CN118126954A - CAR-T cell and application thereof in preparation of AIDS immunotherapy drugs - Google Patents

CAR-T cell and application thereof in preparation of AIDS immunotherapy drugs Download PDF

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CN118126954A
CN118126954A CN202410253599.3A CN202410253599A CN118126954A CN 118126954 A CN118126954 A CN 118126954A CN 202410253599 A CN202410253599 A CN 202410253599A CN 118126954 A CN118126954 A CN 118126954A
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car
cells
cell
fitc
molecule
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张文艳
郑柏松
米执中
王虹
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Jilin University
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Jilin University
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Abstract

The invention is suitable for the technical field of immunotherapy, and provides a preparation method and application of a CAR-T cell capable of killing CD4 + T cells carrying HIV for the immunotherapy of AIDS. The CAR structure of the CAR-T cell of the invention comprises a CAR molecule part which serially expresses a membrane localization signal peptide, an anti-FITC scFv, a CD8 molecule transmembrane region fragment, CD28, 4-1BB and CD3 ζ sequences from amino-terminus to carboxy-terminus. The anti-FITC scFv has high affinity to FITC molecules, and when the anti-FITC scFv is matched with one or more anti-HIV monoclonal or polyclonal antibodies with FITC marks, the CAR-T cells can be specifically targeted to CD4 + T cells carrying HIV, so that immune killing is realized. The invention can provide an effective method for the immune treatment of AIDS.

Description

CAR-T cell and application thereof in preparation of AIDS immunotherapy drugs
Technical Field
The invention belongs to the technical field of immunotherapy, and particularly relates to a CAR-T cell and application thereof in preparation of an AIDS immunotherapy drug.
Background
CAR cell therapies have shown remarkable therapeutic effects in a variety of malignant tumors and cancers by engineering T cells or NK cells of a patient themselves to express a CAR molecule, thereby allowing T cells or NK cells to recognize and eliminate specific tumor cells, rendering them more potent against cancer. However, the traditional CAR-T has antigen drift caused by virus mutation in the clinical application of HIV, so that the targeting of the CAR-T is weakened or disabled, or the efficiency of the same CAR-T cell in the treatment of different individuals is different due to individual differences of HIV-infected persons.
A generic, modular CAR architecture can generally be split into two modules: (1) "signaling module" expressed on T cells or NK cells, including extracellular and intracellular signaling activation domains that can specifically bind to a signaling module; (2) "Signal transduction moiety" generally includes an antibody moiety that specifically binds to a target antigen and a transduction moiety that specifically binds by a CAR cell signaling moiety. The recognition signal of the target antigen is converted into a CAR cell activation signal by the extracellular domain of the "signaling module" specifically binding to the "signal conversion module". According to the designed CAR-T cell for HIV immunotherapy, the CAR molecule of the "signaling module" comprises a single-chain antibody with targeting small molecule FITC, and 2 costimulatory domains CD28 and 4-1BB are integrated at the upstream of CD3 zeta, so that the T cell can kill target cells more effectively, and meanwhile, the production of cytokines is increased, and the survival and the functional activity of the T cell are promoted more effectively. A "signal transduction moiety" is one or more anti-HIV monoclonal or polyclonal antibodies with FITC labels. When in use, the specific monoclonal antibodies can be selected according to different dominant antigen epitopes of HIV latent virus or variant virus, and can mediate CAR-T (NK) cells to kill latent or infected cells specifically under the condition of single or combined use. The advantages are as follows: the activity of the CAR cells can be regulated and controlled by regulating the dosage of the conversion module; modulating the activity of the CAR cell by designing and modulating the affinity of the switch module to the target antigen; blocking CAR cell activity by adding a blocking agent of the switch module; by replacing the conversion module, the target point can be changed without changing the CAR cells; multiple conversion modules can be used simultaneously, so that the simultaneous treatment of the multi-target CAR cells can be realized. The above features may make such CAR-T cell therapy more advantageous in terms of target selection, activity and adverse reaction control. The universal design of the medicine makes the rapid treatment of individual patients possible, and is an innovative application technology for realizing biological treatment of AIDS.
Disclosure of Invention
The invention aims to provide a CAR-T cell and application thereof in preparation of an AIDS immunotherapy medicament, and aims to solve the problems in the background art.
In order to achieve the above purpose, the present invention provides the following technical solutions:
A CAR-T cell comprising a CAR molecule portion that expresses in tandem from amino-terminus to carboxy-terminus a membrane localization signal peptide, an anti-FITC scFv, a CD8 molecule transmembrane region fragment, CD28, 4-1BB, and CD3 ζ sequences.
Further, FITC-labeled protein molecules that bind to the anti-FITC SCFV CAR cells are specific monoclonal or polyclonal antibodies that target HIV.
Further, the amino acid sequence of the anti-FITC scFv is shown as SEQ ID NO. 1; the whole amino acid sequence of the CAR molecule is shown as SEQ ID NO. 2;
The whole amino acid sequence of the CAR molecule is cloned into a pCDH-EF 1-MCS-T2A-copGGFP recombinant vector.
Further, the CAR molecule contains the entire amino acid or is transformed by a recombinant vector.
Further, the CAR molecule is integrated into T cells by lentiviral infection, including but not limited to CD4, CD8, NK cells.
Use of a CAR-T cell as described above in combination with a FITC-labelled antibody for the manufacture of a medicament for the treatment of HIV.
Use of a CAR-T cell as described above, an isolated amino acid as described above and a recombinant vector for the manufacture of a medicament for the treatment of HIV.
The pharmaceutical composition is characterized by comprising the expression vector of the CAR-T cells, the CAR-T cells and pharmaceutically acceptable auxiliary materials.
Compared with the prior art, the invention has the beneficial effects that:
In the invention, after the CAR-T cells are combined with the FITC marked monoclonal or polyclonal antibodies, the capability of identifying and killing HIV infected target cells is stronger, and a more effective method is provided for the clinical stage of cell treatment.
Drawings
FIG. 1 is a schematic structural diagram of the CAR molecule according to example 1.
FIG. 2 is a schematic representation of the coupling of a CAR to FITC-labeled monoclonal antibodies of example 1 that target HIV-infected cells.
Fig. 3 is an expression flow diagram of CAR in CAR-T cells in example 1.
FIG. 4 is a graph showing the results of detection of target cell killing by LDH assay of CAR-T cells conjugated to different FITC-labeled monoclonal antibodies and CAR-T cells unconjugated to antibodies at different potency target ratios of example 1.
FIG. 5 is a graph showing the results of the detection of killing of target cells by P24 experiments of CAR-T cells conjugated with different FITC-labeled monoclonal antibodies and CAR-T cells unconjugated with antibodies at different potency target ratios in example 1.
Detailed Description
The present invention will be described in further detail with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
Specific implementations of the invention are described in detail below in connection with specific embodiments.
One embodiment of the invention provides a universal CAR-T cell with FITC-anti-FITC scFv comprising a CAR molecule portion that expresses in tandem from amino-terminus to carboxy-terminus a membrane-localized signal peptide, an anti-FITC scFv, a CD8 molecule transmembrane region fragment, CD28, 4-1BB and CD3 zeta sequences.
In embodiments of the invention, preferably, each polypeptide of the invention (scFv sequence, CD8 molecule transmembrane region fragment, CD28, 4-1BB, CD3 ζ sequence, membrane localization signal, signal peptide) may be independently selected from the same or different species sources, e.g. murine (mouse, rat), rabbit, sheep, goat, horse, chicken, cow, dog and human.
The CAR-T cells may be selected from any one of helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, MAIT cells and γδ T cells. In addition, there is an alternative option that the CAR-T cells may be from one type of CD3 + T cells, CD3 +/CD4+ T cells or CD3 +/CD8+ T cells.
As a preferred embodiment of the invention, FITC-conjugated monoclonal antibodies that bind to the anti-FITC SCFV CAR cells are one or more of the following, in particular all FITC-labeled proteins or molecules, such as specific monoclonal, polyclonal antibodies or tumor cell surface proteins.
In embodiments of the present invention, it is preferred that the conjugated monoclonal antibodies in other embodiments are not limited to the above, but may be other monoclonal antibodies that can be used as anti-HIV agents, such as 10E8、10E8v4、PG9、PG16、b12、3BNC60、3BNC10 5、3BNC117、3BNC117-LS、VRC01、VRC03、VRC07、VRC07-523、N6、CAP256-VRC26.25、PGT121、PGT124、PGT128、PGT135、PGT145、PGT151、NIH45-46、VRC-CH31、12A12、NIH45-46G54W、NIH45-46LS、PCT64H01、PCT65B02、PGDM1400 and 10-1074.
As a preferred embodiment of the invention, the amino acid sequence of the anti-FITC scFv is shown as SEQ ID NO. 1;
AARPDYKDDDDKASDVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRVSGVPDRFSGSGSGTDFTLKINRVEAEDLGVYFCSQSTHVPWTFGGGTKLEIKSSADDAKKDAAKKDDAKKDDAKKDGGVKLDETGGGLVQPGGAMKLSCVTSGFTFGHYWMNWVRQSPEKGLEWVAQFRNKPYNYETYYSDSVKGRFTISRDDSKSSVYLQMNNLRVEDTGIYYCTGASYGMEYLGQGTSVTVS(SEQ ID NO.1)
The integral amino acid sequence of the CAR molecule is shown as SEQ ID NO. 2.
ATMALPVTALLLPLALLLHAARPAARPDYKDDDDKASDVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRVSGVPDRFSGSGSGTDFTLKINRVEAEDLGVYFCSQSTHVPWTFGGGTKLEIKSSADDAKKDAAKKDDAKKDDAKKDGGVKLDETGGGLVQPGGAMKLSCVTSGFTFGHYWMNWVRQSPEKGLEWVAQFRNKPYNYETYYSDSVKGRFTISRDDSKSSVYLQMNNLRVEDTGIYYCTGASYGMEYLGQGTSVTVSFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRFSVVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO.2)
In the embodiment of the invention, preferably, the linker sequence between the light chain and the heavy chain of the FITC scFv is SGSG.
The whole amino acid sequence of the CAR molecule is cloned into a pCDH-EF 1-MCS-T2A-copGGFP recombinant vector.
As a preferred embodiment of the invention, the CAR molecule contains the entire amino acid or is transformed with a recombinant vector.
As a preferred embodiment of the invention, the CAR molecule is integrated into T cells by lentiviral infection, including but not limited to CD4, CD8, NK cells.
In embodiments of the invention, a variety of different types of recombinant vectors may be employed, including retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, or CRISPR/CAS plasmids. The best choice is a lentiviral vector because it can efficiently integrate the foreign gene into the host chromosome in order to achieve sustained expression of the target sequence. The vector has excellent infection capability, and can efficiently infect various different types of cells such as neuron cells, liver cells, cardiac muscle cells, tumor cells, endothelial cells, stem cells and the like, thereby realizing good gene therapy effect. For cells which are difficult to transfect, such as primary cells, stem cells and undifferentiated cells, the use of lentiviral vectors can significantly improve the delivery efficiency of the gene of interest and increase the probability of integration of the gene of interest into the host cell genome, making it more convenient to achieve long-term and stable gene expression of interest. It is worth noting that these types of vectors are not limited and may be adapted to specific needs. In addition, the vector may include a variety of commonly used genetic engineering regulatory elements such as enhancers, promoters, and other expression control elements, for example, transcription termination signals, polyadenylation signals, and poly-U sequences.
The application of the CAR-T cells combined with FITC labeled antibodies in preparation of medicines for treating HIV is provided in one embodiment of the invention.
The invention provides the application of the CAR-T cells, the isolated amino acids and the recombinant vector in preparation of medicines for treating HIV.
The pharmaceutical composition provided by the embodiment of the invention is characterized by comprising the expression vector of the CAR-T cells, the CAR-T cells and pharmaceutically acceptable auxiliary materials.
In an embodiment of the present invention, preferably, the auxiliary materials include one or more of diluents, preservatives, buffers, disintegrants, antioxidants, suspending agents, colorants, excipients, gelling agents and emulsifiers.
The diluent is selected from one or more of polyethylene glycol, propylene glycol, vegetable oil and mineral oil. The preservative is one or more selected from sorbic acid, methyl sorbate, methyl parahydroxybenzoate, ethyl parahydroxybenzoate, propyl parahydroxybenzoate, butyl parahydroxybenzoate, benzyl parahydroxybenzoate, sodium methyl parahydroxybenzoate, benzoic acid and benzyl alcohol. The buffer is selected from one or more of sodium hydrogen phosphate, sodium dihydrogen phosphate, sodium citrate, sodium tartrate and sodium acetate. The antioxidant is selected from one or more of ethylenediamine tetraacetic acid, disodium ethylenediamine tetraacetate, dibutyl hydroxy toluene, glycine, inositol, ascorbic acid, sodium ascorbate, lecithin, malic acid, hydroquinone, citric acid, succinic acid and sodium metabisulfite.
Pharmaceutically acceptable excipients in the present invention are those that are compatible with the other ingredients of the pharmaceutical formulation and have little or no toxicity, irritation, allergic response, immunogenicity, or other untoward effect upon contact with the tissues or organs of the recipient (e.g., human or animal). The auxiliary materials play a key role in the preparation process, and ensure the quality, stability and safety of the pharmaceutical preparation. The selection and use of these adjuvants requires strict compliance with pharmaceutical regulations and standards to ensure that the final pharmaceutical formulation is safe for the patient without causing any adverse reactions.
Example 1, the invention the following experiments were performed:
1. designing a CAR:
Designing the sequence of the CAR: the expressible scFv sequence was concatenated with the sequences of the human derived CD8 molecule transmembrane region fragment, CD28, 4-1BB and CD3 zeta, and a membrane localization signal was added at the N-terminus, designated "FITC. CAR". The structure of "fitc.car" is shown in figure 1.
2. Packaging of lentiviruses:
After synthesis of the cDNA sequence of FITC. CAR, it was cloned into the lentiviral plasmid pCDH-EF 1-MCS-T2A-copGGFP by restriction endonuclease and T4 DNA ligase. The constructed lentiviral Plasmid, lentiviral packaging Plasmid psPAX2 (available from Addgene, plasmid # 12260) and pMD.2G Plasmid (available from Addgene, plasmid # 12259) were then combined in a mass ratio of 8:3:1, using a cationic polymer transfection reagent PEI to transfect 293T cells, and after plasmid transfection for 48 hours, harvesting culture medium supernatant containing lentivirus, and finally, using ultracentrifugation to centrifugally concentrate the collected supernatant.
3. Preparation of CAR-T cells:
3.1 mL, 10mL of blood sample, more than 99% of CD8 + T cells were isolated using Ficoll's lymphocyte separation liquid and Miltenyl's CD8 beads, and the isolated T cells were cultured in T cell medium at 37℃under 5% CO 2.
3.2, Adding 10-6 cells into 100ul of concentrated lentivirus supernatant, centrifuging at 350g for 90min, culturing for 12h after infection, infecting again for 1 round under the same condition as in step 3.1, and culturing for 24-48h.
3.3, Harvesting the CAT-T cells after culture.
4. CAR-T cell expression detection:
CAR-T cells were centrifuged at 350g for 10 min and then suspended in PBS, after which CAR expression was detected by flow cytometry.
5. LDH experiment:
fitc.car-T cells were conjugated to FITC-conjugated monoclonal antibody (FITC-conjugated monoclonal antibody amount 5 ng/well) in 96-well plates according to a protocol from 1:1 to 1:5 to HIV-infected MT4 target cells, after 24h incubation, the supernatant from each well in the experiment was taken: the killing effect of CAR-T cells on target cells was detected using Roche LDH kit, and the content of viral protein P24 in the supernatant was detected using HIV envelope protein P24 detection kit produced by hebei university of medical science.
6. Characterization data and effect data are as follows:
6.1 flow cytometry detection CARs packaged using the sequence of SEQ ID No.2 of the present invention can be efficiently loaded onto CD8 + T cells, with an efficiency of up to 40% in figure 3.
6.2, LDH experiments prove that FITC. CAR-T cells and FITC-conjugated monoclonal antibodies have stronger killing effect on target cells after being combined.
Cell supernatants from different effector-target ratios were pooled and assayed for LDH values using ELISA, the results are shown in figure 4. In fig. 4, the abscissa indicates the ratio of effector cells to target cells, and the ordinate indicates the killing effect of effector cells on target cells. As can be seen from fig. 4, the ratio of effector cells to target cells is 1:1 to 1:5, the FITC. CAR of the monoclonal antibodies coupled with VRC01, 10E8 and N6 marked by FITC is stronger in killing target cells than the FITC. CAR of the unconjugated antibodies, wherein the ratio of effector cells to target cells is 5: the killing effect is strongest at 1.
6.3 And P24 experiments prove that the FITC. CAR-T cells and the FITC-conjugated monoclonal antibodies have stronger killing effect on target cells after being combined.
Cell supernatants from different effector-target ratios were pooled and tested for P24 values using ELISA, the results are shown in figure 5. In fig. 5, the abscissa represents fitc.car-T cells coupled with different FITC-labeled monoclonal antibodies, and the ordinate represents P24 values. Smaller P24 values represent greater killing of fitc.car-T cells by target cells. As can be seen from fig. 5, the ratio of effector cells to target cells is 1:1 to 1:5, the FITC.CAR-T cells coupled with three FITC labeled monoclonal antibodies of VRC01, 10E8 and N6 have stronger killing effect on target cells than the FITC.CAR-T cells uncoupled with the antibodies, wherein the ratio of effector cells to target cells is 5: the killing effect is strongest at 1.
As can be seen from fig. 4 and 5, fitc.car-T cells coupled with FITC-labeled monoclonal antibodies have a stronger killing effect on target cells.
The foregoing is merely a preferred embodiment of the present invention, and it should be noted that modifications and improvements can be made by those skilled in the art without departing from the spirit of the present invention, and these should also be considered as the scope of the present invention, which does not affect the effect of the implementation of the present invention and the utility of the patent.

Claims (8)

1. A CAR-T cell comprising a portion of a CAR molecule that expresses in tandem from amino-terminus to carboxy-terminus a membrane-localized signal peptide, an anti-FITC scFv, a CD8 molecule transmembrane fragment, CD28, 4-1BB, and CD3 ζ sequences.
2. The CAR-T cell of claim 1, wherein the FITC-labeled protein molecule that binds to the anti-FITC SCFV CAR cell is a specific monoclonal or polyclonal antibody that targets HIV.
3. The CAR-T cell of claim 2, wherein the amino acid sequence of the anti-FITC scFv is shown in SEQ ID No. 1; the whole amino acid sequence of the CAR molecule is shown as SEQ ID NO. 2;
The whole amino acid sequence of the CAR molecule is cloned into a pCDH-EF 1-MCS-T2A-copGGFP recombinant vector.
4. A CAR-T cell according to claim 3, characterized in that it comprises the whole amino acid of the CAR molecule or is transformed by a recombinant vector.
5. The CAR-T cell of claim 1, wherein the CAR molecule is integrated into T cells by lentiviral infection, including but not limited to CD4, CD8, NK cells.
6. Use of a CAR-T cell according to any one of claims 1to 5 in combination with a FITC-labelled antibody for the manufacture of a medicament for the treatment of HIV.
7. Use of a CAR-T cell according to any one of claims 1 to 5, a CAR molecule as a whole amino acid according to claim 3 and a recombinant vector in the manufacture of a medicament for the treatment of HIV.
8. A pharmaceutical composition comprising the CAR-T cell expression vector of any one of claims 1-5, the CAR-T cell of any one of claims 1-5, and a pharmaceutically acceptable adjuvant.
CN202410253599.3A 2024-03-06 2024-03-06 CAR-T cell and application thereof in preparation of AIDS immunotherapy drugs Pending CN118126954A (en)

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