CN117229424B - Chimeric antigen receptor targeting CD55 and application thereof - Google Patents

Chimeric antigen receptor targeting CD55 and application thereof Download PDF

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CN117229424B
CN117229424B CN202311503117.7A CN202311503117A CN117229424B CN 117229424 B CN117229424 B CN 117229424B CN 202311503117 A CN202311503117 A CN 202311503117A CN 117229424 B CN117229424 B CN 117229424B
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antigen receptor
chimeric antigen
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CN117229424A (en
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张茜
常新霞
彭燕
李扬清
唐奇
汪秀星
冯振卿
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Nanjing Medical University
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Nanjing Medical University
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Abstract

The invention relates to the technical field of biological medicines, and discloses a specific chimeric antigen receptor capable of targeting CD55, a modified T cell thereof, a related composition and application. The chimeric antigen receptor CD55 includes an antigen binding domain, a transmembrane domain, and an intracellular signaling domain. The invention also discloses an amino acid sequence and a nucleic acid sequence of the CD55 specific chimeric antigen receptor. The chimeric antigen receptor is used for modifying T lymphocytes, and the modified T lymphocytes can be used for treating tumors such as glioblastoma and other related applications aiming at CD55 high expression.

Description

Chimeric antigen receptor targeting CD55 and application thereof
Technical Field
The invention relates to the field of biological medicine, in particular to a chimeric antigen receptor targeting CD55 and application thereof.
Background
Tumor treatment has undergone the progress of various means such as surgical excision, radiotherapy and chemotherapy, etc., and the treatment effect of these methods on some tumors is still very limited. With the development of molecular biology, new biotechnology is continuously applied to clinic. Adoptive cellular immunotherapy (adoptive cellular immunotherapy, ACI) is used in clinic as a new anti-tumor immunotherapy through intensive research, bringing new hopes to cancer patients. ACI kills tumor cells without compromising the patient's immune system and its function and can avoid tumor immune escape. Chimeric antigen receptor T cell (chimeric antigen receptor T cell, CAR-T) therapy is an important immunotherapeutic approach, whose mechanism of action is by constructing a gene expression vector of chimeric antigen receptor (chimeric antigen receptor, CAR) that specifically recognizes tumor antigens, transfecting T cells to induce cell surface expression of CAR, further inducing antigen-antibody response in vivo, releasing granzyme, perforin, and recruiting immune cells, specifically recognizing killer tumor cells.
Glioblastoma (GBM) is the most common and most malignant central nervous system tumor. Standard treatment regimens for surgical resection, radiation therapy, and chemotherapy have increased the median survival of adults to 15 months. Tumor genomic profile (TCGA) glioblastomas were divided into three subtypes based on transcriptional cluster analysis: anterior, classical and mesenchymal. Mesenchymal subtypes are the most invasive and aggressive subtypes. Glioblastoma Stem Cells (GSCs) are key drivers of GBM heterogeneity and malignancy. GSCs have the ability to self-renew, proliferate, differentiate, metastasize, and treat resistance, leading to heterogeneity, progression, and aggressiveness of GBM. During the proliferation of GSCs, angiogenesis and central necrosis occur sequentially, thereby causing inhibition of anti-tumor immune responses and resistance to cytotoxic therapies.
CD55, also known as DAF, is a membrane complement regulator protein anchored by phosphatidylinositol, protecting cells from complement-mediated lysis, and is the primary regulator of the classical pathway of complement activation. CD55 inhibits early complement activation by accelerating the degradation of C3 convertase, a central molecule that regulates the complement cascade, and prevents C3b deposition and inhibits the formation of membrane attack complexes. Among GBM surface markers screened by TCGA database, CD55 was expressed more highly in the most malignant mesenchymal GBM than in the other two subtypes of glioblastoma, and its sustained high expression was likely closely related to malignant progression of GBM. At present, CD55 is not reported as a target point of immunotherapy of malignant glioma.
Disclosure of Invention
The invention aims to construct a specific chimeric antigen receptor of CD55, prepare a T lymphocyte modified by the specific chimeric antigen receptor of targeted CD55, and the T lymphocyte modified by the chimeric antigen receptor can specifically identify and kill CD55 positive tumor cells and is applied to the immunotherapy of tumors.
In a first aspect of the invention, there is provided a chimeric antigen receptor comprising a CD55 antigen binding domain, a transmembrane domain and an intracellular signaling domain; the CD55 antigen binding domain comprises a heavy chain variable region comprising the amino acid sequence shown as SEQ ID No.9 and a light chain variable region comprising the amino acid sequence shown as SEQ ID No. 13.
Preferably, the CD55 antigen binding domain is an anti-CD 55 single-chain antibody, and the amino acid sequence of the anti-CD 55 single-chain antibody is shown as SEQ ID NO. 3. Wherein, a connecting peptide (Linker) is arranged between the heavy chain variable region and the light chain variable region of the anti-CD 55 single chain antibody, and the amino acid sequence of the connecting peptide is shown as SEQ ID NO. 11.
Preferably, the intracellular signaling domain comprises a CD137 intracellular region and a CD3 zeta intracellular region in tandem, the amino acid sequence of the CD137 intracellular region is shown in SEQ ID No.17, and the amino acid sequence of the CD3 zeta intracellular region is shown in SEQ ID No. 19.
Preferably, the transmembrane domain comprises the human CD8 transmembrane region CD 8. Alpha. Finger, the amino acid sequence of which is shown in SEQ ID NO. 15.
Preferably, the chimeric antigen receptor consists of a CD8 alpha signal peptide (CD 8 alpha SP), a CD55 antibody heavy chain variable region (CD 55 scFv-VH), a Linker region, a CD55 antibody light chain variable region (CD 55 scFv-VK), a human CD8 alpha Hinge region (CD 8 alpha Hinge), CD137, and an immunoreceptor tyrosine activation motif CD3 zeta tandem. The amino acid sequence of the chimeric antigen receptor CD8 alpha SP-CD55 scFv VH-Linker-CD55 scFv VK-CD8 alpha finger-CD 137-CD3 zeta is shown as SEQ ID NO. 1.
In a second aspect of the invention, there is provided a gene encoding the chimeric antigen receptor described above.
Preferably, the nucleotide sequence of the gene encoding the chimeric antigen receptor comprises the sequence shown as SEQ ID NO. 2.
Wherein the nucleotide residue sequence of the code CD8 alpha signal peptide (CD 8 alpha SP) is SEQ ID NO.8. The nucleotide residue sequence of the CD55 scFv is SEQ ID NO.4; more specifically, the nucleotide residue sequence of CD55 scFv VH is SEQ ID NO.10; the nucleotide residue sequence of the Linker is SEQ ID NO.12; the nucleotide residue sequence of CD55 scFv VK is SEQ ID NO.14. The nucleotide residue sequence of the CD8 alpha transmembrane region (CD 8 alpha finger) is SEQ ID NO.16. The nucleotide residue sequence of the CD137 intracellular signal region is SEQ ID NO.18. The nucleotide residue sequence of CD3 zeta is SEQ ID NO.20.
In a third aspect of the invention, there is provided a method of modifying a T cell by said chimeric antigen receptor comprising transfecting a nucleotide sequence encoding said chimeric antigen receptor into a T cell to obtain a CD 55-targetable chimeric antigen receptor modified T cell; the transfection is by any one or more combination of viral vectors, eukaryotic expression plasmids or mRNA sequences into T cells.
In a fourth aspect of the present invention, there is provided a vector comprising the nucleotide sequence of the above gene.
Preferably, the vector comprises a viral vector comprising one or more combinations of lentiviral, retroviral or adenoviral vectors.
In a fifth aspect of the present invention, there is provided a recombinant lentivirus obtained by cotransfecting mammalian cells with the above-described viral vector of a chimeric antigen receptor and a packaging helper plasmid.
Preferably, the packaging helper plasmid comprises pSPAX2, pMD2.G, and the mammalian cell is a LentiX-293T cell.
In a sixth aspect of the invention, there is provided an immune cell expressing the chimeric antigen receptor described above. The immune cells include T cells.
In a seventh aspect of the invention, there is provided a composition comprising a chimeric antigen receptor-modified T cell, vector, recombinant lentivirus or T cell expressing a chimeric antigen receptor as described above, prepared by the method described above.
In an eighth aspect, the present invention provides the use of the chimeric antigen receptor described above or the composition described above in the preparation of a medicament for the treatment of a neoplastic disease.
Preferably, the neoplastic disease is one that is positive for the expression of the detected CD55 gene.
Preferably, the neoplastic disease comprises brain glioma, prostate cancer, cervical cancer, breast cancer, colorectal cancer, lung cancer, thyroid cancer, chronic Lymphocytic Leukemia (CLL), acute Lymphoblastic Leukemia (ALL), chronic Myelogenous Leukemia (CML) or Acute Myeloid Leukemia (AML).
The chimeric antigen receptor CD55 CAR provided by the invention has a CD55 antigen binding domain, and the T lymphocyte modified by the chimeric antigen receptor CD55 CAR can specifically identify and kill CD55 positive tumor cells. Furthermore, the chimeric antigen receptor provided by the invention is connected in series by CD8 alpha SP-CD55 scFv-CD8 alpha finger-CD 137-CD3 zeta, and a recombinant lentiviral vector of the chimeric antigen receptor capable of targeting CD55 is prepared by a lentiviral technology, so that T lymphocytes can be infected in vitro. The obtained T lymphocyte modified by the chimeric antigen receptor of the CD55 can specifically identify the tumor cells expressing the CD55, such as brain glioma stem cells through the single-chain antibody part of the CAR, and simultaneously activate the CAR-T cells to release various cytokines such as IFN-gamma, TNF and the like to kill the tumor cells. The CD55 CAR and the prepared CD55 CAR-T cell can specifically target the GSCs of CD55, inhibit the GBM progression, slow down GBM malignant tumor, break the maintenance of mesenchymal GBM subtype and provide a new method for GBM clinical treatment. At present, CD55 is not reported as a target point of immunotherapy of malignant brain glioma, and the invention provides application foundation and technical support for development of related disease immunotherapy technology.
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FIG. 1 shows the SDS-PAGE results of humanized anti-CD 55 whole-molecule IgG antibodies in one embodiment. Each band number in the figure indicates: 1. protein markers; 2. 293F cell culture supernatant after transfection of antibody expression plasmids; 3. purified CD55 IgG;4. ultrafiltration treatment of the fluid passing liquid; 5. protein purification fluid passing; 6. 293F cell culture supernatant without antibody plasmid transfection; 7. human control IgG.
FIG. 2 shows the results of an ELISA assay for anti-CD 55 antibodies in one embodiment.
FIG. 3 shows the result of immunoblotting of anti-CD 55 antibody and CD55 protein in one embodiment. Each band number in the figure indicates: 1.2907 cell total protein; mes20 cell total protein; post ip 2907 cell protein; post-ip MES20 cell protein.
FIG. 4 shows the result of anti-CD 55 IgG immunofluorescence assay in one embodiment.
FIG. 5 is a schematic diagram of the structure of a CD55 CAR and a Unrelaged CAR in one embodiment. Fig. 5 (a) is a schematic diagram of the structure of a CD55 CAR, and fig. 5 (b) is a schematic diagram of the structure of a Unrelated CAR.
FIG. 6 is a graph showing the results of expression verification of the CD55 CAR plasmid in one embodiment.
Figure 7 is a graph showing the results of a CD55 CAR lentiviral package titer assay in one embodiment.
FIG. 8 is a flow cytometric assay of the infection efficiency of CD55 CAR-T cells in one embodiment.
FIG. 9 is a flow cytometric view of a CD55 CAR-T cell phenotype in one embodiment.
FIG. 10 is a graph showing the killing effect of CD55 CAR-T cells on different wild-type target cells for a fixed effective target ratio in one embodiment.
FIG. 11 is a graph showing the killing effect of CD55 CAR-T cells on CD 55-positive brain glioma cells with varying effective target ratios in a specific embodiment.
FIG. 12 is a graph showing cytokine detection during killing of tumor cells by CD55 CAR-T cells at a fixed effective target ratio in one embodiment.
Description of the embodiments
Example 1: preparation of anti-CD 55 whole-molecule IgG antibody
1) The CD55 antigen is used for carrying out six rounds of 'adsorption-elution-amplification' enrichment screening in a human Fab phage library to obtain the Fab antibody of the anti-CD 55, and then the variable region sequence of the Fab antibody is obtained by PCR amplification sequencing.
2) Primers were designed based on the heavy and light chain variable region sequences of the obtained antibodies.
3) The anti-CD 55 antibody heavy and light chains were amplified.
4) The heavy chain and light chain genes of the whole molecular human antibody were amplified by the above-prepared humanized Fab template using the above-mentioned upstream and downstream primers for the heavy chain and light chain, respectively.
(1) And (2) PCR: the reaction system is as follows:
the reaction conditions were as follows:
(2) 2% agarose gel electrophoresis, observing the target band under ultraviolet, and cutting and recovering.
(3) And (5) purifying the target DNA fragment by using the gel recovery kit, and eluting with deionized water.
(4) Double-digested IgG expression plasmid: the IgG expression plasmids pFUSE-CHIg-hG1, pFUSE-CLIg-hk (available from Invivogen) contained heavy and light chain (Lambda) constant region base coding sequences of human origin of the IgG1 type. The double cleavage reaction system for pFUSE-CHIg-hG1 and pFUSE-CLIg-hk template vectors is as follows:
the reaction conditions are as follows: 37. the mixture was digested overnight at room temperature. And (3) carrying out agarose gel electrophoresis with the concentration of 1%, and recovering ultraviolet undercut gel. And (5) purifying the target DNA fragment by using the gel recovery kit, and eluting with deionized water. Information PCR recombinant expression plasmid
The reaction system is as follows:
the reaction conditions are as follows: incubate at 50℃for 15min.
And 5 mu L of reaction liquid is taken to transform competent bacteria, the competent bacteria are paved on a plate with corresponding resistance, and the clone is selected and sent to sequencing the next day. And (5) cloning and preserving strains with correct sequencing results, performing expansion culture, and extracting plasmids.
5) Expression of anti-CD 55 antibodies
(1) Taking 50 mu g of recombinant heavy chain plasmid in 1mL of Opti-MEM culture medium, taking 50 mu g of light chain plasmid in 1mL of Opti-MEM culture medium, taking 200 mu L of 293Fectin in 2.8mL of Opti-MEM culture medium, and standing the three mixed solutions at room temperature for 5min;
(2) And then uniformly mixing the two plasmid mixed liquids, supplementing 500 mu L of Opti-MEM culture medium, uniformly mixing, directly adding the mixed liquid of the transfection reagent 293Fectin, uniformly mixing, and standing for 20min. During the treatment of 293F cells, the 293F cells were centrifuged and resuspended in 293F Expression Medium, then counted and the cell viability ratio was calculated by trypan blue staining and 1X10 aspiration was performed 8 The individual cells were in culture flasks and fixed to 94mL volume with 293F Expression Medium;
(3) After 20min, 6mL of the complex of DNA and 293Fectin is added into the prepared 293F cells;
(4) The cells were cultured in a shaker incubator at 8% CO2, 120rmp,37℃and after 6 days the cell supernatants were collected.
6) Purification of anti-CD 55 antibodies
The collected cell supernatants were filtered through a 0.22 μm filter, while the equilibration and eluate were filtered through the filter. Purification was performed using an AKATA purifier according to the standard procedure for Protein A purification, loading at 1mL/min and eluting at 1.5 mL/min.
Results successful expression and purification of the anti-CD 55 antibody. The purified anti-CD 55 antibody was subjected to SDS-PAGE, and the results are shown in FIG. 1. As can be seen from FIG. 1, the purified antibody had a high purity and the effect of purification by using Protein A column was good.
Example 2: anti-CD 55 antibody biological activity assay
1) ELISA method
Coating ELISA 96-well plates with coating solution (0.1M carbonate buffer, pH 9.6) of CD55 protein to 2 [ mu ] g/mL, adding 100 [ mu ] L of each well, and standing overnight at 4 ℃; PBST (PBS containing 0.5% Tween 20) was blocked with 5% skimmed milk-wash buffer, and incubated at 37deg.C for 2h; after PBST is washed for 5 times, 100 mu L of CD55 antibody (initial concentration of 2 mu g/mL, 14 concentration gradient dilutions) is added into each hole for 2 hours at 37 ℃; 1, the method comprises the following steps: adding 100 mu L of 5000 diluted goat anti-human secondary antibody into the hole, and incubating at 37 ℃ for 1h; the reaction was stopped with 2M sulfuric acid after 10 minutes at room temperature at 100. Mu.L/well of peroxidase substrate color development liquid, and OD values were measured on-line using two wavelengths of 450 nm/690 nm.
The results are shown in FIG. 2, and as can be seen from FIG. 2: the anti-CD 55 antibody is capable of reacting with an antigen-antibody of CD55 protein.
2）Western blot
Protein was extracted from human glioma stem cells 2907 and Mes20 cells expressing CD55 protein, and cellular proteins were extracted after IP enrichment, subjected to 10% sds-PAGE vertical electrophoresis and electrotransferred onto nitrocellulose membranes, which were incubated with 2 μg/mL CD55 antibody for 1h at room temperature, 1:5000 dilution of HRP-goat anti-human IgG (Peking China fir) and ECL luminescence kit (Pierce Co., USA) were exposed to gel imaging system (Bio-Rad Co.).
The results are shown in FIG. 3: the anti-CD 55 antibody specifically binds to the CD55 protein expressed by 2907 and Mes 20.
3) Immunofluorescence method
Human glioma stem cells 2907, CW738 and GSC23 expressing CD55 protein are taken as cell climbing sheets, 0.3% of PBST (prepared by PBS and containing 0.3% of Triton X-100) is penetrated for 10 min/time for 3 times, and then a sealing liquid is used for sealing for 2-4h at room temperature. After the sealing is completed, adding a primary antibody incubation liquid, and stably placing the primary antibody incubation liquid in a refrigerator at 4 ℃ overnight. After the next day of taking out, rinsing with 0.1% PBST for 3 times, 10min each time, adding the corresponding secondary fluorescent antibody after washing the primary antibody, and incubating for 1-2h at room temperature in dark place. The solution was washed 3 times with PBS for 10min each, and finally with 0.1% PBST.
Sealing piece: dripping DAPI-containing anti-fluorescence quenching agent on the glass slide, sealing the glass slide, sucking redundant liquid with filter paper, and storing in a cassette. The stained film is photographed and observed under an Olympic Games laser confocal microscope and a common fluorescence microscope.
The results are shown in FIG. 4, in which CD55 IgG specifically recognizes the naturally expressed CD55 protein on the surface of tumor cells.
Example 3: construction of CD 55-specific chimeric antigen receptor lentiviral vector
The chimeric antigen receptor CAR provided by the embodiment of the invention is formed by connecting a CD8 alpha signal peptide (CD 8 alpha SP), CD55 scFv, a CD8 transmembrane region (CD 8 alpha finger), a CD137 intracellular signal region and a CD3 zeta intracellular signal region in series, and the structure is shown in (a) in figure 5. The anti-CD 55 scFv gene sequence is from a monoclonal antibody sequence constructed in the laboratory, and codon optimization is carried out, and the information of each gene or amino acid sequence is shown in SEQ ID NO. 1-20.
In the embodiment of the invention, SEQ ID NO. 1-20 are respectively:
the amino acid sequence of the coded chimeric antigen receptor is SEQ ID NO.1;
the nucleotide sequence of the coded chimeric antigen receptor is SEQ ID NO.2;
the amino acid residue sequence of the CD55 scFv is SEQ ID NO.3;
the nucleotide residue sequence of the CD55 scFv is SEQ ID NO.4;
the amino acid residue sequence of the intracellular signal peptide fragment (CD 8 alpha finger-CD 137-CD3 zeta) of the chimeric antigen receptor is SEQ ID NO.5;
the nucleotide residue sequence of the intracellular signal peptide segment (CD 8 alpha finger-CD 137-CD3 zeta) of the chimeric antigen receptor is SEQ ID NO.6;
the amino acid residue sequence of the coded CD8 alpha signal peptide (CD 8 alpha SP) is SEQ ID NO.7;
the nucleotide residue sequence of the coded CD8 alpha signal peptide (CD 8 alpha SP) is SEQ ID NO.8;
the amino acid residue sequence of CD55 scFv VH is SEQ ID NO.9;
the nucleotide residue sequence of CD55 scFv VH is SEQ ID NO.10;
the amino acid residue sequence of Linker is SEQ ID NO.11;
the nucleotide residue sequence of the Linker is SEQ ID NO.12;
the amino acid residue sequence of the CD55 scFv VK is SEQ ID NO.13;
the nucleotide residue sequence of the CD55 scFv VK is SEQ ID NO.14;
the amino acid residue sequence of the CD8 alpha transmembrane region (CD 8 alpha finger) is SEQ ID NO.15;
the nucleotide residue sequence of the CD8 alpha transmembrane region (CD 8 alpha finger) is SEQ ID NO.16;
the amino acid residue sequence of the CD137 intracellular signal region is SEQ ID NO.17;
the nucleotide residue sequence of the CD137 intracellular signal region is SEQ ID NO.18;
the amino acid residue sequence of CD3 zeta is SEQ ID NO.19;
the nucleotide residue sequence of CD3 zeta is SEQ ID NO.20.
Splicing of each nucleotide structural fragment of the CAR in this example was completed using information PCR to obtain CD55 CAR and Unrelated CAR (CD 19 CAR). The structure of the Unrelaged CAR (UnCAR) is shown in FIG. 5 (b). Addition of the 5' -terminal of the CAR StructureXbaI enzyme cutting site, 3' end addingNotI cleavage site. Lentiviral vector pCDH-CMV-MCS-EF1a-CopGFPXbaI/NotI was double digested, and then CD55 CAR and Unrelaged CAR fragments were ligated with PCDH-CMV-MCS-EF1a-CopGFP vectors, respectively, using In-fusion PCR.
E.coli (DH 5 alpha) competence is respectively transformed from the products obtained by connection, and after monoclonal culture is selected, plasmids are extracted and sequenced to obtain pCDH-CD55 CAR and pCDH-Un CAR plasmids.
Example 4: identification of CD 55-specific chimeric antigen receptor expression
Culture of Lenti-X in a 10cm Petri dish TM 293T cells, when the density was about 80%, were transfected and 24h was replaced before transfectionDMEM medium without antibiotics (containing 10% fbs).
Plasmid transfection of pCDH-CD55 CAR and pCDH-Un CAR into Lenti-X, respectively TM 293T cells transfected with 15ug of plasmid per dish, untransfected Lenti-X TM 293T cells served as negative controls. GFP fluorescence expression was observed by fluorescence microscopy 48h after transfection and Lenti-X was collected 72h after transfection TM 293T cells and culture supernatants, and total cell proteins were extracted. The Western Blot method detects exogenous CD3 zeta expression. After separation of the extracted proteins by 10% SDS-PAGE, constant flow (300 mA,1 h) was transferred to PVDF membrane, incubated with anti-CD 3 zeta (1:1000) antibody, and incubated overnight at 4 ℃. After 3 washes with PBST, the mice were incubated with HRP sheep anti-mouse secondary antibody (1:5000) for 1h at room temperature. After ECL was added for development, imaging analysis was performed with ChemiDoc XRS System from Bio-Rad corporation.
As shown in FIG. 6, each CAR plasmid had exogenous CD3 ζ expression, and the protein size was consistent with the theoretical CAR protein size, i.e., about 80KD with the yang reference CD19, whereas untransfected 293T cells had no band. The results indicate that the prepared CAR plasmid can be successfully expressed in cells.
Example 5: packaging, concentration and titre determination of CD55 specific chimeric antigen receptor lentiviruses
Packaging of lentiviruses: 2X 10 will be 24h before transfection 6 Lenti-X TM 293T cells were inoculated into 10cm cell culture dishes and cultured in antibiotic-free DMEM medium (containing 10% FBS) until cell density reached about 80% for transfection.
Co-transfecting pCDH-CD55 CAR, pCDH-Un CAR plasmid and packaging plasmid pSPAX2, pMD2.G to Lenti-X, respectively TM The 293T cells were used to prepare lentiviruses with a mass ratio of pMD2.G to psPAX2 and recombinant expression plasmid of 1:3:4. After 48h of transfection, GFP expression was observed by fluorescence microscopy, cell culture supernatants were collected after 48h and 72h of transfection, respectively, centrifuged at 3000rpm for 10min, and filtered through a 0.45 μm filter to obtain pCDH-CD55 CAR and pCDH-Un CAR virus stocks, respectively.
Concentration of lentiviruses: the virus supernatant was placed in an Amicon Ultra 100 kD ultrafiltration tube and centrifuged at 4000rpm at 4℃for 30min, 1:100 DMEM dissolves lentiviral precipitate, split charging and storing at-80 deg.C to obtain lentiviral concentrate.
Lentiviral titer assay: lenti-X is pre-applied TM 293T cells were seeded into 96-well plates, 1X10 per well 4 cells were cultured overnight. The next day, virus concentrate is diluted according to a ratio of 1:10, 9 concentration gradient virus dilutions are prepared, culture medium in a 96-well plate is sucked, the virus dilutions are sequentially added into the 1 st to 9 th wells, 100 [ mu ] L of each well is taken as a blank control group, 100 [ mu ] L of DMEM complete culture medium is added into the 10 th well, and polybrene (with a final concentration of 10 [ mu ] g/mL) is added into each well. After culturing for 48 hours, the fluorescent expression was observed with a fluorescent microscope, and the calculation of virus titer was performed by using the LASER method.
The results are shown in FIG. 7, with a CD55 CAR lentivirus titre of 10 8 PFU/ml。
Example 6: preparation of CD 55-specific chimeric antigen receptor-modified T lymphocytes
1) Preparation of T lymphocytes
Fresh anticoagulants were collected from 20mL healthy volunteers, and Peripheral Blood Mononuclear Cells (PBMCs) were isolated with lymph isolation (GE). The isolated cells were stimulated with CD3 and CD28 plates for 48h, with T lymphocyte medium GT-T551 (TAKARA Co.) plus 1:5000 IL2 is induced and cultured to obtain T lymphocyte.
2) Preparation of CAR-T cells
A24-well plate of a non-tissue culture plate was coated with 50. Mu.g/mL retroNectin (TAKARA Co.) and 500. Mu.L of each well was added overnight at 4 ℃. mu.L of virus supernatant was added to each well and incubated at 37℃for 30min. The viral supernatant was removed, 500. Mu.L of viral supernatant was added, incubated at 37℃for 30min, the viral supernatant was removed, 1.5mL of viral supernatant was added per well, and 0.5mL of diluted T lymphocytes were added. Thus, CD 55-specific CAR-T cells were obtained, and fluorescence microscopy revealed that the T cells transfected for 72 hours expressed GFP fluorescence.
Constructing CAR-T by using the prepared lentivirus transfected T cells, and detecting the transfection efficiency of the lentivirus by using an FCM method at 72h after transfection, wherein the result shows that the transfection efficiency of the CD55 CAR-T cells is 52.48% respectively (figure 8); for CAR-T cell phenotypeThe results of the assays performed showed CD4 for the CD55 CAR-T cell group and Activated T cell group + 、CD8 + There is no statistical significance between the T cell phenotype ratioP> 0.05), suggesting that lentiviral transfection did not have an effect on T cell differentiation (figure 9).
Example 7: killing of glioma stem cells by CD 55-specific CAR-T cells
Glioma stem cells 2907, CW738, MNK1, 839 and ENSA cells and breast cancer MCF7 cells (CD 55 positive expression) were transfer cultured into 96 well plates (2X 10) 5 3 multiple wells per well), and after cell attachment, according to the number of effector T cells: the number of target cells (effective target ratio) was 10:1, and CD55 CAR-T cells, un CAR-T cells and activated cultured T cells were added, respectively. After co-culturing for 18 hours, the 96-well plate is centrifuged at 1500rpm for 5 minutes, the supernatant is collected, and the LDH release method is adopted to detect the specific killing effect of the CAR-T cells (see figure 10), and the result shows that the killing effect of the CD55 CAR-T cells is positively correlated with the expression abundance of the CD55 gene in cancer cells.
Glioma stem cells 2907, CW738, MNK1, 839 and ENSA cells and breast cancer MCF7 cells (CD 55 positive expression) were transfer cultured into 96 well plates (2X 10) 5 3 compound wells are arranged per well), and after the cells are attached, respectively adding the CD55 CAR-T cells, the Un CAR-T cells and the activated and cultured T cells according to the effective target ratio of 20:1, 10:1,5:1 and 2:1. After co-cultivation for 18 hours, the 96-well plate was centrifuged at 1500rpm for 5 minutes, and the supernatant was collected and tested for CAR-T cell specific killing by LDH release (see fig. 11), which indicated that the killing effect of CD55 CAR-T cells was positively correlated with the number of CAR-T cells.
Cell killing rate calculation (LDH release assay kit):
1) The medium in the 96-well plate was aspirated and replaced with DMEM-H medium with 5% FBS. And respectively adding 50 mu L effector cell suspensions with various concentrations into the sample group and the sample Blank group, and co-culturing for 18 hours.
2) Adding 10 MuL of Lysis Buffer into the high control hole and the high control Blank hole, and adding 5% CO 2 Culturing in an incubator at 37 ℃ for 30min.
3) 100 [ mu ] L Working Solution was added to each well and incubated for 15min in the dark.
4) 50 mu.m LStop Solution was added to each well and the absorbance at 490 nm was measured.
5) And (3) calculating a killing value: a (sample well-sample Blank well), B (high control well-high control Blank well), and C (low control well-background Blank well). The formula is as follows: cell killing (%) = [ (a-C)/(B-C) ]x100%.
Example 8: cytokine detection during glioma stem cell killing by CD55 specific CAR-T cells
Glioma stem cells 2907, CW738, MNK1, 839 and ENSA cells and breast cancer MCF7 cells were cultured at 1X10 5 The cells are inoculated into a 96-well plate, and after the cells are attached, CD55 CAR-T cells, un CAR-T cells and activated and cultured T cells are respectively added according to the effective target ratio of 10:1. After 24 hours of co-culture, supernatants were collected and the levels of IL-2 and IFN-gamma in each supernatant were assayed by ELISA (see FIG. 12), which indicated that the CD55 CAR-T cell line resulted in increased release of both IL-2 and IFN-gammap<0.05）。
1) Target cells: digestive glioma cells 2907, CW738, MNK1, 839, ENSA, breast cancer cells MCF7, adjusting cell concentration to 1x10 5 100 mu L of cell suspension is sucked into each mL, a 96-well plate is paved, 3 compound wells are arranged, and the culture is carried out overnight.
2) Effector cells: cell count was performed on each group of CAR-T cells to adjust the cell density to 1X10 6 100 mu L of CAR-T cells of each group are respectively added into corresponding holes, cell culture supernatant is collected after co-culture for 24 hours, and the mixture is centrifuged at 1000 rpm/min for 5min to remove sediment.
3) Capture Antibody was diluted using a 1 Xcoating Buffer, 100 μl was added to each well, and the sealing film was sealed overnight at 4deg.C.
4) Liquid is sucked and abandoned, 250 mu L of PBST is added into each hole, the liquid is soaked for 1min, the residual liquid is sucked and abandoned by the absorbent paper, and the absorbent paper is rinsed for 3 times.
5) 200 μl 1×ELISA/ELISPOT unit was added to each well for blocking and incubated for 1h at room temperature.
6) ELISA/ELISPOT components were blotted and washed 3 times with PBST solution.
7) The standard is dissolved and diluted with 1X Diultent according to a ratio of 1:2, 8 concentration gradients are prepared, 100 MuL of the standard is added into each hole, 3 compound holes are arranged in each group, and the standard is incubated for 2 hours at room temperature in a dark place.
8) The liquid was pipetted off and PBST washed 5 times. 100 mu L of diluted Detection Antibody is added to each well of a 96-well plate, and the wells are incubated for 1h at room temperature.
9) PBST was washed 5 times, diluted Avidin-HRP was added to each well, 100. Mu.L/well, and incubated for 30min at room temperature.
10 Repeating the step 4, soaking for 2min each time, and cleaning for 6 times.
11 1×tmb, 100 μl/well, was added to the 96-well plate and incubated for 15min at room temperature.
12 Opening the microplate reader, adding 100 mu L Stop Solution into each hole, and detecting the absorbance at 450 nm.
The invention has been described in detail in connection with the specific embodiments and exemplary examples thereof, but such description is not to be construed as limiting the invention. It will be understood by those skilled in the art that various equivalent substitutions, modifications or improvements may be made to the technical solution of the present invention and its embodiments without departing from the spirit and scope of the present invention, and these fall within the scope of the present invention. The scope of the invention is defined by the appended claims.

Claims (16)

1. A chimeric antigen receptor comprising a CD55 antigen binding domain, a transmembrane domain, and an intracellular signaling domain; the chimeric antigen receptor is formed by sequentially connecting CD8 alpha SP, CD55 scFv VH, linker, CD55 scFv VK, CD8 alpha finger, CD137 and CD3 zeta in series, and the amino acid sequence of the chimeric antigen receptor is shown as SEQ ID NO. 1.
2. A gene encoding the chimeric antigen receptor of claim 1.
3. The gene according to claim 2, characterized in that the nucleotide sequence of the gene comprises the sequence shown as SEQ ID No. 2.
4. The gene of claim 2, wherein the CD55 antigen binding domain is an anti-CD 55 single chain antibody, and the nucleotide sequence encoding the anti-CD 55 single chain antibody is set forth in SEQ ID No. 4.
5. The gene of claim 2, wherein the nucleotide sequence encoding said CD8 a SP is set forth in SEQ ID No.8.
6. The gene of claim 2, wherein the transmembrane domain is the human CD8 transmembrane region cd8αrange; the nucleotide sequence for encoding the CD8 alpha finger is shown as SEQ ID NO.16.
7. The gene of claim 2, wherein the intracellular signaling domain comprises a CD137 intracellular region and a CD3 zeta intracellular region in tandem; the nucleotide sequence of the CD137 intracellular region is shown as SEQ ID NO.18; the nucleotide sequence for encoding the CD3 zeta intracellular area is shown as SEQ ID NO.20.
8. A method of modifying T cells using a chimeric antigen receptor according to claim 1, wherein a nucleotide sequence encoding the chimeric antigen receptor is transfected into T cells to obtain CD 55-targetable chimeric antigen receptor modified T cells; the transfection is by any one or more combination of viral vectors, eukaryotic expression plasmids or mRNA sequences into T cells.
9. A vector comprising the nucleotide sequence of the gene of any one of claims 2-7.
10. The vector of claim 9, wherein the vector comprises a viral vector comprising a combination of one or more of a lentiviral vector, a retroviral vector, or an adenoviral vector.
11. A recombinant lentivirus obtained by co-transfecting a mammalian cell with the viral vector of the chimeric antigen receptor of claim 1 and a packaging helper plasmid.
12. The recombinant lentivirus of claim 11, wherein the packaging helper plasmid comprises pSPAX2, pmd2.G, and the mammalian cell is a Lentix-293T cell.
13. A T cell expressing the chimeric antigen receptor of claim 1.
14. A composition comprising the chimeric antigen receptor-modified T cell produced by the method of claim 8, the vector of claim 9 or 10, the recombinant lentivirus of claim 11 or 12, or the T cell of claim 13.
15. Use of the chimeric antigen receptor of claim 1 or the composition of claim 14 for the preparation of a medicament for the treatment of a brain glioma disease.
16. Use of the chimeric antigen receptor of claim 1 or the composition of claim 14 for the manufacture of a medicament for the treatment of a neoplastic disease, wherein the neoplastic disease is glioblastoma of the mesenchymal subtype.
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CN115806625A (en) * 2021-09-15 2023-03-17 广州百暨基因科技有限公司 Chimeric antigen receptor with limited expression of T cells and application thereof

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EP1591456A1 (en) * 1999-03-01 2005-11-02 Genentech Inc. Antibodies for cancer therapy and diagnosis
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