CN111154791B - Recombinant CD6 gene, T cell modified by same, preparation method and application - Google Patents
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Abstract
The invention provides a recombinant CD6 gene, a T cell modified by the recombinant CD6 gene, a preparation method and application, wherein the recombinant CD6 gene comprises a signal peptide nucleic acid artificial sequence, an SRCR-D3 nucleic acid artificial sequence, a Stalk nucleic acid artificial sequence, a transmembrane-costimulatory structural domain nucleic acid artificial sequence and a cytoplasmic nucleic acid artificial sequence which are connected in sequence. The T cell modified by the recombinant CD6 gene strictly targets a brain tumor region and does not affect other normal brains and body tissues, so that the T cell modified by the recombinant CD6 gene has the advantages of breaking through a blood brain barrier, accurately targeting a brain tumor and being high in safety.
Description
Technical Field
The invention relates to the technical field of genes, in particular to a recombinant CD6 gene, a T cell modified by the same, a preparation method and application.
Background
Brain glioma is the most common malignant tumor of the central nervous system, accounts for 40% -50% of craniocerebral tumors, has complex pathogenesis, has high lethality in the second place of ten common tumors, and has high disability rate in the fourth place. At present, the treatment means of glioma mainly comprises a comprehensive treatment mode of surgery, radiotherapy and/or chemotherapy. Surgery is the first choice treatment strategy for glioma, and because glioma grows invasively, total resection is difficult to achieve, and postoperative recurrence is easy; the most common chemotherapy treatment is X-knife and gamma-knife treatment, but the treatment scope is limited due to the tumor position, the tumor size (generally limited to less than 3 cm) and the sensitivity of the tumor to rays; the chemotherapy drugs are limited to blood brain barriers and toxic and side effects of the drugs, the curative effect is not certain, and the effective rate of the commonly used chemotherapy drugs of BCNU, CCNU and VM-26 is only below 30%. The treatment methods all bring great challenges to the treatment of glioma, and glioma is easy to relapse, high in fatality rate and not optimistic in treatment effect.
With the rise of tumor immunotherapy in recent years, a new idea is provided for the treatment of brain glioma. Among them, adoptive immunotherapy, as an emerging field, has shown great potential in clinical trials of cancer, infection and autoimmune disease. However, effective homing of therapeutic T cells to the target site remains a major limiting factor, especially for brain tumors. Because tumor endothelial cells express high levels of leukocyte cell adhesion molecules (ALCAM), but their cognate ligand CD6 (naturally expressed on T cells) cannot mediate sufficient transendothelial migration (TEM) of tumor cells, T cell immunotherapy faces a significant challenge in the treatment of brain gliomas — the blood-brain barrier, blocking the entry of T cells into the brain. The blood brain barrier is beneficial to the brain under normal conditions, but it prevents T cells from reaching the brain glioma, thereby rendering immunotherapy ineffective.
ALCAM (also known as CD166), a member of the class I transmembrane protein and immunoglobulin superfamily, plays an important role in triggering T cell infiltration. The process of T cells passing through the blood brain barrier depends on the combination of a ligand molecule CD6 on the surface of the T cells and cell adhesion molecules such as ALCAM, intercellular adhesion molecule 1(ICAM1) and vascular cell adhesion protein 1(VCAM1) on endothelial cells, and once the T cells reach the adhesion threshold by combining with the cell adhesion molecules, the T cells can leave blood vessels and enter the brain. However, the conventional CD6 molecule cannot be directly introduced or introduced into T cells in an effective amount, so that the therapeutic effect of this method on brain gliomas is greatly reduced.
Therefore, the development of a novel recombinant CD6 gene, a T cell modified by the same, a preparation method and application not only have urgent research values, but also have good economic benefits and industrial application potentials, which is the basis and the motivation for the completion of the invention.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a recombinant CD6 gene, a T cell modified by the recombinant CD6 gene, a preparation method and application thereof, so as to improve the treatment effect on brain glioma.
In a first aspect, the invention provides a recombinant CD6 gene, wherein the recombinant CD6 gene comprises a signal peptide nucleic acid artificial sequence, an SRCR-D3 nucleic acid artificial sequence, a Stalk nucleic acid artificial sequence, a transmembrane-costimulatory domain nucleic acid artificial sequence and a cytoplasmic nucleic acid artificial sequence which are connected in sequence.
In the present invention, as a preferred embodiment, the transmembrane-stimulating domain is selected from the group consisting of CD6, CD8, CD27, CD28, CD137/4-1BB, CD134/OX40, all or a partial fragment of an ICOS molecule.
In the present invention, as a preferred embodiment, the transmembrane-costimulatory domain nucleic acid artificial sequence is a CD6 TM nucleic acid artificial sequence.
In the present invention, as a preferable technical means, the above
The nucleic acid human sequence of signal peptide is SEQ ID NO. 4;
the nucleic acid sequence of the SRCR-D3 nucleic acid human process is SEQ ID NO. 5;
the Stalk nucleic acid human process sequence is SEQ ID NO. 6;
the CD6 TM nucleic acid human procedure is set as SEQ ID NO. 7;
the cytoplasmic nucleic acid human sequence is SEQ ID NO. 8.
In a second aspect, the invention provides T cells modified with a recombinant CD6 gene, which T cells were infected with a lentivirus co-expressing an anti-EGFRvIII-CAR and a recombinant CD6 gene.
In the present invention, as a preferred technical scheme, the T cell includes autologous or transgenic T cell, NK cell, cytotoxic T lymphocyte or regulatory T cell, memory T cell, bispecific T cell, CIK cell.
In the invention, as a preferable technical scheme, the lentivirus is obtained by cotransfecting 293T cells by adopting a calcium phosphate transfection method through adopting a recombinant plasmid and a lentivirus packaging plasmid.
In the invention, as a preferable technical scheme, the recombinant plasmid is obtained by synthesizing an EGFRvIII-CAR nucleic acid artificial sequence, a self-cleavage polypeptide T2A nucleic acid artificial sequence, a signal peptide nucleic acid artificial sequence, an SRCR-D3 nucleic acid artificial sequence, a Stalk nucleic acid artificial sequence, a CD6 TM nucleic acid artificial sequence and a cytoplasmic nucleic acid artificial sequence into a whole expression frame, inserting a pLent-C-GFP vector, transforming to E.coli, and extracting by using a plasmid extraction kit after the sequencing is correct.
In a third aspect, the present invention provides a method for producing T cells, comprising the steps of:
1) synthesizing an EGFRvIII-CAR nucleic acid artificial sequence, a self-cleavage polypeptide T2A nucleic acid artificial sequence, a signal peptide nucleic acid artificial sequence, an SRCR-D3 nucleic acid artificial sequence, a Stalk nucleic acid artificial sequence, a CD6 TM nucleic acid artificial sequence and a cytoplasmic nucleic acid artificial sequence into a whole expression frame, inserting a pLent-C-GFP vector, transforming the vector into E.coli, extracting a recombinant plasmid by using a plasmid extraction kit after correct sequencing to obtain a recombinant expression vector pLent-Anti-EGFRvIII-CAR 3 HS;
2) taking peripheral blood, and separating peripheral blood mononuclear cells; after induction culture by using a culture medium of recombinant interferon alpha 2a, adding IL-2, OKT-3 and autologous plasma of a patient for induction continuous culture; adding liquid at a certain ratio every two days, culturing to 14 days, detecting that the positive rate of CD3+ in the T cells is more than 80% and the positive rate of CD3+ CD56+ in the T cells is more than 20% by flow cytometry, judging that the T cells are successfully induced, and reserving the T cells to treat virus infection;
3) recovering the 293T cell, and transfecting the 293T cell with the recombinant expression vector pLent-Anti-EGFRvIII-CAR 3HS obtained in the step 1) and a lentivirus packaging plasmid by adopting a calcium phosphate transfection method to obtain a pLent-Anti-EGFRvIII-CAR 3HS lentivirus;
4) infecting the Anti-EGFRvIII-CAR 3HS T cells (namely the T cells modified by the recombinant CD6 gene) prepared in the step 2) with the pLent-Anti-EGFRvIII-CAR 3HS lentivirus obtained in the step 3).
In a fourth aspect, the invention provides an application of a recombinant CD6 gene, which means that the recombinant CD6 gene is applied to the preparation of drugs for treating solid tumors such as GBM, non-small cell lung cancer, head and neck cancer, breast cancer, ovarian cancer, prostate cancer and the like.
In the invention, as a preferable technical scheme, the recombinant CD6 gene is particularly applied to the preparation of the medicine for treating the brain tumor.
Due to the adoption of the technical scheme, the invention has the following beneficial effects:
in the research, the inventor finds that in glioblastoma, the cerebrovascular system is changed, i.e. ALCAM, ICAM1 and VCAM1 are underexpressed or not expressed, so that T cells cannot enter and cross a blood brain barrier, and then brain tumors cannot be attacked. If a synthesized CD6 molecule is introduced into T cells to make the T cells express more molecular protein, once enough ligand molecule CD6 is combined with ALCAM, SLP-76 protein on the T cells is activated, and the SLP-76 protein can promote LFA-1 protein to move to the cell surface and combine with a small amount of ICAM-1 molecule on endothelial cells to further enhance the adhesion between the T cells and the endothelial cells. These molecular changes activate FAK proteins, enabling T cells to cross the blood brain barrier and successfully enter the brain, thereby allowing T cells to act to attack brain gliomas (fig. 1). Therefore, ALCAM binding is optimized by rational recombination of CD6 to create a system that directs T cells to home to brain cancer, allowing T cells to enter the brain through the blood-brain barrier, through the otherwise restricted tumor endothelial cells, with important implications for brain tumors and metastatic brain tumors.
Based on the thought, the inventor creatively recombines a CD6 gene segment, and the CD6 molecule can be connected with a glioma high-expression target point, such as GD2, HER2, EGFRvIII, TGF-beta 2 and other CAR sequences, so that the accurate targeted killing effect on glioma is enhanced. The invention firstly proposes that a ligand molecule CD6 on the surface of a T cell is introduced into the T cell by using retrovirus, so as to enhance the anchoring of the T cell to ALCAM, compensate the reduction of ICAM1 expression, promote the occurrence of migration, and promote the T cell to pass through a blood brain barrier and enter the brain, thereby achieving the effect of resisting brain tumor. The T cell provided by the invention can carry a CD6 molecule and CAR of a common brain glioma target, and the CD6 molecule enables the T cell to pass through endothelial cells and enter the brain, guides the T cell to home to the brain tumor, and plays a role in homing of CD6 (HOING SYSTEM, HS); and the modified T cells are strictly targeted to the brain tumor region without influencing other normal brains and body tissues by adding a target point aiming at the brain glioma, so that the method has the advantages of breaking through the blood brain barrier, accurately targeting the brain tumor and having high safety.
In conclusion, the T cell provided by the invention strictly targets the brain tumor region, and does not affect other normal brains and body tissues, so that the T cell has the advantages of breaking through the blood brain barrier, accurately targeting the brain tumor and having high safety.
Drawings
FIG. 1 is a schematic representation of the binding conductance of CD6 molecule to ALCAM.
FIG. 2 is the recombinant gene design diagram of EGFRvIII-CAR-CD6-CD6-CD 6.
FIG. 3 is a schematic representation of modules co-expressing anti-EGFRvIII-CAR and CD 6.
FIG. 4 shows that the efficiency of flow cytometry to detect CAR expression of Anti-EGFRvIII-CAR 3HS T cells was 36.8%.
FIG. 5 is a graph showing the in vivo killing results of Anti-EGFRvIII-CAR 3HS T cells.
FIG. 6 is a comparison graph of median survival of the EGFRvIII-CAR 3HS T cells, EGFRvIII-CAR-T cells and blank controls of the EGFRvIII-CAR 3HS T cells of the invention, wherein the median survival of the EGFRvIII-CAR 3HS T cells is significantly higher than that of the other two groups.
Detailed Description
The technical solution of the present invention will be described in detail with reference to specific examples. The following examples are only for illustrating the technical solutions of the present invention more clearly, and therefore are only examples, and the protection scope of the present invention is not limited thereby.
Example 1
The recombinant CD6 gene comprises a signal peptide nucleic acid artificial sequence, an SRCR-D3 nucleic acid artificial sequence, a Stalk nucleic acid artificial sequence, a transmembrane-costimulatory domain nucleic acid artificial sequence and a cytoplasmic nucleic acid artificial sequence which are connected in sequence. Wherein the nucleic acid human process sequence of the signal peptide is SEQ ID NO. 4; the nucleic acid sequence of the SRCR-D3 nucleic acid human process is SEQ ID NO. 5; the Stalk nucleic acid human process sequence is SEQ ID NO. 6; the CD6 TM nucleic acid human procedure is set as SEQ ID NO. 7; the cytoplasmic nucleic acid human sequence is SEQ ID NO. 8.
Because the present invention uses recombinant CD6 gene in conjunction with the expression of anti-EGFRvIII-CAR gene fragments, the following examples are described together with the co-expression of anti-EGFRvIII-CAR and CD6 gene fragments.
Example 2
Preparation of Co-expression anti-EGFRvIII-CAR and CD6 Gene fragment
The modules co-expressing anti-EGFRvIII-CAR and CD6 are schematically shown in FIG. 3 (see appendix SEQ ID NO.1 for the complete nucleic acid sequence).
The sequence of each module of Anti-EGFRvIII-CAR-CD6 is as follows (wherein the sequence of each module of CD6 molecule is (3) - (7)):
EGFRvIII-CAR nucleic acid artificial sequence (SEQ ID NO.2)
Self-cleavage polypeptide T2A nucleic acid artificial sequence (SEQ ID NO.3)
Signal peptide (Signal peptide region) nucleic acid artificial sequence (SEQ ID NO.4)
SRCR-D3 nucleic acid artificial sequence (SEQ ID NO.5)
Stalk (handle region) nucleic acid Artificial sequence (SEQ ID NO.6)
CD6 TM (transmembrane-costimulatory region) nucleic acid artificial sequence (SEQ ID NO.7)
cytoplasmic nucleic acid artificial sequence (SEQ ID NO.8)
EGFRvIII-CAR nucleic acid artificial sequence (SEQ ID NO.2), self-cleaving polypeptide T2A nucleic acid artificial sequence (SEQ ID NO.3), signal peptide nucleic acid artificial sequence (SEQ ID NO.4), SRCR-D3 nucleic acid artificial sequence (SEQ ID NO.5), staged nucleic acid artificial sequence (SEQ ID NO.6), CD6 TM nucleic acid artificial sequence (SEQ ID NO.7), cytoplasmic nucleic acid artificial sequence (SEQ ID NO.8) (wherein the signal peptide nucleic acid artificial sequence-cytoplasmic nucleic acid artificial sequence is repeated 3 times in total, the recombinant gene map is shown in FIG. 2) Consortium Biotechnology (Shanghai) Limited synthesizes the entire expression cassette thereof and inserts onto a standard vector pUC, thus named pUC-AnRvIII-pUC 3HS, the Anti-EGFRvIII-3 CAR was subjected to Fast Digest SI (from ThermoI) and Fisher ThermoI double digestion CAR (SEQ ID NO.3), the enzyme was cleaved at 37 ℃ for 20 min. The 100. mu.l enzyme system is: 10 × buffer: 10 mu l of the mixture; 6 mu g of DNA; AsiSI enzyme: 3 mu l of the solution; NotI enzyme: 3 mu l of the solution; deionized water to make up the volume. Cutting off the agar parts containing the DNA fragments of Anti-EGFRvIII-CAR 3HS by using agar electrophoresis, placing the cut agar parts into two centrifuge tubes, dissolving DNA out of agar by using a DNA extraction kit (purchased from ThermoFisher company) and concentrating, firstly adding 500ml DF buffer into the centrifuge tubes, acting for 10 minutes at 55 ℃, shaking every 2-3 minutes until the agar is completely dissolved. The agar solution was then aspirated into the DF Column and covered with the Collection Tube (Collection of filtrate). Centrifuge at 8000rpm for 1 minute and pour off the filtrate. 500ml of Wash Buffer was added thereto, and the mixture was centrifuged at 8000rpm for 1 minute, and the filtrate was decanted. Centrifugation at 12000rpm for 2 minutes ensured that ethanol was removed. And finally transferring the DF Column to another clean micro-centrifuge tube, adding 25 mu l of Elution Buffer, standing for 2 minutes at room temperature, centrifuging for 2 minutes at 14000rpm, and obtaining the purified Anti-EGFRvIII-CAR 3HS DNA fragment from the liquid in the micro-centrifuge tube.
Example 3
Preparation of recombinant plasmid
EGFRvIII-CAR nucleic acid artificial sequence (SEQ ID NO.2), self-cleaving polypeptide T2A nucleic acid artificial sequence (SEQ ID NO.3), signal peptide nucleic acid artificial sequence (SEQ ID NO.4), SRCR-D3 nucleic acid artificial sequence (SEQ ID NO.5), staged nucleic acid artificial sequence (SEQ ID NO.6), CD6 TM nucleic acid artificial sequence (SEQ ID NO.7), cytoplasmic nucleic acid artificial sequence (SEQ ID NO.8) (wherein signal peptide nucleic acid artificial sequence-cytoplasmic nucleic acid artificial sequence is repeated 3 times in total, the recombinant gene map is shown in FIG. 2) the entrusted Biotech (Shanghai) Limited company synthesizes the entire expression cassette and inserts it into a standard vector pUC, thus named pUCnFRvIII-3 HS, while Fastn-AnRvIII-3 and pLent-Dic-DistFastFastFastFastThergi are purchased from GFP Thermor Inc. (FisherSI) and FisherI, the enzyme was cleaved at 37 ℃ for 20 min. The 100. mu.l enzyme system is: 10 × buffer: 10 mu l of the mixture; 6 mu g of DNA; AsiSI enzyme: 3 mu l of the solution; NotI enzyme: 3 mu l of the solution; deionized water to make up the volume. And cutting the agar parts of the DNA fragment containing Anti-EGFRvIII-CAR 3HS and the linearized pLent-C-GFP DNA fragment by using agar electrophoresis, and placing the cut sections into two centrifuge tubes.
The DNA was dissolved from the agar using a DNA extraction kit (available from ThermoFisher Co.) and concentrated by first adding 500ml DF buffer to the centrifuge tube and allowing to act at 55 ℃ for 10 minutes, shaking every 2-3 minutes until the agar was completely dissolved. The agar solution was then aspirated into the DF Column and covered with the Collection Tube (Collection of filtrate). Centrifuge at 8000rpm for 1 minute and pour off the filtrate. 500ml of Wash Buffer was added thereto, and the mixture was centrifuged at 8000rpm for 1 minute, and the filtrate was decanted. Centrifugation at 12000rpm for 2 minutes ensured that ethanol was removed. And finally transferring the DF Column to another clean micro-centrifuge tube, adding 25 mu l of Elution Buffer, standing for 2 minutes at room temperature, centrifuging for 2 minutes at 14000rpm, and obtaining the liquid in the micro-centrifuge tube, namely the purified Anti-EGFRvIII-CAR 3HS DNA fragment and the linearized pLent-C-GFP DNA fragment.
The two DNA fragments were ligated overnight at 16 ℃ to form the pLent-Anti-EGFRvIII-CAR 3HS CAR plasmid. The connecting system is as follows: 10 × buffer: 1 mul; t4 ligase: 1 mul; Anti-EGFRvIII-CAR 3HS DNA: 4 mu l of the solution; linearized pLent-C-GFP DNA: 4 μ l.
Coli (DH5 α) was transformed with the above-described pLent-Anti-EGFRvIII-CAR 3 HS. Screening and identifying positive clones, selecting the positive clones at 37 ℃, shaking at 250rpm (12h-16h), and extracting a pLent-Anti-EGFRvIII-CAR 3HS plasmid according to a plasmid extraction and purification kit (purchased from Invitrogen company), wherein the specific steps are described in the specification. The preparation method of the pLent-Anti-EGFRvIII-CAR plasmid is the same as that of the pLent-Anti-EGFRvIII-CAR 3HS plasmid.
Sequencing the pLent-Anti-EGFRvIII-CAR 3HS plasmid and the pLent-Anti-EGFRvIII-CAR plasmid by committing the Biotechnology engineering (Shanghai) Co., Ltd. Sequencing is carried out correctly for later use.
Example 4
Lentiviral packaging
The lentivirus packaging cell line 293T is inoculated in a culture dish containing DMEM + 10% FBS10cm, cultured under the conditions of 37 ℃ and 5% CO2, and transfected after the anchorage rate is 70% -80%. The recombinant plasmid (about 10. mu.g) and the empty plasmid (about 10. mu.g) in example 1 were CO-transfected into 293T cells with the lentiviral vector plasmid by calcium phosphate transfection, gently mixed, cultured in a 5% CO2 incubator at 37 ℃ for 12 hours, added with 8mL of DMEM liquid medium containing 10% FBS, and cultured for another 48 hours. After 48h, the expression of green fluorescent protein in the cells was observed under an inverted fluorescence microscope. And after 72h, collecting the supernatant, removing cell fragments, harvesting viruses, and concentrating to obtain concentrated EGFRvIII-CAR 3HS virus solution and EGFRvIII-CAR virus solution, and storing in a low-temperature refrigerator at-70 ℃ for later use.
According to Lenti-XTMGoStixTMThe kit (product of Beijing Huaxia ocean technology Co., Ltd.) determines the virus titer, and the result shows that the titer of the recombinant lentivirus is divided intoIs 3.36 × 106pfu/mL and 3.32X 106pfu/mL。
Example 5
Preparation of T cells
50ml of patient autologous peripheral blood was taken and peripheral blood mononuclear cells were isolated using TBD sample density separation medium (purchased from Tianjin tertiary ocean organisms). After induction culture with a medium (purchased from CORNING corporation, 88-551-CM) containing 1000IU/ml of recombinant interferon alpha 2a (purchased from Shenyang Sansheng), 1000IU/ml of IL-2 (purchased from Shenyang Sansheng), 50ng/ml of OKT-3 and 5% of autologous patient plasma were added for further culture for 24 hours. Adding liquid at a doubling ratio every two days, culturing to the 14 th day, and detecting CD3 in T cells by flow cytometry+、CD56+Positive expression rate (CD3-FITC, CD16/CD56-PE antibody from BECKMAN, A07735). CD3+Positive rate>80％，CD3+CD56+Double positive rate>20% of the cells were considered successful in T cell induction and were left for viral infection.
Example 6
Lentiviral-infected T cells and expansion culture of CAR-T cells
After T cells were activated, 1X 10 cells were removed6And (3) adding concentrated EGFRvIII-CAR 3HS virus solution and EGFRvIII-CAR virus solution into each cell, and uniformly mixing, wherein the MOI is 6. After one week, the transfected T cells were individually tested for correlation. And detecting the expression of the chimeric antigen receptor by a fluorescence microscope, wherein the positive cells for detecting the GFP are positive cells for expressing CAR and CD6 due to the co-expression of the GFP and the CAR.
FIG. 3 shows a flow cytometry detection of the EGFRvIII-CAR 3HS T cell vector constructed by the present invention
Expression of the body on the surface of T cells. The result shows that 36.8% of cells are positive to GFP (GFP protein is expressed by the lentivirus vector), which indicates that the EGFRvIII-CAR 3HS constructed by the invention can be expressed on the surface of T cells, and the transfection efficiency is 36.8% respectively.
Example 7
Anti-EGFRvIII-CAR 3HS T cell killing in vitro
EGFRv III + U87 cells as target cells, and effector cells are Anti-EGFRvIII-CAR 3HS T cells, Anti-EGFRvIII-CAR T cells and untransfected T cells.
According to E: t (effector cell to target cell ratio) is 5: 1, adding 1X 106Collecting Anti-EGFRvIII-CAR 3HS T cells and untransfected T cells after the cells are completely attached to the wall, and respectively adjusting the cell concentration to be 1.36 multiplied by 107Each well was incubated for 12 hours with 100. mu.L of 5% CO2 at 37 ℃. The supernatant was discarded, 20. mu.L of diluted CCK8 (from MCE) was added thereto, the mixture was incubated for 4 to 6 hours, and the absorbance of OD450 was measured by a microplate reader. Killing rate ═ 1- (effector cell + target cell well OD value-OD value of effector cell alone)/OD value of target cell alone]X 100%. The killing efficiency of Anti-EGFRvIII-CAR 3HS T cells to EGFRv III + U87 cells is 98.66% (p)<0.02) is obviously higher than the killing efficiency of EGFRvIII-CAR T cells and untransfected T cells (the killing efficiency of the EGFRvIII-CAR T cells and the untransfected T cells to the EGFRv III + U87 cells is 52.36 percent respectively (p)<0.02) and 18.39% (p)<0.02)). Therefore, the Anti-EGFRvIII-CAR 3HS T cells prepared by the invention have the capacity of efficiently killing EGFRv III + U87 cells.
Example 8
Anti-EGFRvIII-CAR 3HS T cell in vivo killing
EGFRv III + U87 cells were cultured in high-glucose DMEM medium (containing 10% FBS) at 37 ℃ in a 5% CO2 incubator. When the medium reaches log phase, the medium is digested with 0.25% trypsin, collected in a 15ml centrifuge tube, the cells are washed with high-sugar DMEM medium, and the supernatant is discarded. Resuspend to 1X 10 cell density with sterile PBS6A single cell suspension in ml is ready for use. The nude mice (weight 17-20g, 6-8 weeks old) are in a prone type, puncture points are sterilized by 75% ethanol, needles are vertically inserted for 2.5mm, the puncture points are equivalent to deep white matter of right apical leaves (namely, occiput of the nude mice), 10ul of cell suspension is injected, the needle holes are pressed for a moment after the needles are pulled out, and the nude mice are fed back to an SPF (specific pathogen free) feeding room for feeding after inoculation. After inoculation, the mice were observed daily for diet, defecation, mental status, etc. One week later, if the mouse is found to have exostosis of the apical bone and an increase in intracranial pressure, and is examined by MRI imaging, it is judged that the mouse brain is exostosis and forms a tumor tissue. When the tumor volume reaches about 600mm3Then, the U87-GBM mouse model is successfully established, and the mouse is obtainedThe groups were randomly divided into 3 groups of 10 individuals. Cells were dissolved in 1ml PBS and injected into mice via tail vein on days 6 and 12, respectively, as follows:
experimental group a group: 1X 107Anti-EGFRvIII-CAR 3HS T cells;
experimental group B: 1X 107Anti-EGFRvIII-CAR-T cells;
control group C: 1X 107Anti-GCC-CAR-T cells (unrelated target CAR targeting other molecules);
blank control group D: only 1ml PBS was added.
After infusion, the mice were observed as above, daily for mental, dietary, and bowel movements, weighed, examined for red swelling and ulceration at each injection site, and tumor length and width, and tumor volume were assessed by MRIMRI imaging for a total of 60 days. When the tumor volume reaches>2000mm3At that time, mice were sacrificed. The data of each group were counted and plotted (see FIG. 5 for the results).
Tail vein injection experimental group a: Anti-EGFRvIII-CAR 3HS T cells and experimental group B: compared with the Anti-EGFRvIII-CAR-T cells, after 18d, intracranial tumors of mice injected with the Anti-EGFRvIII-CAR 3HS T cells are gradually reduced and directly and completely disappear, so that the experimental group A can induce the regression of formed tumors of intracranial brain gliomas of the mice, while the experimental group B can only temporarily slow down the growth of the tumors; tail vein injection experimental group a and control group C: compared with the Anti-GCC-CAR-T cells (irrelevant target CAR targeting other molecules) and the blank control group D, after 20 days, mice in the irrelevant target control group C and the blank control group D have the phenomena of obvious decrease in feeding, slow action, listlessness, accelerated tumor growth speed and the like, and have no tumor regression.
In the experiment, we also found that the median survival time of mice receiving the experimental group a cell therapy was about 60 days (the median survival time of mice in experimental group B and blank control group D was only 21 days or 18 days). Therefore, Anti-EGFRvIII-CAR 3HS T cells constructed according to the invention can delay the survival of mice (see FIG. 6).
Therefore, the Anti-EGFRvIII-CAR 3HS T cells can penetrate through a blood brain barrier, play a role in resisting cancer and delay the survival period.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; such modifications and substitutions do not depart from the spirit and scope of the present invention, and they should be construed as being included in the following claims and description.
Sequence listing
<110> Shandong Xingyi Biotechnology Ltd
<120> recombinant CD6 gene, T cell modified by using same, preparation method and application
<130> 2018
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cccaggctcc tcatctatgg tgcatccacc agggccactg gtgtcccagc caggttcagt 660
ggcagtgatt ctgggacaga gttctctctc accatcagca gcctgcagtc tgaagatttt 720
gcagtttatt tctgtcagca gtataaggac tggcccttca ctttcggccc agggaccaag 780
gtggagatca aaaccacgac gccagcgccg cgaccaccaa caccggcgcc caccatcgcg 840
tcgcagcccc tgtccctgcg cccagaggcg tgccggccag cggcgggggg cgcagtgcac 900
acgagggggc tggacttcgc ctgtgatatc tacatctggg cgcccttggc cgggacttgt 960
ggggtccttc tcctgtcact ggttatcacc ctttactgca acagaaggag aaggagagag 1020
agaagagatc tatttacaga gtcctgggat acacagaagg cacccaataa ctatagaagt 1080
cccatctcta cctctcaacc taccaatcaa tccatggatg atacaagaga ggatatttat 1140
gtcaactatc caaccttctc tcgcagacca aagactagag ttaagagagg ccggaagaag 1200
ctgctgtaca tcttcaagca gcccttcatg cggcccgtgc agaccaccca ggaagaggac 1260
ggctgcagct gtcggttccc cgaggaagaa gaaggcggct gcgaactgag agtgaagttc 1320
agcaggagcg cagacgcccc cgcgtaccag cagggccaga accagctcta taacgagctc 1380
aatctaggac gaagagagga gtacgatgtt ttggacaaga gacgtggccg ggaccctgag 1440
atggggggaa agccgagaag gaagaaccct caggaaggcc tgtacaatga actgcagaaa 1500
gataagatgg cggaggccta cagtgagatt gggatgaaag gcgagcgccg gaggggcaag 1560
gggcacgatg gcctttacca gggtctcagt acagccacca aggacaccta cgacgccctt 1620
cacatgcagg ccctgccccc tcgcgaaggc cgagggagcc tgctgacatg tggcgatgtg 1680
gaggaaaacc caggaccaat gtggctcttc ttcgggatca ctggattgct gacggcagcc 1740
ctctcaggtt ggcgcctgac agggggcgct gaccgctgcg aggggcaggt ggaggtacac 1800
ttccgagggg tctggaacac agtgtgtgac agtgagtggt acccatcgga ggccaaggtg 1860
ctctgccagt ccttgggctg tggaactgcg gttgagaggc ccaaggggct gccccactcc 1920
ttgtccggca ggatgtacta ctcatgcaat ggggaggagc tcaccctctc caactgctcc 1980
tggcggttca acaactccaa cctctgcagc cagtcgctgg cagccagggt cctctgctca 2040
gcttcccgga gtttgcacaa tctgtccact cccgaagtcc ctgcaagtgt tcagacagtc 2100
actatagaat cttctgtgac agtgaaaata gagaacaagg aatctcggga gctaatgatc 2160
ccctccatcg ttctgggaat tctcctcctt ggctccctca tcttcatagc cttcatcctc 2220
ttgagaatta aaggaaaata tgccctcccc gtaatggtga accaccagca cctacccacc 2280
accatcccgg cagggagcaa tagctatcaa ccggtcccca tcaccatgtg gctcttcttc 2340
gggatcactg gattgctgac ggcagccctc tcaggttggc gcctgacagg gggcgctgac 2400
cgctgcgagg ggcaggtgga ggtacacttc cgaggggtct ggaacacagt gtgtgacagt 2460
gagtggtacc catcggaggc caaggtgctc tgccagtcct tgggctgtgg aactgcggtt 2520
gagaggccca aggggctgcc ccactccttg tccggcagga tgtactactc atgcaatggg 2580
gaggagctca ccctctccaa ctgctcctgg cggttcaaca actccaacct ctgcagccag 2640
tcgctggcag ccagggtcct ctgctcagct tcccggagtt tgcacaatct gtccactccc 2700
gaagtccctg caagtgttca gacagtcact atagaatctt ctgtgacagt gaaaatagag 2760
aacaaggaat ctcgggagct aatgatcccc tccatcgttc tgggaattct cctccttggc 2820
tccctcatct tcatagcctt catcctcttg agaattaaag gaaaatatgc cctccccgta 2880
atggtgaacc accagcacct acccaccacc atcccggcag ggagcaatag ctatcaaccg 2940
gtccccatca ccatgtggct cttcttcggg atcactggat tgctgacggc agccctctca 3000
ggttggcgcc tgacaggggg cgctgaccgc tgcgaggggc aggtggaggt acacttccga 3060
ggggtctgga acacagtgtg tgacagtgag tggtacccat cggaggccaa ggtgctctgc 3120
cagtccttgg gctgtggaac tgcggttgag aggcccaagg ggctgcccca ctccttgtcc 3180
ggcaggatgt actactcatg caatggggag gagctcaccc tctccaactg ctcctggcgg 3240
ttcaacaact ccaacctctg cagccagtcg ctggcagcca gggtcctctg ctcagcttcc 3300
cggagtttgc acaatctgtc cactcccgaa gtccctgcaa gtgttcagac agtcactata 3360
gaatcttctg tgacagtgaa aatagagaac aaggaatctc gggagctaat gatcccctcc 3420
atcgttctgg gaattctcct ccttggctcc ctcatcttca tagccttcat cctcttgaga 3480
attaaaggaa aatatgccct ccccgtaatg gtgaaccacc agcacctacc caccaccatc 3540
ccggcaggga gcaatagcta tcaaccggtc cccatcacc 3579
<210> 2
<211> 1644
<212> DNA
<213> ethnic species (Homo sapiens)
<400> 2
atggcactgc cagtgaccgc cctgctgctg cctctggccc tgctgctgca cgcagccaga 60
ccccaggtta ccttgaagga gtctggtcct gtgctggtga aacccacaga gaccctcacg 120
ctgacctgca ccgtctctgg gttctcactc aataatgcta gaatgggtgt gagctggatc 180
cgtcagcccc cagggaaggc cctggagtgg tttgcacaca ttttttcgac tgacgaaaaa 240
tccttcagaa catctctgcg gagcaggctc accctctcca aggacacctc caaaagccag 300
gtggtcctta ccatgaccaa catggaccct gtggacacag ccacatatta ctgtgcacgg 360
gattctagca attacgaagg ttactttgac tactggggcc agggaattct ggtcaccgtc 420
tcgagcggag gtgggggcag tggtggcggg ggatctggag gtggaggttc cgaaatagtg 480
atgacgcagt ctccagccac cctgtctgtg tctccagggg aaagagccac cctctcctgc 540
agggccagtc agagtgttag caataactta gcctggtacc agcagaaacc tggccaggct 600
cccaggctcc tcatctatgg tgcatccacc agggccactg gtgtcccagc caggttcagt 660
ggcagtgatt ctgggacaga gttctctctc accatcagca gcctgcagtc tgaagatttt 720
gcagtttatt tctgtcagca gtataaggac tggcccttca ctttcggccc agggaccaag 780
gtggagatca aaaccacgac gccagcgccg cgaccaccaa caccggcgcc caccatcgcg 840
tcgcagcccc tgtccctgcg cccagaggcg tgccggccag cggcgggggg cgcagtgcac 900
acgagggggc tggacttcgc ctgtgatatc tacatctggg cgcccttggc cgggacttgt 960
ggggtccttc tcctgtcact ggttatcacc ctttactgca acagaaggag aaggagagag 1020
agaagagatc tatttacaga gtcctgggat acacagaagg cacccaataa ctatagaagt 1080
cccatctcta cctctcaacc taccaatcaa tccatggatg atacaagaga ggatatttat 1140
gtcaactatc caaccttctc tcgcagacca aagactagag ttaagagagg ccggaagaag 1200
ctgctgtaca tcttcaagca gcccttcatg cggcccgtgc agaccaccca ggaagaggac 1260
ggctgcagct gtcggttccc cgaggaagaa gaaggcggct gcgaactgag agtgaagttc 1320
agcaggagcg cagacgcccc cgcgtaccag cagggccaga accagctcta taacgagctc 1380
aatctaggac gaagagagga gtacgatgtt ttggacaaga gacgtggccg ggaccctgag 1440
atggggggaa agccgagaag gaagaaccct caggaaggcc tgtacaatga actgcagaaa 1500
gataagatgg cggaggccta cagtgagatt gggatgaaag gcgagcgccg gaggggcaag 1560
gggcacgatg gcctttacca gggtctcagt acagccacca aggacaccta cgacgccctt 1620
cacatgcagg ccctgccccc tcgc 1644
<210> 3
<211> 54
<212> DNA
<213> ethnic species (Homo sapiens)
<400> 3
gaaggccgag ggagcctgct gacatgtggc gatgtggagg aaaacccagg acca 54
<210> 4
<211> 51
<212> DNA
<213> ethnic species (Homo sapiens)
<400> 4
atgtggctct tcttcgggat cactggattg ctgacggcag ccctctcagg t 51
<210> 5
<211> 291
<212> DNA
<213> ethnic species (Homo sapiens)
<400> 5
tggcgcctga cagggggcgc tgaccgctgc gaggggcagg tggaggtaca cttccgaggg 60
gtctggaaca cagtgtgtga cagtgagtgg tacccatcgg aggccaaggt gctctgccag 120
tccttgggct gtggaactgc ggttgagagg cccaaggggc tgccccactc cttgtccggc 180
aggatgtact actcatgcaa tggggaggag ctcaccctct ccaactgctc ctggcggttc 240
aacaactcca acctctgcag ccagtcgctg gcagccaggg tcctctgctc a 291
<210> 6
<211> 117
<212> DNA
<213> ethnic species (Homo sapiens)
<400> 6
gcttcccgga gtttgcacaa tctgtccact cccgaagtcc ctgcaagtgt tcagacagtc 60
actatagaat cttctgtgac agtgaaaata gagaacaagg aatctcggga gctaatg 117
<210> 7
<211> 63
<212> DNA
<213> ethnic species (Homo sapiens)
<400> 7
atcccctcca tcgttctggg aattctcctc cttggctccc tcatcttcat agccttcatc 60
ctc 63
<210> 8
<211> 105
<212> DNA
<213> ethnic species (Homo sapiens)
<400> 8
ttgagaatta aaggaaaata tgccctcccc gtaatggtga accaccagca cctacccacc 60
accatcccgg cagggagcaa tagctatcaa ccggtcccca tcacc 105
Claims (4)
1. T cells modified with the recombinant CD6 gene, characterized in that: the T cell is obtained by infecting a lentivirus which expresses an anti-EGFRvIII-CAR gene and a recombinant CD6 gene together;
the nucleic acid sequences of the EGFRvIII-CAR-resistant and recombinant CD6 gene are shown as SEQ ID NO.1 in the sequence table.
2. The T cell modified with the recombinant CD6 gene of claim 1, wherein: the T cells include autologous or transgenic T cells.
3. The T cell modified with the recombinant CD6 gene of claim 2, wherein: the T cell is one of cytotoxic T lymphocyte or regulatory T cell, memory T cell, bispecific T cell and CIK cell.
4. The use of a T cell according to claim 1, wherein: the recombinant CD6 gene modified T cell is applied to the preparation of anti-glioma drugs.
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| WO2023081878A2 (en) * | 2021-11-08 | 2023-05-11 | Baylor College Of Medicine | Engineered immune cells and methods for use |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018071058A1 (en) * | 2016-10-15 | 2018-04-19 | Baylor College Of Medicine | Platform for enhanced targeted cell delivery |
| CN109265561A (en) * | 2018-09-25 | 2019-01-25 | 山东兴瑞生物科技有限公司 | The safety-type Chimeric antigen receptor of anti-EGFRv III, preparation method, the NK cell using its modification and application |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018071058A1 (en) * | 2016-10-15 | 2018-04-19 | Baylor College Of Medicine | Platform for enhanced targeted cell delivery |
| CN109265561A (en) * | 2018-09-25 | 2019-01-25 | 山东兴瑞生物科技有限公司 | The safety-type Chimeric antigen receptor of anti-EGFRv III, preparation method, the NK cell using its modification and application |
Non-Patent Citations (2)
| Title |
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| A cellular platform enables targeted brain delivery of T cells;H Samaha等;《Cytotherapy》;20170531;第19卷(第5期);第S13页 * |
| A homing system targets therapeutic T cells to brain cancer;Samaha H等;《NATURE》;20180920;第561卷(第7723期);第331-337页 * |
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