CN117802048A - CAR-T cell expressing enhancement factors and application thereof in tumor treatment - Google Patents
CAR-T cell expressing enhancement factors and application thereof in tumor treatment Download PDFInfo
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Abstract
The invention provides an engineering T cell which can express chimeric antigen receptor CAR protein and LCK kinase protein simultaneously, wherein genes expressing the CAR protein and the LCK kinase protein comprise GS-P2A sequences connected between the CAR gene and the LCK gene, and the nucleotide sequences are shown as SEQ ID NO:1 or SEQ ID NO:2. And application of engineering T cells in preparing antitumor drugs. According to the invention, the engineering nucleic acid molecule CAR-GS-P2A-LCK is introduced into the immune T cell in a virus transfection mode, so that the engineering T cell capable of simultaneously expressing the chimeric antigen receptor CAR protein and the LCK kinase protein is obtained, has a stable expression effect, and has prolonged survival time and long-acting tumor inhibition effect in a tumor-bearing mouse through verification of glioma and lymphoma mouse models. Meanwhile, the CAR-T is not expressed outside the cell, so that the systematic safety risk is not caused, and the cost is not increased.
Description
Technical Field
The invention relates to the technical field of biological medicine, in particular to a CAR-T cell expressing an enhancement factor and application thereof in tumor treatment.
Background
Chimeric antigen receptor-T (CAR-T) cells are used as a means of current cancer immunotherapy, achieve better therapeutic effects in hematological tumors, achieve disease control for more than 5 years in some patients, and can continuously detect reinfused CAR-T cells in the peripheral blood of such patients. In contrast, some patients undergo CAR-T treatment, and then tumor recurrence occurs in a short time, but CAR-T cells in the patients cannot be detected when tumor recurrence occurs. The above results indicate that the persistence of CAR-T cells in patients is critical to ensuring the efficacy of CAR-T. Meanwhile, in the treatment of solid tumors, the returned CAR-T cells can reach the tumor part after circulating in peripheral blood, so that the continuous viability of the CAR-T cells in the peripheral blood circulation also has an important influence on the control of the solid tumors. In summary, improving the persistence of CAR-T cells themselves is critical to the clinical outcome of CAR-T treatment.
There are a number of methods reported in the literature to enhance the persistence of CAR-T cells. The intracellular region of the co-stimulatory molecule CD28 or 4-1BB is added in the CAR structure of the second generation CAR-T, so that compared with the first generation CAR-T, the persistence of cells is obviously improved, the tumor treatment effect is improved, and the CAR structure adopted in most CAR-T products at present is also adopted. In addition, the memory T cells are screened and CAR gene modification is carried out, so that the prepared CAR-T cells have more memory T cells, and the survival time of the CAR-T cells in vivo can be prolonged; the IL-7 and IL-15 cytokines are used for preparing and amplifying the CAR-T cells, so that the proportion of the central memory T cells can be effectively increased, and the persistence of the CAR-T cells in the body and the tumor killing effect are enhanced; the secretory or membrane-bound IL-15 is synchronously expressed in the CAR-T, which is also helpful for improving the survival and proliferation capacity of the CAR-T cells, and has better tumor control effect compared with the traditional CAR-T. Recently, the document reports that mRNA vaccine injected with antigen regularly can effectively stimulate proliferation and survival of Claudin 6CAR-T in vivo, and a brand new means is provided for improving the persistence of CAR-T cells.
However, the existing method for enhancing the persistence of the CAR-T is mainly realized by screening the memory cells or cytokines which are helpful for proliferation of the memory cells to increase the proportion of the central memory T cells, however, the screening method improves the preparation difficulty of the memory CAR-T cells due to the smaller proportion and weaker expansion capacity of the memory cells; whereas the use of CAR-T cells to express cytokines often results in other side effects, increasing the risk of CAR-T treatment, due to the circulation of CAR-T and cytokines around the periphery. While the above problems can be avoided by periodic vaccine injection, the combination of the two drugs can increase the potential risk and cost of treatment and limit their use.
The method of enhancing persistence of CAR-T by IL-15-like molecules is primarily to promote the downstream memory signaling pathway by activating cytokines and JAK/STAT signaling pathways in T cells, so other molecules can also achieve enhanced persistence of T cells by affecting this pathway. LCK acts as a critical signaling molecule for T cell pathways and is critical for T cell activation, expansion, tumor killing, and cytokine secretion. LCK is a tyrosine kinase that activates downstream signaling pathways by phosphorylating downstream substrate CD 3. LCK can also affect T cell memory pathways by phosphorylating STAT molecules, thereby effecting an effect on T cell persistence. The present invention co-expresses LCK molecules in CAR-T cells to achieve enhancement of CAR-T cell killing ability and persistence.
Disclosure of Invention
It is an object of the present application to provide a CAR-T cell expressing an enhancement factor, which aims to solve the above-mentioned problems in the prior art.
Embodiments of the present application provide an engineered T cell capable of simultaneously expressing a chimeric antigen receptor CAR protein and an LCK kinase protein.
Further, it is capable of overexpressing chimeric antigen receptor CAR proteins and LCK kinase proteins.
Further, the genes expressing the CAR protein and the LCK kinase protein comprise a CAR gene and an LCK gene linked together by an engineering nucleic acid molecule, and the expression sequence of the CAR gene is higher than that of the LCK gene, the CAR gene is from a targeted CSPG4 or a targeted CD19, and the expressed amino acid sequence is as shown in SEQ ID NO:3 or SEQ ID NO:4 is shown in the figure; the amino acid sequence expressed by the LCK gene is shown as SEQ ID NO: shown at 5.
An engineering nucleic acid molecule, which is formed by splicing GS-P2A sequences connected between a CAR gene and an LCK gene through overlapping PCR, is named as CAR-GS-P2A-LCK, and has nucleotide sequences respectively shown in SEQ ID NO:1 or SEQ ID NO:2 is shown in the figure; where GS is a linker molecule, P2A recognizes an internal ribosome entry site or ribosome codon jump site for initiation of independent translation of a second molecule.
A nucleic acid construct comprising an engineered nucleic acid molecule and further comprising other acceptable ligands for the nucleic acid construct.
A vector virus comprising in its genetic sequence the nucleotide sequence SEQ ID NO:1 or the nucleotide sequence SEQ ID NO:2.
a pharmaceutical composition having tumor resistance for use in the treatment of a tumor disease comprising said engineered T cell, said engineered nucleic acid molecule, said nucleic acid construct or said vector virus.
Further, a pharmaceutically acceptable carrier thereof is also included.
The use of said engineered T-cell, said engineered nucleic acid molecule, said nucleic acid construct, said vector virus or said pharmaceutical composition for the preparation of a medicament for the diagnosis, treatment or prevention of a tumor.
The beneficial effects of the invention are as follows: according to the invention, the engineering nucleic acid molecule CAR-GS-P2A-LCK is introduced into the immune T cell in a virus transfection mode, so that the engineering T cell capable of simultaneously expressing the chimeric antigen receptor CAR protein and the LCK kinase protein is obtained, has a stable expression effect, and has prolonged survival time and long-acting tumor inhibition effect in a tumor-bearing mouse through verification of glioma and lymphoma mouse models. Meanwhile, the CAR-T is not expressed outside the cell, so that the systematic safety risk is not caused, and the cost is not increased.
Drawings
FIG. 1 is a block diagram of four different single vectors co-expressing CAR and LCK.
Fig. 2 is a graph showing the expression levels of transduced CSPG4CAR-T cell surface CAR after flow cytometry detection of different vector-produced viruses.
FIG. 3 shows the expression level of LCK of FLAG tag in CSPG4CAR-T cells transduced after virus preparation by western blot detection of different vectors.
In fig. 4, panel a is the expression level of the glioma cell line LN229 surface antigen CSPG 4; panel B shows the expression levels of transduced CSPG CAR-T cell surface CAR following viral preparation with three different vectors.
Fig. 5 shows control NT for flow cytometry detection and three different vector transduced CSPG4CAR-T cells with tumor cells LN229 according to e:t=1: 1 ratio of residual T cells and tumor cells after two rounds of co-culture.
Fig. 6 shows control NT for flow cytometry detection and three different vector transduced CSPG4CAR-T cells with tumor cells LN229 according to e:t=1: 5 ratio of residual T cells and tumor cells after co-culture.
FIG. 7 is a graph showing the killing of gliomas by T cells transduced with control NT, CSPG4 CAR-only and CAR-GS-P2A-LCK vectors in the cranium of tumor-bearing mice.
In fig. 8, panel a shows the expression level of the cell surface antigen CD19 of the B lymphoma cell line Daudi; panel B shows the expression levels of T cell surface CARs transduced with CD19 CAR-only and CD19 CAR-GS-P2A-LCK vectors; panel C is the expression levels of LCK and CAR in control NT and transduced CAR-T cells.
FIG. 9 is a graph showing the killing of lymphomas in tumor bearing mice by control NT, CD19 CAR-only, and CD19 CAR-GS-P2A-LCK transduced T cells.
Figure 10 is a T cell count statistic for control NT and two CD19 CAR-T at different time points in lymphoma mice.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example one, cell line
Cell lines used in the present invention include 293T cells cultured in IMDM medium containing 10% FBS,1% glutamax,1% P/S. The culture medium of glioma cell line LN229 and B lymphoma cell line Daudi was RPMI640 containing 10% FBS and 1% P/S, in which GFP positive tumor cells were prepared by transfection with GFP-encoding retrovirus and the tumor cell line of the mouse experiment was obtained by transduction with a retrovirus encoding Luciferase. T cells were cultured in X-VIVO 15 medium containing 5% FBS, and IL-7 and IL-15 were added at a final concentration of 10ng/mL and 5ng/mL simultaneously.
Example two construction of chimeric antigen receptor and LCK expression vector
The invention uses a single virus vector to carry out gene modification on T cells, so that two protein molecules of CAR and LCK are co-expressed in the same T cell. To select the appropriate vector construction mode, the present invention was designed based on a CSPG 4-targeting CAR.
Wherein the CSPG 4-targeting CAR sequence is derived from patent CN114249832A, the CD 19-targeting CAR sequence is derived from document "doi 10.1182/blood-2010-04-281931", and the sequences are obtained by gene synthesis. The gene sequence of LCK is derived from Genbank ID 3932, and is obtained from a T cell cDNA library by PCR method, and FLAG tag (amino acid sequence DYKDDDDK) is added at C end of LCK protein. And then connecting the CAR and LCK genes by using P2A or GS-P2A sequences by using overlapped PCR to obtain 4 different expression structures of the CAR and LCK, namely CAR-P2A-LCK, CAR-GS-P2A-LCK, LCK-P2A-CAR and LCK-GS-P2A-CAR, as shown in figure 1. The full length product was cloned into murine leukemia retroviral vector SFG and the sequence accuracy was confirmed by testing.
Of the 4 structures described above, P2A was used to initiate independent translation of the second molecule, while the added GS linker was used to enhance the efficiency of initiation of the P2A molecule, while a FLAG tag was fused to LCK for subsequent detection of its expression level.
Example III transduction and expansion of T cells
Peripheral blood donated by healthy persons was used to isolate mononuclear cells. Mononuclear cells were isolated using lymphocyte density gradient separation. After activation of isolated mononuclear cells by murine anti-human CD3 and CD28 antibodies, T cells were transduced with retroviruses packaged with the 4 expression constructs of example two. The chimeric antigen receptor transduced T cells are then expanded for detection of chimeric antigen receptor expression and tumor killing.
Example IV, chimeric antigen receptor expression detection
CSPG-targeted CARs were detected using CSPG4 protein extracellular domain and human antibody Fc fusion proteins. And harvesting part of the amplified CSPG4CAR-T cells, incubating the CSPG4CAR-T cells with the CSPG4 extracellular region-Fc fusion protein on ice for 30 minutes, adding an APC fluorescent dye-labeled antibody Fc binding protein after PBS washing, and detecting the expression of the CSPG4CAR on the surface of the T cells by using flow cytometry after 30 minutes of dyeing. CD 19-targeting CARs use the extracellular domain of the CD19 protein and Fc fusion proteins, the expression of which was detected using a similar method.
Results as shown in fig. 2, CAR-preceding vectors (CAR-P2A-LCK and CAR-GS-P2A-LCK) expressed CSPG4CAR at similar levels in T cells as CAR-alone-expressing vectors, while CAR expression levels in T cells of the other two vectors (LCK-P2A-CAR and LCK-GS-P2A-CAR) were significantly reduced. Since the T cell surface CAR molecule level determines its killing effect on tumors, the first two vectors of LCK were discarded.
Example five, detection of expression of chimeric antigen receptor and LCK in T cells
Expression of chimeric antigen receptor and LCK in T cells was examined using western blot to assess the efficiency of P2A priming.
Partially expanded T cells were collected by centrifugation and denatured at 95 ℃ for 5 minutes after lysis with SDS lysates. The samples were then separated by SDS-PAGE and the proteins transferred to PVDF membrane. PVDF membranes were blocked with 5% nonfat milk powder for 1 hour and rinsed twice with TBST, then incubated with anti-human CD3 antibodies (for CAR detection) or anti-FLAG-tagged antibodies (for LCK detection) overnight at 4 ℃. After three rinsing with TBST, goat anti-mouse or rabbit secondary antibody was added and incubated at room temperature for 1.5 hours. After three more rinsing passes with TBST, development was performed by ECL.
The results are shown in FIG. 3, in addition to the failure to detect LCK in the LCK-GS-P2A-CAR group, there are still more fusion proteins in the CAR-P2A-LCK group, while the CAR and LCK in the other two groups can be effectively detected near their molecular weights, indicating that the CAR-P2A-LCK cannot effectively separate the two proteins, thus the CAR-GS-P2A-LCK was selected as the target vector for subsequent evaluation.
Example six detection of antigen expression in tumor cell lines
To compare the killing capacity and persistence of CAR-T after increasing LCK expression, the present invention compares the killing capacity of T cells expressing CAR alone and co-expressing CAR and LCK to tumor cells. Glioma cell LN229 was used as target tumor cell, after collection of log phase grown tumor cells, washed 3 times with 1 XPBS, then incubated with anti-CSPG 4 or anti-CD 19 specific antibodies on ice for 30 min, after 1 XPBS washing, APC or PE conjugated secondary antibodies were added, incubated for 20 min at room temperature, after 1 XPBS washing, the expression levels of CSPG4 or CD19 on the corresponding tumor cells were detected by flow cytometry.
As a result, as shown in FIG. 4A, there was a high level of CSPG4 antigen expressed on the cell surface. The invention synchronously prepares only CSPG4CAR (CAR only), three different CAR-T cells of CAR-P2A-LCK and CAR-GS-P2A-LCK, and detects the expression level of the CAR by using CSPG4-Fc fusion protein and flow cytometry. The results are shown in FIG. 4B, where the results of the previous tests are consistent, the CAR only T cells have the highest level of CAR expression, the CAR-GS-P2A-LCK times, and the CAR-P2A-LCK has the lowest level of CAR expression.
Example seven Co-culture of tumor cells with T cells
Three T cells were tested for their ability to kill and persist in tumors by co-culturing with tumor cells. The tumor cells are LN229 glioma cells.
At 24 holesInoculating 1-2×10 wells per plate 5 Is a GFP-labeled tumor cell according to T cells after 24 hours: tumor cells = 1:1 or 1:5, and adding corresponding quantity of T cells. Cells in wells were harvested 3-7 days later and residual cells were detected using flow cytometry. Wherein T cells are detected using CD3 and tumor cells are detected using GFP.
Wherein, at CAR-T: tumor cells were 1:1, tumor cells were killed in two consecutive rounds using the same CAR-T cells, for evaluation of the ability of CAR-T to sustain killing after repeated antigen stimulation. Residual cells were collected after the second round of co-culture and the residual ratio of CAR-T and tumor cells was detected using flow cytometry. As shown in fig. 5, T cells of the CAR only group and CAR-P2A-LCK group can still effectively kill tumors, but a high proportion of tumor cells remain after two rounds of co-culture compared to control non-transduced T cells (NTs); compared with the CAR-GS-P2A-LCK group, a significantly smaller proportion of tumor cell residues are found, which indicates that the CAR-GS-P2A-LCK group can better control the recurrence of tumors.
In CAR-T, tumor cells were 1:5, and assessing the ability of three different CAR-T cells to kill tumor cells continuously under antigen-sustained stimulation, residual cell types and ratios were also detected using flow cytometry. The results are shown in fig. 6, although CAR only and CAR-P2A-LCK had less tumor cell remnants than the control NT group, the significantly lower proportion of tumor cells in the CAR-GS-P2A-LCK group, indicating that CAR-GS-P2A-LCK group T cells can control tumor proliferation more permanently and more effectively.
Example eight, xenograft tumor mice experiments
In order to confirm the control effect of the cell line on tumors in vivo, an animal model of the tumors is established in a nude mouse by utilizing a LN229 glioma cell line, and then CAR-T cells are injected into the cranium of the mouse to evaluate the control effect of the cell line on the tumors.
Glioma-transplanted mice experiments were completed in 6-8 week old nude mice. GFP-FFLuc labeled LN229 tumor cells were first implanted in nude mice intracranially by stereotactic, and after 2-3 weeks, control non-transduced T cells (NTs) or CSPG4CAR-T cells were injected into the nude mice ventricle after confirmation of tumor graft success by an in vivo imaging system. After imaging once a week, T cells were observed for tumor killing. B cell lymphoma mice experiments were completed in NSG mice for 6-8 weeks. The GFP-FFLuc labeled Daudi tumor cells were first injected by tail vein, and after 1 week of tumor cell reinfusion, the control NT or CD19 CAR-T cells were injected by tail vein. Then imaging by using an in-vivo imaging system every 3-7 days, and observing and recording the killing condition of T cells on lymphoma.
The results are shown in fig. 7, where CAR-T cells of the CAR-GS-P2A-LCK group effectively controlled tumor progression early compared to tumor progression in the control NT and CAR only groups, indicating that co-expression of LCK enhances killing and persistence of CAR-T cells.
Example nine, quantitative detection of T cells in NSG mice
Lymphoma NSG mice were collected weekly by 100-200 μl venous blood through the submaxillary vein before and after T cell injection. After the collected venous blood was lysed by erythrocytes, it was incubated with fluorescein-labeled murine anti-human CD45, CD3 and CD19 antibodies for 30 minutes at 4℃and then 10. Mu.l of counting microspheres were added and the absolute numbers of T cells and Daudi tumor cells in the venous blood were detected and quantified by flow cytometry.
Since the intracranial tumor model of mice was unable to assess the persistence of CAR-T cells in vivo, the present invention constructed a B lymphoma model in NSG mice using Daudi cells (expressing CD19, fig. 8A), and then assessed the effect of LCK on CAR-T cell killing ability and persistence in vivo using CD19 CAR-T. The invention also constructs a CD19 CAR-GS-P2A-LCK vector, prepares retrovirus-transduced T cells, and then detects the expression of the CAR by using CD19 protein and flow cytometry. The results are shown in FIG. 8B, where both CD19 CAR only and CD19 CAR-GS-P2A-LCK groups had some level of CAR expression.
The expression of LCK in the CD19 CAR-GS-P2A-LCK group was also confirmed using WesternBlot (FIG. 8C). The mice were then imaged 1-2 times weekly to assess tumor progression 7 days after intravenous injection of luciferase labeled Daudi cells into the tail of NSG mice, followed by injection of control NT, CD19 CAR-only and CD19 CAR-GS-P2A-LCK T cells.
The results are shown in fig. 9, the control NT group tumor continued to progress, and all mice died from the tumor by day 23, while both other groups were effective in controlling tumor progression. In contrast, on day 13, the CD19 CAR-GS-P2A-LCK group had been more effective in controlling tumors than the CD19 CAR-only group; on day 68, however, tumor recurrence occurred in the CD19 CAR-only group, while no tumor signal was shown in the CD19 CAR-GS-P2A-LCK group.
Meanwhile, in order to determine the survival and proliferation of CAR-T cells in vivo, the present invention collects peripheral blood of a small number of mice weekly and detects the number of human T cells remained therein using flow cytometry. The results are shown in FIG. 10, in which T cells in the CD19 CAR-only group and the CD19 CAR-GS-P2A-LCK group proliferate significantly in the first week, and the decrease occurs after tumor control; while the T cells of the CD19 CAR-only group are continuously maintained at a lower level, the T cells of the CD19 CAR-GS-P2A-LCK group continuously survive in vivo and display a certain proliferation capacity, and are consistent with a better tumor control effect in vivo, so that LCK can effectively increase the persistence of the CAR-T cells in vivo and further enhance the control capacity of the LCK on tumors.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.
Claims (9)
1. An engineered T cell capable of simultaneously expressing a chimeric antigen receptor CAR protein and an LCK kinase protein.
2. The engineered T-cell of claim 1, wherein it is capable of overexpressing chimeric antigen receptor CAR proteins and LCK kinase proteins.
3. The engineered T-cell of claim 2, wherein the genes expressing the CAR protein and the LCK kinase protein comprise a CAR gene and an LCK gene linked together by an engineered nucleic acid molecule, and wherein the CAR gene is expressed in a sequence that is preferentially over the LCK gene, the CAR gene being derived from either the targeted CSPG4 or the targeted CD19, and wherein the expressed amino acid sequence is as set forth in SEQ ID NO:3 or SEQ ID NO:4 is shown in the figure; the amino acid sequence expressed by the LCK gene is shown as SEQ ID NO: shown at 5.
4. An engineering nucleic acid molecule is characterized in that the engineering nucleic acid molecule comprises a GS-P2A sequence connected between a CA R gene and an LCK gene, which is spliced by overlapping PCR, and is named as CAR-GS-P2A-LCK, and the nucleotide sequences of the engineering nucleic acid molecule are respectively shown as SEQ ID NO:1 or SEQ ID NO:2 is shown in the figure; where GS is a linker molecule, P2A recognizes an internal ribosome entry site or ribosome codon jump site for initiation of independent translation of a second molecule.
5. A nucleic acid construct comprising the engineered nucleic acid molecule of claim 4, further comprising an additional acceptable ligand for the nucleic acid construct.
6. A vector virus, characterized in that the genetic sequence of the vector virus comprises the nucleotide sequence of SEQ ID NO:1 or the nucleotide sequence SEQ ID NO:2.
7. a pharmaceutical composition having tumor resistance for use in the treatment of a tumor disease comprising the engineered T cell of any one of claims 1-3, the engineered nucleic acid molecule of claim 4, the nucleic acid construct of claim 5, or the vector virus of claim 6.
8. The pharmaceutical composition of claim 7, further comprising a pharmaceutically acceptable carrier thereof.
9. Use of an engineered T cell of any one of claims 1-3, an engineered nucleic acid molecule of claim 4, a nucleic acid construct of claim 5, a vector virus of claim 6 or a pharmaceutical composition of claim 7 or 8 for the preparation of a medicament for the diagnosis, treatment or prevention of a tumor.
The sequence table needs to be provided again: (none include tag sequences)
SEQ ID NO：1：CSPG4-CAR-GS-P2A-LCK
Nucleotide sequence:
SEQ ID NO：2：CD19-CAR-GS-P2A-LCK
nucleotide sequence:
SEQ ID NO：3：CSPG4-CAR
amino acid sequence:
SEQ ID NO：4：CD19-CAR
amino acid sequence:
SEQ ID NO：5：LCK。
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