CN113980139A

CN113980139A - Chimeric antigen receptor cell of autocrine TREM2scFv and preparation method and application thereof

Info

Publication number: CN113980139A
Application number: CN202111062454.8A
Authority: CN
Inventors: 黄曦; 朱天川; 郑葳
Original assignee: Qinyuan Regenerative Medicine Zhuhai Co ltd
Current assignee: Qinyuan Regenerative Medicine Guangdong Co ltd
Priority date: 2021-09-10
Filing date: 2021-09-10
Publication date: 2022-01-28
Anticipated expiration: 2041-09-10
Also published as: CN113980139B

Abstract

The invention belongs to the technical field of biological medicines, and discloses an autocrine TREM2scFv chimeric antigen receptor cell, a preparation method and an application thereof. Through TREM2scFv secreted in a tumor microenvironment, the antigen presenting capacity of dendritic cells and macrophages can be enhanced, the activation of CAR-T cells is promoted, the failure of the CAR-T cells is reduced, the CAR-T cells are further promoted to secrete killing substances such as granzyme and perforin, and the purpose of improving the anti-tumor capacity of the CAR-T cells is finally achieved.

Description

Chimeric antigen receptor cell of autocrine TREM2scFv and preparation method and application thereof

Technical Field

The invention relates to the technical field of biological medicines, and in particular relates to an autocrine TREM2scFv chimeric antigen receptor cell and a preparation method and application thereof.

Background

CAR-T cell therapy, i.e., chimeric antigen receptor T cell therapy, utilizes genetic engineering techniques to express synthetic Chimeric Antigen Receptors (CARs) on T cells, thereby enabling T cells to recognize and be highly activated by specific cancer cells, thereby better killing tumor cells. CAR has four major components of modular elements: an antigen binding domain, a hinge, a transmembrane domain, and an intracellular signaling domain. These elements all have unique functions and the optimal molecular design of the CAR can be achieved through variation of the constituent elements.

Currently, two CAR-T cell products targeting CD19, yescata and kymeriah, have been approved in europe and the united states. The greatest difference between the two products is in their co-stimulatory domains: yescata uses CD28, while Kymriah uses 4-1 BB. Kymeriah has been approved for use in relapsed or refractory B-cell malignancies, such as Acute Lymphoblastic Leukemia (ALL) in patients under 25 years of age, and two or more relapsed or refractory diffuse large B-cell lymphoma (DLBCL) systemic therapies; while yescata is mainly approved for DLBCL and primary mediastinal large B-cell lymphoma (PMBCL). To date, adult ALL patients showed the best rate of Complete Remission (CR) (83% -93%) using CAR-T therapy. It is noteworthy, however, that this high remission rate may not be entirely attributable to CAR-T therapy, since patients typically require non-myeloablative chemotherapy prior to CAR-T therapy. In addition, the complete remission rate was also higher in pediatric ALL patients, between 67% and 90%. In contrast, the CR rate in patients with DLBCL is between 43% and 54%, and that in Chronic Lymphocytic Leukemia (CLL) patients is between 21% and 29%. After CAR-T cell treatment, the risk of disease recurrence was high in all patients, and 41% of patients had relapsed within 12 months as shown in one study.

Furthermore, CAR-T cells are currently less effective in solid tumors. CAR-T cells have many limitations when treating solid tumors, such as cytokine release syndrome, tumor escape due to antigen loss, insufficient chemokine receptor profile, CAR-T cell failure due to tonic signaling or depletion, and the like. CAR-T cells, once they enter the tumor microenvironment, are inhibited in both their cytotoxicity and their proliferative capacity on tumor cells. The inhibition may be by contact-dependent means or may be mediated by cytokines secreted by the tumor and the suppressor cell. TGF- β, IDO, immune checkpoints, and inhibitory immune cells (myeloid-derived suppressor cells) can all inhibit CAR-T cells. In general, dendritic cells are key regulators of T cell responses. In a pro-inflammatory environment, T cells are effectively stimulated upon encountering dendritic cells, thereby eliciting an immune response against target cells; however, in environments lacking inflammation, T cells may not have an immune response. For example, IL-10 may not only impair the anti-tumor function of dendritic cells, but also deprive CAR-T cells of the support of the endogenous immune system by down-regulating the expression of TAP1 and TAP2 in tumor cells, thereby preventing antigen processing and presentation. TFG- β is another ubiquitous cytokine produced by both tumor cells and Treg cells that inhibits T cell activation, proliferation and differentiation. CTLA-4 and PD-1 can render T cells immune-unresponsive by driving them into an unresponsive state. In addition, physical factors such as hypoxia, nutritional deficiencies, low pH also cause infiltrating immune cells to malfunction. Therefore, there is a need for further modifications to CAR-T cells to enhance the tumor killing effect of CAR-T cells.

TREM2 has been previously documented as a receptor that interacts with a variety of ligands, many of which are hallmarks of tissue damage. TREM2 is expressed by tumor-infiltrating macrophages, and there is increasing evidence that TREM2 plays a role in tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs). A recent study found that TREM2 was significantly upregulated on peripheral blood mononuclear cells and TAMs in lung cancer patients and tumor-bearing mice compared to healthy controls. Furthermore, TREM2 levels of macrophages surrounding tumor cells are positively correlated with tumor progression. In summary, there is increasing evidence that TREM2 plays an important role in promoting a tumor immunosuppressive microenvironment. Therefore, TREM2 may be an ideal target for targeted treatment of tumor marrow infiltration and enhanced immune checkpoint immunotherapy.

Disclosure of Invention

The technical problem to be solved by the present invention is to overcome the above-mentioned drawbacks described in the prior art, and firstly to provide a chimeric antigen receptor of autocrine TREM2 scFv.

It is a second object of the present invention to provide a gene sequence encoding a chimeric antigen receptor that autocrine TREM2 scFv.

It is a third object of the present invention to provide a cell expressing the above chimeric antigen receptor.

The fourth purpose of the invention is to provide a medicine for targeted therapy of tumors.

The purpose of the invention is realized by the following technical scheme:

an autocrine TREM2scFv chimeric antigen receptor comprising a signal peptide, a single chain variable fragment capable of binding a tumor antigen, a hinge region, a transmembrane domain, a costimulatory molecule, an immunoreceptor tyrosine activation motif, a self-cleaving peptide fragment, a single chain variable fragment of an anti-TREM2 antibody.

Preferably, the tumor antigen is selected from AFP, BCMA, B7H4, CD52, CD56, CD80, CDK4/m, CEA, CT, DAM, EGFR, ErbB3, ELF2M, EMMPRIN, EpCam, G250, GAGE, GnTV, HAGE, HER2, HPVE7, HSP70, hTERT, iCE, IGF1R, IL2R, IL5, LAGE, LDLR/FUT, MAGE, MART1, MART2, Mesothelin, MUCl, PRAME, PSMA, RAGE, SAGE, TGFf3, TPI/m, VEGF, WTL, MLSN.

TREM2 effectively inhibits secretion of proinflammatory cytokines by monocytes. In addition, TREM2 can also inhibit the antigen-presenting function of macrophages and dendritic cells in the tumor microenvironment, thereby inhibiting T cell activation. In order to further optimize CAR design and overcome the problems that tumor immunosuppression microenvironment and blood tumor are easy to relapse, TREM2scFv is added on an original CAR expression vector, so that the designed CAR-T can express and secrete TREM2scFv, and the TREM2scFv secreted in the tumor microenvironment can enhance the antigen presenting capacity of dendritic cells and macrophages, promote the activation of CAR-T cells and reduce the failure of the CAR-T cells. The chimeric antigen receptor T cell provided by the invention can obviously improve the anti-tumor capacity of the CAR-T cell in vivo.

More preferably, the tumor antigen is MLSN.

The invention also provides a gene sequence of the chimeric antigen receptor for encoding the autocrine TREM2scFv, which is shown as SEQ ID NO: 1 is shown.

The invention also provides a lentivirus vector containing the gene sequence.

The invention also provides cells expressing the chimeric antigen receptor, wherein the cells are T cells or cell groups containing the T cells.

The present invention also provides a method for producing the above cell, which comprises the steps of:

(1) obtaining a nucleic acid sequence of a chimeric antigen receptor which can specifically secrete TREM2scFv and targets a tumor antigen;

(2) recombining a nucleic acid sequence of a chimeric antigen receptor which can specifically secrete TREM2scFv and target tumor antigen into a lentivirus expression vector to obtain a lentivirus expression plasmid;

(3) transfecting the expression plasmid into 293T cells; packaging to obtain virus particles, and performing centrifugal concentration to obtain a lentivirus concentrated solution;

(4) infecting the T lymphocytes with the lentivirus concentrate to obtain chimeric antigen receptor modified T lymphocytes that can be derived from TREM2scFv and targeted to a tumor antigen.

The invention also provides a medicine for targeted therapy of tumors, which contains the chimeric antigen receptor or can express the chimeric antigen receptor.

Preferably, the tumor is a solid tumor or a hematological tumor.

In a particular embodiment of this use provided herein, the tumor is non-small cell lung cancer. Notably, the CAR of the invention is characterized in that it comprises a secretory TREM2 scFv.

Compared with the prior art, the invention has the following beneficial effects:

the chimeric antigen receptor cell capable of autocrine TREM2scFv can specifically recognize and kill tumor cells highly expressing tumor-associated antigens, and at the same time autocrine TREM2 scFv. Through TREM2scFv secreted in a tumor microenvironment, the antigen presenting capacity of dendritic cells and macrophages can be enhanced, the activation of CAR-T cells is promoted, the failure of the CAR-T cells is reduced, further, CAR-T is promoted to secrete killing substances such as granzyme and perforin, and finally, the purpose of improving the anti-tumor capacity of the CAR-T cells is achieved.

Drawings

FIG. 1 is a design of the molecular structure of MSLN CAR-TREM2-T cells;

FIG. 2 shows that MSLN CAR-TREM2-T cells produce large amounts of anti-TREM2 scFv;

FIG. 3 shows that the lysis rate of PC-9 cells by MSLN CAR-TREM2-T cells is higher than that of MSLN CAR-T cells;

FIG. 4 shows that MSLN CAR-TREM2-T cells can increase survival of PC-9 tumor-bearing mice.

Detailed Description

The following further describes the embodiments of the present invention. It should be noted that the description of the embodiments is provided to help understanding of the present invention, but the present invention is not limited thereto. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

The test methods used in the following experimental examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.

Example 1 construction of CAR plasmid

The chimeric antigen receptor provided by the invention is formed by connecting a CD8 alpha signal peptide, MSLN scFv, a CD8 hinge region, a CD8 transmembrane region, a 4-1BB intracellular signal region, a CD3 zeta intracellular signal region, a T2A self-splicing sequence, an antibody secretion signal peptide and TREM2scFv in series, and the structure is shown in figure 1; the gene sequence of the anti-TREM2 single-chain antibody (anti-TREM 2 scFv) is from a monoclonal antibody sequence constructed in the laboratory, and is subjected to codon optimization to ensure that the gene is more suitable for expression of T cells under the condition of no change of an encoding amino acid sequence, and the gene sequence information is shown as a sequence table SEQ ID NO. 1. After the sequence was designed, the Shanghai Czeri Bio Inc. was commissioned for synthesis.

After completion of the synthesis of MSLN CAR-TREM 2scFv, it was subcloned into the plasmid of pCDH-CMV-MCS-EF1-copGFP containing CD19-CAR-T, replacing the anti-CD 19 scFv in CD19 CAR, to obtain a plasmid of pCDH-CMV-MCS-EF1-copGFP containing MSLN CAR-TREM2 scFv.

Example 2 packaging and titration of CAR lentiviruses

Using the plasmid of pCDH-CMV-MCS-EF1-copGFP containing MSLN CAR-TREM 2scFv constructed above, a lentivirus comprising the sequence of MSLN CAR-TREM 2scFv was further prepared.

Lentiviruses were packaged using a three plasmid system for subsequent experiments. The three plasmid system was the plasmid of pCDH-CMV-MCS-EF1-copGFP, the plasmid of psPAX2 and the plasmid of pMD2.G, respectively, containing MSLN CAR-TREM2 scFv. In the present invention, 293T cells are used for packaging lentiviruses. The virus in the supernatant of 293T medium is collected, and then the virus can be used for infection of target cells through ultra-high speed centrifugation, heavy suspension and subsequent lentivirus titer titration. The target cells for infection were 293T cells: the 293T cells were plated in microwell plates until they grew to a density of 70% -80%.

The specific operation of packaging lentiviruses in this example is as follows:

(1) 293T packaging cells at 1.3-1.5X10⁵The cells are seeded at a concentration per ml in the seeding medium in the culture plate. Cells were incubated overnight. After about 24 hours, the cells should be about 70% confluent.

(2) Transfection of packaging cells: a mixture of 3 transfection plasmids was prepared: pmd2.g plasmid, psPAX2 plasmid, recombinant plasmid, and OptiMEM medium.

(3) Lipofectamine 2,000 diluted with OptiMEM: 10 μ l Lipofectamine +90 μ l OptiMEM. Lipofectamine reagent is added dropwise and mixing is performed by rotating the tip or flicking the tube (not by pipetting or vortexing); incubate at room temperature for 5 minutes.

(4) The 3 plasmid mixtures were added drop-wise to the diluted Lipofectamine reagent and mixed by rotating the tip or flicking the tube.

(5) The transfection mixture was incubated at room temperature for 5-40 minutes.

(6) Carefully transferring the transfection mixture into packaging cells in an inoculation medium, which may be sensitive to perturbation; care was taken not to remove the cells from the culture dish.

(7) Cells were incubated overnight and the medium was changed to remove transfection reagents.

(8) Cells were incubated overnight.

(9) The lentivirus-containing medium was harvested approximately 40 hours after transfection. The medium is transferred to a storage tube.

(10) Repeating the virus harvesting every 12-24 hours, and replacing with 6ml of harvest culture medium, wherein the virus titer tends to decrease in the later harvest period; discarding the packaging cells after the final harvest; the viral harvests can be combined as desired.

(11) The virus-containing medium was spun at 1,250rpm for 5 minutes to pellet all packaging cells collected during harvest.

(12) The virus can be stored at 4 ℃ for short periods (hours to days), but should be frozen at-20 ℃ or-80 ℃ for long-term storage. To reduce the number of freeze/thaw cycles, please dispense large-scale virus preparations into smaller storage tubes prior to long-term storage.

By the above-described lentivirus packaging, we obtained lentiviruses containing the gene of interest. Since different organelles are infected at different multiplicity, it is necessary to further determine the titer of the lentiviral vector. Since lentiviruses are fluorescently labeled, the percentage of fluorescent cells and thus lentivirus titer can be determined using a microscope. The specific procedure for titration of lentivirus titres in this example is as follows:

(1) 293T cells were seeded into each well of a 6-well dish.

(2) Cells were incubated overnight.

(3) If freshly harvested virus is used, filtration through a 0.45 μm polyethersulfone filter may be performed to remove cells and debris.

(4) In freeze-thaw cycles, lentivirus titers decrease. If the virus is to be frozen and aliquoted, titers must be determined from the frozen stock to account for the titer loss associated with freeze-thawing.

(5) If frozen virus is used, an aliquot of the lentivirus is rapidly thawed at 37 ℃ with agitation in warm water.

(6) Lentiviral dilutions were prepared in DMEM containing 10. mu.g/mL polyethylene. The diluent was mixed well.

(7) The medium was gently aspirated from the cells.

(8) To each well was added 1.5mL of virus diluent (one well per well, one well remaining).

(9) The cells in the remaining wells were counted, and cell counts were required to calculate titers.

(10) And incubating for 48-72 hours.

(11) The medium was gently aspirated and replaced with 1mL PBS.

(12) The percentage of fluorescence positive cells in each well was calculated.

When calculating titers, only wells with less than 40% fluorescence positive cells were considered. The titration method assumes 1 integration event per unit. When the percentage exceeds 40%, cells with multiple integration events may be counted, which may result in underestimating true titer.

Example 3 preparation of CAR immune cells

After the titer of the lentivirus is determined through the steps, the lentivirus can be used for infecting immune cells, and then the immune cells which stably express the chimeric antigen receptor can be obtained; in this example, T cells are preferred as the infected cells. The specific steps for infecting cells with lentiviruses are as follows:

(1) coating of 48-well plate: in order to allow the lentiviruses to infect T cells more easily, the plates were pre-coated with Retronection in this experiment. Retronection was diluted to 15 μ g/ml using PBS, then 150 μ l was pipetted into 48 well plates and incubated overnight at 4 ℃. The next day, the Retronection solution was aspirated and washed 2 times with PBS for use.

(2) T cells activated for 48 hours using CD3 and CD28 were collected into 15ml centrifuge tubes and then resuspended using serum-free media. Add resuspended T cells to the above-described coated 48-well plate and adjust the T cell density to 1-5X10⁶Individual cells/ml.

(3) Adding the packaged CAR-T lentivirus into a micropore plate according to the MOI of 5, and gently mixing uniformly; the plate was then centrifuged at 300Xg for 60 minutes using a centrifuge. After centrifugation, the plate was returned to the incubator for culture.

(4) After the T cells continued to be cultured for about 2 days, the old medium was changed to new, and the CAR-T cells were tested for positive rate at different time points of the subsequent culture of T cells.

Example 4 detection of secretion of TREM2scFv by CAR-T cells

To compare TREM2scFv secretion from T, MSLN CAR-TREM2-T cells, T, MSLN CAR-TREM2-T prepared in example were cultured for 3 days, the supernatant was recovered, and TREM2scFv was assayed by ELISA. The detection result shows that the TREM2scFv is not detected by the control group T and the MSLN CAR-T, and the TREM2 cytokine is detected by the MSLN CAR-TREM2-T and is more than 300ng/ml (figure 2). It was also further verified that MSLN CAR-TREM2-T can secrete TREM2 scFv.

Example 5 detection of CAR-T cell tumor killing Capacity

To compare the differences in killing capacity of T, MSLN CAR-TREM2-T cells against tumor cells, we used CytoTox

And (3) detecting the release amount of LDH in the supernatant after the incubation of the CAR-T cells and the tumor wash white by a non-radioactive cytotoxicity detection method. Cytotox

The non-radioactive cytotoxicity detection is a detection method based on a colorimetric method, and can quantitatively measure Lactate Dehydrogenase (LDH). LDH is a stable cytosolic enzyme that is released upon cell lysis. The more LDH released into the supernatant, the more cells lysed. In addition, in this example, PC-9 cells are preferred as the cells to be killed. The specific operation steps are as follows:

(1) comparison: experimental controls included: effector cell spontaneous LDH release: correcting the LDH released by effector cells spontaneously; target cell spontaneous LDH release: correcting the spontaneous release of LDH from the target cells; maximum LDH release from target cells: this control is required for calculation to determine 100% LDH release; volume correction control: correcting for volume changes due to addition of lysate (10 ×); media background control: correcting for background uptake by LDH activity produced by serum in the medium and phenol red; LDH positive control: 2ul of positive control was added to 10ml PBS + 1% BSA.

(2) Experiment hole: a fixed number of tumor cells were added to a 96-well plate, followed by different numbers of effector cells or exosomes. The final volume is not less than 100 ul/hole; then, the cells are centrifuged for 4min at a rotation speed of 250g, so that the effective target cells are fully contacted.

(3) Culturing the culture system for 4-8 hours; at 45min before collection, 10ul of lysate was added to the maximum LDH control wells of the target cells.

(4) After 4 hours, centrifuge at 250g for 4 min.

(5) Then, 50ul of the supernatant was aspirated and added to a new 96-well plate.

(6) Adding 50ul of substrate, wrapping with tinfoil paper, and incubating at room temperature for 30 min.

(7) 50ul of stop solution was added.

(8) Puncture large bubbles and detect in 492nm within 1 h.

(9) And (3) calculating: cytotoxicity ═ (experiment-effector cells spontaneous-target cells white hairs)/(target cells maximal one target cell spontaneous) X100%. The results showed that MSLN CAR-TREM2-T cells were the most potent for lysis of PC-9 cells (FIG. 3).

Example 6 anti-tumor Effect of CAR-T cells in vivo

To compare the differences in the anti-tumor effects of T, MSLN CAR-TREM2-T cells in vivo, we implanted PC-9 cells into mice, infused different cells into different groups of mice, respectively, and observed the differences in survival rates of the mice. The specific operation is as follows:

(1) cell preparation: culturing PC-9 lung cancer cell to logarithmic growth phase, when lung cancer cell grows to 90% fusion degree, digesting with pancreatin, collecting cell, washing with PBS 3 times, and densifying cell with PBSAdjusted to 1x10⁷One/ml for standby.

(2) Animal preparation: 5-6 week-old BalB/c mice were divided into groups: t cell panel, MSLN CAR-TREM2-T cell panel.

(3) Using a 1ml injection needle, 100. mu.l of lung cancer cells were inoculated into the left lower groin of the mouse. The volume size of the mouse tumor was measured daily. When the tumor volume reaches 50 to 100mm³Thereafter, the mice were given an injection of the drug. Tumor volume 1/2x length (mm) x width (mm)²。

(4) Using an insulin needle, 200. mu.l of the corresponding cells were injected into the T cell group, the MSLN CAR-T cell group, and the MSLN CAR-TREM2-T cell group, respectively.

(5) The day of drug injection was recorded as day 0, and the mice were observed daily for changes in body weight and the like, and their death was recorded.

The results showed that mice injected with MSLN CAR-TREM2-T cells had the lowest mortality rate (FIG. 4).

The embodiments of the present invention have been described in detail, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, and the scope of protection is still within the scope of the invention.

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Claims

1. An autocrine TREM2scFv chimeric antigen receptor comprising a signal peptide, a single chain variable fragment capable of binding a tumor antigen, a hinge region, a transmembrane domain, a costimulatory molecule, an immunoreceptor tyrosine activation motif, a self-cleaving peptide fragment, a single chain variable fragment of an anti-TREM2 antibody.

2. The chimeric antigen receptor autocrine of TREM2scFv according to claim 1, wherein the tumor antigen is selected from AFP, BCMA, B7H4, CD52, CD56, CD80, CDK4/m, CEA, CT, DAM, EGFR, ErbB3, ELF2M, EMMPRIN, EpCam, G250, GAGE, GnTV, HAGE, HER2, HPVE7, HSP70, hTERT, iCE, IGF1R, IL2R, IL5, LAGE, LDLR/FUT, MAGE, MART1, MART2, Mesothelin, MUCl, PRAME, PSMA, RAGE, SAGE, TGFf3, TPI/m, VEGF, WTL, MLSN.

3. The gene sequence encoding the chimeric antigen receptor of autocrine TREM2scFv of claim 1, characterized by the amino acid sequence as set forth in SEQ ID NO: 1 is shown.

4. A lentiviral vector comprising the gene sequence of claim 3.

5. The cell expressing the chimeric antigen receptor of claim 1, wherein said cell is a T cell or a population of T cell-containing cells.

6. The method for producing the cell according to claim 5, comprising the steps of:

7. A medicament for the targeted treatment of tumours, said medicament comprising or capable of expressing a chimeric antigen receptor according to claim 1.

8. The medicament of claim 7, wherein the tumor is a solid tumor or a hematological tumor.