWV
SEQ ID NO:7(CD28)：
RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS
SEQ ID NO:8(CD3_zeta)：
RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
SEQ ID NO:9：
MALPVTALLLPLALLLHAARPQVQLVESGGGLVQPGGSLRLSCAASGFTLDTFDYYNIGWFRQAPGKEREEVSCISSNDASTNYANSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAARKHYYCPMYGCCNEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSQVQLQESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVGWIYPGNGDTSYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARUYGCCGYYYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
Claims (10)
1. A dual-target chimeric antigen receptor that targets CD70 and B7H3 simultaneously, characterized in that: the dual-target chimeric antigen receptor comprises an antigen binding domain, a hinge region, a transmembrane domain, and a signaling domain;
the antigen binding domain comprises the binding domains of CD70 and B7H 3.
2. A dual-target chimeric antigen receptor simultaneously targeting CD70 and B7H3 according to claim 1, wherein: the double-target chimeric antigen receptor comprises a CD8Leader, a CD 70-targeting nanobody, a B7H 3-targeting nanobody, a hinge region CD8a, a transmembrane region CD28, an intracellular co-stimulatory domain CD28 and an intracellular signal transduction domain CD3 zeta which are sequentially connected in series to form CD8a
L-VHH1-I-VHH2-H-CD28 TM CD28-CD3 zeta fusion protein;
wherein VHH1 is an antigen binding domain targeting CD70 and VHH2 is an antigen antibody domain targeting B7H 3.
3. A dual-target chimeric antigen receptor simultaneously targeting CD70 and B7H3 according to claim 2, wherein: the CD8a L-VHH1-I-VHH2-H-CD28 TM In the CD28-CD3 zeta fusion protein, the amino acid sequence of the CD8Leader is shown in SEQ ID NO: 1.
4. A dual-target chimeric antigen receptor simultaneously targeting CD70 and B7H3 according to claim 2, wherein: the CD8a L-VHH1-I-VHH2-H-CD28 TM In the CD28-CD3 zeta fusion protein, the amino acid sequence of the anti-CD70 VHH is shown in SEQ ID NO: 2.
5. A dual-target chimeric antigen receptor simultaneously targeting CD70 and B7H3 according to claim 2, wherein: the CD8a L-VHH1-I-VHH2-H-CD28 TM In the CD28-CD3 zeta fusion protein, the amino acid sequence of CD8a range is shown as SEQ ID NO. 5.
6. A dual-target chimeric antigen receptor simultaneously targeting CD70 and B7H3 according to claim 2, wherein: the CD8a L-VHH1-I-VHH2-H-CD28 TM In the CD28-CD3 zeta fusion protein, the amino acid sequence of CD28TM is shown as SEQ ID NO. 6.
7. A dual-target chimeric antigen receptor simultaneously targeting CD70 and B7H3 according to claim 2, wherein: the CD8a L-VHH1-I-VHH2-H-CD28 TM In the CD28-CD3 zeta fusion protein, the amino acid sequence of CD28 is shown as SEQ ID NO. 7, and the amino acid sequence of CD3 zeta is shown as SEQ ID NO. 8.
8. A dual-target chimeric antigen receptor simultaneously targeting CD70 and B7H3 according to claim 2, wherein: the CD8a L-VHH1-I-VHH2-H-CD28 TM The amino acid sequence of the CD28-CD3 zeta fusion protein is shown as SEQ ID NO. 9.
9. A dual-target chimeric antigen receptor CAR-T cell that targets CD70 and B7H3 simultaneously, characterized in that: a dual-target chimeric antigen receptor comprising the simultaneous targeting of CD70 and B7H3 of any one of claims 1-8.
10. The use of a dual-target chimeric antigen receptor CAR-T cell simultaneously targeting CD70 and B7H3 according to claim 9, wherein: the application in preparing antitumor drugs.
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| CN202311765118.9A CN117843809A (en) | 2023-12-21 | 2023-12-21 | Double-target chimeric antigen receptor for simultaneously targeting CD70 and B7H3, chimeric antigen receptor CAR-T cell and application thereof |
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| CN202311765118.9A CN117843809A (en) | 2023-12-21 | 2023-12-21 | Double-target chimeric antigen receptor for simultaneously targeting CD70 and B7H3, chimeric antigen receptor CAR-T cell and application thereof |
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| CN (1) | CN117843809A (en) |
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