CN109134467B

CN109134467B - Linker arm for controlling activation/inhibition of chimeric antigen receptor T cells and application thereof

Info

Publication number: CN109134467B
Application number: CN201810900635.5A
Authority: CN
Inventors: 张博; 张烜; 李凌君; 周德敏; 王妍; 黄申龙
Original assignee: Peking Union Medical College Hospital Chinese Academy of Medical Sciences
Current assignee: Peking Union Medical College Hospital Chinese Academy of Medical Sciences
Priority date: 2018-08-09
Filing date: 2018-08-09
Publication date: 2022-01-21
Anticipated expiration: 2038-08-09
Also published as: CN109134467A; CN114805356A

Abstract

The invention discloses a connecting arm for controlling CAR-T activation/inhibition, which comprises a targeting molecule capable of recognizing target cells at one end, a part with biological orthogonality capable of being recognized by specific CAR-T cells at the other end, and a chemical group capable of being subjected to biological orthogonal fragmentation is coupled in the middle. The invention can realize the rapid and flexible conversion of the CAR-T cells from an activated state to a resting state, and has systematicness and high efficiency on the inhibition of the CAR-T cells. The regulation is reversible, does not damage or kill the existing CAR-T cells, can realize the flexible conversion of CAR-T cells from activation to deactivation to activation, and does not affect the overall effect of the treatment.

Description

Linker arm for controlling activation/inhibition of chimeric antigen receptor T cells and application thereof

Technical Field

The invention belongs to the field of cellular immunotherapy, and relates to a technology capable of regulating chimeric antigen receptor T cells (CAR-T) in a switching mode and an application method.

Background

CAR-T technology has become a critical technology for cellular immunotherapy and has promising applications in such areas as cancer, autoimmune diseases, and antiviral therapies. In normal production, T cells can effectively activate T cells to kill tumor cells by firstly using MHC-I molecules to present tumor cell surface specific markers as first signals and then using co-stimulation such as CD28 and the like as second signals for recognizing the tumor cells. This condition often fails to effectively activate immune T cells, thereby making tumor cells susceptible to immune escape. The CAR-T cell technology is to combine an antibody capable of recognizing a certain tumor antigen with the intracellular part of CD3 zeta chain or fcsri gamma to form a fusion gene, and then transfect the patient's own T cells by gene transduction to express a Chimeric Antigen Receptor (CAR). After the patient's T cells are transduced, a large number of tumor-specific T cells, CAR-T cells, will be generated to fight the tumor cells.

The first generation CARs consisted of a single chain antibody recognizing a tumor surface antigen and an immunoreceptor tyrosine activation sequence (CD3 zeta chain or fcsri gamma). First generation CARs, lacking costimulatory signals for T cell activation, are not very effective in mimicking endogenous T cell activation processes, and therefore cause only transient T cell proliferation and low levels of cytokine secretion, and do not provide long lasting T cell expansion and anti-tumor effects. To more effectively mimic the activation process of endogenous T cells, second and third generation CARs were subsequently developed that introduced the signal sequence of costimulatory molecules, such as CD28 and 4-1BB, in the design. The designed CAR-T cells have greatly improved in vivo proliferation, survival time, cytokine release capacity and cytotoxicity, and can produce in vivo lasting and effective tumor cell scavenging effect.

Although CAR-T technology has achieved good clinical efficacy, controllability is a major application problem. When the CAR-T cell contacts with a target cell in vivo, the CAR-T cell can cause obvious cytotoxic effect and a large amount of cytokine secretion, and is manifested as side reaction phenomena such as high fever, nervous disorder and the like, and can endanger life in severe cases. How to effectively activate and close the CAR-T cells is a key problem to be solved urgently in the field. The existing research mainly focuses on the following three aspects: (1) introduction of suicide gene. CAR-T can be effectively switched off by introducing a suicide gene. The existing suicide genes comprise HSV-TK genes, caspase9 genes and the like, the genes and CAR genes are introduced into T cells together to construct CAR-T cells capable of inducing apoptosis by small molecules, and when the T cells are excessively reacted, the excessively activated CAR-T cells are immediately killed by adding an inducer, so that toxic and side effects are reduced (Brown et al, 2014); (2) and (4) introducing a switch connector. The intermediate linker arm is designed such that the CAR-T cells recognize one end of the linker arm and the other end of the linker arm recognizes the target cell (Cartellieri et al, 2016; Rodgers et al, 2016; Tamada et al, 2012; Urbanska et al, 2012). The activation of the CAR-T cell under the design depends on the addition of the connecting arm, and is dose-dependent, and the CAR-T cell can be effectively regulated and controlled in the aspect of activation through the intermediate connecting arm; (3) and (3) introducing an antibody. Blocking of specific cytokines, such as IL-6 and IL-1, with specific antibodies may be effective against Cytokine Release Syndrome (CRS) by CAR-T cells in some clinical cases.

In practical applications, the ideal CAR-T cell regulatory pattern should include efficacy, immediacy, and reversibility. Although the above method for introducing suicide gene can effectively close CAR-T cells, irreversible cell loss can bring inevitable damage to the therapeutic effect, thereby limiting the clinical application; on the other hand, the introduction of a switch linker as described above, while very effective in controlling the activation of CAR-T cells, the turning off of CAR-T cells is dependent on the metabolism of the switch compound. In the presence of target cells, the switch compounds preferentially bind to the target cells, thereby delaying their metabolism in vivo, while antibody-based switch compounds themselves have a longer half-life. Thus, this approach, while very effective in controlling CAR-T cell activation, does not provide immediate and effective shut-down of CAR-T cells; antibody-based control methods are clinically more favored over the two approaches described above. However, the CRS effect caused by CAR-T cells is caused by the co-release of multiple factors, so that the effect of controlling one factor alone cannot achieve the systemic CRS inhibition effect. In conclusion, although some studies have been made to solve the CAR-T control problem to some extent, they have only improved (turned off or activated) one way and have not yet achieved an effective, immediate and reversible CAR-T cell control effect. There is currently no theoretical approach to effectively achieve the above regulatory effects on CAR-T cells.

Disclosure of Invention

In order to solve the above problems, the present invention provides a linker arm for controlling CAR-T activation/inhibition.

The invention provides a connecting arm for controlling CAR-T activation/inhibition, which comprises a targeting molecule capable of recognizing target cells at one end, a part with biological orthogonality capable of being recognized by specific CAR-T cells at the other end, and a chemical group capable of being cleaved by biological orthogonality in the middle.

In one embodiment of the invention, the targeting molecule recognizes a target cell. Preferably, the targeting molecules capable of recognizing the target cells are macromolecular or small molecular compounds with cell targeting. More preferably, the targeting molecule includes, but is not limited to, folic acid, antibodies, antibody fragments, and tumor targeting polysaccharides, peptides, etc. Alpha-folate receptors are expressed in about 50% of tumor cells, and especially highly expressed in breast, lung, uterine and brain cancer-related cells, but less expressed in normal tissues. But tumor marker-based screens, such as Her2 antibodies and fragments thereof specific for breast cancer cells; antibodies and antibody fragments against EGFR of cells such as colorectal cancer and head and neck squamous cell carcinoma; and antibodies and fragments thereof against B cell surface specific CD19, CD20, and the like.

In one embodiment of the invention, the linker arm contains a moiety with bio-orthogonality that is recognized by specific CAR-T cells and forms a specific orthogonal pair with the extracellular region of the CAR-T cell. Fluorescein Isothiocyanate (FITC) and its specific antibody have been demonstrated to have good specificity and orthogonality in vivo, and further, biotin/avidin or biotin antibody, azide/azide specific antibody or alkyne compound, nitrobenzene/nitrobenzene specific antibody, or the rest of the reactive pairs having good specificity and orthogonality in biological systems can be used as the specific recognition region for linking arm molecules and T cells. The part can be specifically and orthogonally recognized by the extracellular region of the constructed T cell, and the specific T cell can also code the antibody or other affinity fragments of the recognized part. Such moieties include, but are not limited to, FITC, biotin, azide, small molecules of nitrobenzene, and small peptides, proteins, nucleic acids, or biological macromolecules of polysaccharides. Correspondingly, the T cells include, but are not limited to, antibodies, antibody fragments, and other molecules with specific affinity that specifically recognize FITC or other bio-orthogonal moieties.

In one embodiment of the invention, the chemical group that can be cleaved is any chemical group that can be artificially induced to cleave. The chemical groups which can be broken have breaking reaction of various principles, and can be broken by being excited by ultraviolet, infrared, blue light and the like with different wavelengths; fragmentation can also be induced by small molecules, acid-base pH changes, temperature changes, etc. The fracture can also be induced by ultrasound, nuclear magnetism, radiation and the like. Such as o-nitrobenzyl ester, which can cleave two intermolecular bonds within the group under UV light at 365nm, thereby separating the chemical groups on either side of the cleaved group. Although the cleavable chemical group used in the present invention is ortho-nitrobenzyl ester, the cleavable chemical group principle described in the present invention includes, but is not limited to, ultraviolet cleavable, infrared cleavable, blue light irradiation cleavable, and small molecule cleavable, etc.; specific cleavable chemical groups include, but are not limited to, ortho nitrobenzyl esters cleaved by ultraviolet light, tetrazines induced cleaved trans cyclooctene, and the like. Specific cleavable chemical groups include, but are not limited to, ortho nitrobenzyl esters cleaved by ultraviolet light, tetrazines induced cleaved trans cyclooctene, and the like.

The invention also provides therapeutic agents containing said linker arms for controlling CAR-T activation/inhibition.

The invention also provides application of the connecting arm for controlling CAR-T activation/inhibition in preparation of medicines for treating diseases.

The invention also provides a method for controlling CAR-T activation/inhibition, which comprises coupling CAR-T cells and target cells through the connecting arm so as to activate the CAR-T cells; through a controllable fragmentation reaction, the CAR-T cell and a target cell are subjected to instant and effective fragmentation, and side effects of the CAR-T cell are further inhibited.

According to the invention, the bioorthogonal fragmentation technology is introduced into the design and application of CAR-T, and the bifunctional connecting arm containing the orthogonal fragmentation group is synthesized, so that the flexibility and the effectiveness in CAR-T cell control are improved, and the CAR-T cell can be controlled in an on-off manner. One end of the designed bifunctional connecting arm contains a target molecule which can identify target cells, such as small molecule target heads of antibody molecules or folic acid and the like; the other end is designed to contain a group with bio-orthogonality for recognition by specific CAR-T cells, such as Fluorescein Isothiocyanate (FITC) or biotin; the intermediate is coupled with a chemical group that can be cleaved bioorthogonally, such as a UV cleavable group or a small molecule cleavable group. Thus, activation of CAR-T will depend on the addition of a linker molecule, and the shutting down of CAR-T can also be terminated by specific chemical reactions.

Advantages of the invention over other approaches may be realized in one or more of the following:

the regulation has the characteristics of instantaneity, high efficiency and flexibility, and can realize the rapid and flexible conversion of the CAR-T cells from an activated state to a resting state;

the inhibition of CAR-T cells is systemic and highly effective. Directly separating the CAR-T cells from the target cells, and further performing systemic inhibition on the activation effect generated by the contact of the CAR-T cells and secreted cytokines.

The regulation has reversibility, does not damage or kill the existing CAR-T cells, can realize the flexible conversion of the CAR-T cells from activation to shutdown to activation, and does not influence the overall effect of treatment;

by means of the characteristics of real-time, high efficiency and reversibility, the 'switch' type ideal regulation and control mode of the CAR-T cell can be realized.

Drawings

FIG. 1 shows a scheme for the synthesis of Folate-NBE-FITC.

FIG. 2 shows Folate-NBE-FITC targeting validation. Screening FR positive cell strains; Folate-NBE-FITC targeting FR high expression cell line validation.

FIG. 3 shows the anti-FITC CAR-T cell construction. A. Construction of a second generation CAR-T lentiviral vector; validation of car structural expression; CAR-T specific recognition Folate-NBE-FITC validation.

FIG. 4 shows the validation of the effect of fragmentation at the cellular level of Folate-NBE-FITC.

FIG. 5 shows the optimization of the fragmentation conditions at the cellular level of Folate-NBE-FITC. A. The fragmentation efficiency was optimized with time conditions, with KB cells above and CAR-T cells below; B. the trypan blue method detects the influence of the illumination time on the cell viability.

FIG. 6 shows Folate-NBE-FITC-mediated binding and dissociation of CAR-T cells to KB cells. A. Flow cytometry results of binding and dissociation; B. fluorescence image and bright field image of double positive cell cluster.

FIG. 7 shows Folate-NBE-FITC-mediated CAR-T cell activation. CAR-T cells can be dose-dependently activated by Folate-NBE-FITC; car-T cell activation surface marker detection; Folate-NBE-FITC-mediated IFN- γ (left) and IL-2 (right) release assays from CAR-T cells; detection of Folate-NBE-FITC-mediated CAR-T cell aggregation.

FIG. 8 shows Folate-NBE-FITC-mediated modulation of CAR-T cell activity in vitro. A. A model design flow chart; b.0-31 hour cytotoxic effect profile; c.0-31 h IL-2 accumulation trend graph; d.0-31 h IFN-gamma accumulation trend graph; detection of the CAR-T cell aggregation Effect mediated by Folate-NBE-FITC addition/fragmentation.

FIG. 9 shows Folate-NBE-FITC-mediated anti-tumor effect of CAR-T cells in vivo in diffuse tumor mice.

FIG. 10 shows Folate-NBE-FITC-mediated anti-tumor effect of CAR-T cells in vivo in diffuse tumor mice. A. A model design flow chart; B. a mouse bioluminescence imaging result; C. a time trend change graph of the tumor-bearing volume of the mouse; D. UV transdermal cleavage Folate-NBE-FITC molecular validation.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

Example 1

A controlled-cleavage bifunctional linker arm based on FITC, Folate, and o-nitrobenzyl ester (Folate-NBE-FITC) was synthesized, which consists essentially of three parts: (1) FITC acts as a linking ligand, binding to CAR-T cells that specifically recognize FITC. FITC, as a fluorescent molecule, has been proven to have good bio-orthogonality and biocompatibility in vivo; (2) folic acid is used as a target head part and targets folic acid high-expression tumor cells. The existing results show that the alpha type folate receptor is fully expressed on about 50 percent of tumor cells, but is rarely expressed in normal tissues, so that the folate molecule can be used as an ideal target for model verification; (3) ortho-nitrobenzyl esters (NBE) with controlled cleavage by UV light. The group can be rapidly and efficiently broken under the condition of biocompatible ultraviolet illumination (UV365nm), and the model verification is facilitated.

As shown in FIG. 1, a 200ml round bottom flask was charged with 1, 4-dibromobutane (21.6g, 10mmol, 1eq), 50ml DMF was added, and sodium azide I (16.5g, 21mmol, 2.1eq dissolved in 25ml water) was added. The reaction was then transferred to 80 ℃ for 20 hours. After the reaction was completed, the reaction mixture was diluted with 200ml of brine and extracted with n-hexane (200 ml. times.3), and then the organic phase was dried over anhydrous sodium sulfate. The solvent was evaporated to dryness to give 2(1, 4-diazidebutane) (13.72g, 98%). 1H NMR (400MHz, CDCl3) δ 3.30(t, J ═ 6.8Hz,2H),2.74(t, J ═ 6.7Hz,2H), 1.70-1.60 (m,2H),1.53(dt, J ═ 14.2,7.0Hz,2H).

A200 mL round bottom flask was charged with 1, 4-diazidebutane (4.2g, 30mmol, 1eq), 1M HCl (60mL), diethyl ether (20mL), ethyl acetate (20mL) was added, then cooled to 0 deg.C and triphenylphosphine (7.86g, 30mmol, 1eq) was added slowly over a period of one hour. The reaction was then transferred to 21 ℃ and stirred for 20 hours. After the reaction was completed, the aqueous phase and the organic phase were separated, the aqueous phase was washed with ether (100 ml. times.3) to remove the generated triphenoxyphosphorus oxide, the aqueous phase was adjusted to pH 13 with a newly prepared aqueous solution of sodium hydroxide, and then extracted with dichloromethane (200 ml. times.3), and the organic phase was dried over anhydrous magnesium sulfate and evaporated to give 4-azidobutylamine as a pale yellow oily liquid 3(2.74g, 24mmol, 80%). 1H NMR (400MHz in CDCl 3). delta.3.29 (s,2H),1.64(s,2H).

A25 mL round bottom flask was charged with fluorescein isothiocyanate (100mg, 0.25mmol, 1eq)5ml DMSO, 4-azidobutylamine (34.2mg, 0.3mmol, 1.2eq) DIPEA (64.63mg, 0.5mmol, 2 eq). Stirring is then carried out for 6 hours at room temperature, TLC monitoring is carried out, DMSO is dried in a spinning mode after the reaction is finished, and then the pure compound bright yellow solid 4(40 percent) is obtained through column chromatography

A50 ml flask was weighed with 4-methoxy-3-hydroxyacetophenone (3.32g, 20mmol, 1eq), added with solvent DMF 15ml, added with potassium carbonate (2.76g, 20mmol, 1eq) bromopropyne (2.38g, 20mmol, 1eq) and then reacted at room temperature for 1 hour. After completion of the reaction the solvent was evaporated to dryness and extracted with dichloromethane (200 ml. times.3), the organic phase was dried over anhydrous sodium sulfate, and the yield 6(4.0g 98%) of the dried white solid was evaporated

A100 mL round bottom flask was taken and 10mL of concentrated nitric acid was added, followed by 5mL of acetic anhydride with ice bath and stirring for 30min 6(2.17g, 12mmol) dissolved in 2.5mL of acetic anhydride was slowly added to the solution. The reaction was stirred for an additional 5 hours and then poured into 200ml of ice water. Then extracted 3 times with dichloromethane and the resulting organic solution was dried over anhydrous sodium sulfate and concentrated in vacuo. Recrystallization from ethanol gave product 7(1.63g, 61%).

A50 mL flask was taken, 7(1.2g, 4.9mmol) was weighed into THF (10mL) solution to neutralize methanol (20mL), then the reaction was transferred to an ice bath, sodium tetrahydroborate (1.2g, 34mmol) was slowly added to the solution and stirred for 3 hours. After the reaction is finished, the solvent is removed by vacuum concentration, and then H is added₂O (50mL) and 2M HCl (5mL) were added and the solution was extracted with chloroform (3X 100 mL). The organic phase was dried over anhydrous sodium sulfate and the solvent was removed under reduced pressure to give (99%) light yellow solid 8.

A100 mL round-bottomed flask was taken, and compound 8(1.25g, 5mmol) was weighed, followed by addition of pyridine (5mL), and transfer to an ice bath where 4-nitrophenyl chloroformate (1.2g, 6mmol) was added. The reaction was then transferred to room temperature and stirred for 6 hours, then with NaHCO₃(10mL) quench. The reaction mixture was extracted with ethyl acetate, and the organic layer was washed with brine (10mL), MgSO₄Drying and vacuum concentrating. Purification by column chromatography on silica gel then gave 9(1.35g, 65%).

A dry 50ml round bottom flask was taken and ethylenediamine (180mg, 3mmol) and DIPEA (623ul, 4mmol) were added and then 9(832mg, 2mmol) was weighed out and dissolved in 5ml dichloromethane. After stirring in the room the temperature was overnight and the reaction mixture was concentrated to dryness in vacuo. The residue was purified by silica gel chromatography (EtOAc/hexanes ═ 1/1) to give 10(573mg, 84%)

A dried 50ml round bottom flask was taken, folic acid (300mg, 0.68mmol, 1eq) was weighed out and added to dry DMSO solvent under nitrogen protection, then NHS (86mg, 0.748mmol, 1.1eq) DCC (154.22mg, 0.748mmol, 1.1eq) was added and the reaction was stirred under conditions for 4 hours. After the reaction was completed, the reaction mixture was filtered to remove DCU formed, and then 10(229.16mg, 0.68mmol, 1eq) was added to the filtrate and stirred under nitrogen for 28 hours in the dark. After the reaction, the reaction solution was dropwise added to a solution prepared by cooling to 0 ℃ in ether/acetone (7/3) to precipitate an orange-yellow precipitate, which was then filtered and washed three times with dichloromethane to remove unreacted 10, twice with distilled water, and then dried. After drying was completed, the mixture was placed in a round-bottom flask, dissolved by adding dry DMSO10ml, and then added with 1(343.4mg, 0.68mmol, 1eq), copper sulfate pentahydrate (17mg, 0.068mmol, 0.1eq) sodium ascorbate (53.85mg, 0.272, 0.4eq) and reacted under nitrogen for two days. After the reaction was completed, the reaction solution was dropwise added to a solution which was cooled to 0 degrees with ethyl ether/acetone (7/3), washed three times with dichloromethane to remove unreacted 1, washed twice with distilled water, and then purified by HPLC to give 11.

Folate-NBE-FITC targeting was verified. First, Folate Receptor (FR) high-expression tumor cell lines KB and SKOV3, and FR low-expression cell lines A549 and 4T1 (FIG. 2-A) were screened by flow cytometry. And then incubating the Folate-NBE-FITC with cells, washing, labeling with an APC (antigen-binding plasma) fluorescence labeled secondary antibody for identifying FITC molecules, and detecting that the Folate-NBE-FITC specifically identifies the FR high-expression tumor cell strain, but rarely or not combined with the FR low-expression cell strain through flow cytometry (figure 2-B), thereby proving that the Folate-NBE-FITC has targeting property. On the other hand, it was also demonstrated that after the Folate-NBE-FITC molecule binds to the target cell, the FITC molecule is exposed and can bind to the antibody as well as the CAR-T cell.

Example 2

CAR-T cells recognizing FITC molecules (anti-FITC-CAR-T) were constructed. The constructed gene fragment was constructed into a lentiviral vector by fusing the FITC-recognizing single chain antibody gene sequence to the CD8 hinge and transmembrane regions, 4-1BB and CD3 zeta region (CAR construct) (FIG. 3-A). And infecting human CD3+ primary T cells by the packaged lentivirus vector to enable CD3+ T cells to express CAR structures, thereby obtaining CAR-T cells which can specifically recognize FITC molecules and can be specifically activated by the FITC molecules. We examined the expression of CAR structure on the cell surface with APC-labeled Anti-mouse-Fab antibody (FIG. 3-B), and confirmed that Anti-FITC-scFv was correctly folded on the T cell surface by double labeling with Anti-mouse-Fab-APC antibody, and successfully generated CAR-T cells specifically recognizing Folate-NBE-FITC (FIG. 3-C).

Folate-NBE-FITC was demonstrated to be cleavable at the CAR-T cell and target cell levels, confirming that it can be efficiently cleaved by UV illumination at 365nm on KB cells (FIG. 4-A) as well as CAR-T cells (FIG. 4-B).

By optimizing the fragmentation conditions of Folate-NBE-FITC at the cellular level, optimal conditions for fragmenting the linker arm at the cellular level to switch off CAR-T cell activity were obtained. By exploring the photodisruption conditions on CAR-T cells and KB cells with different light intensities and different illumination times (fig. 5), it was confirmed that at low uv power (milliwatt level), within a short time (CAR-T cells 10 min, KB cells 20 min), a fast and efficient disruption of Folate-NBE-FITC could be achieved at the cellular level, and the reaction was biocompatible.

It was demonstrated that a cleavable small molecule-based switch could mediate the binding and dissociation of CAR-T cells to target cells. By co-incubating the APC-labeled CAR-T cells and the PE-labeled KB cells and by an imaging flow system, it is visually shown that the CAR-T cells and the KB cells can be combined by adding Folate-NBE-FITC; and both cells were dissociated by breaking the Folate-NBE-FITC by light (FIG. 6-A). By fluorescence analysis of the cell clusters, CAR-T cell binding to KB cells could be clearly seen, and images of dissociation achieved by illumination (fig. 6-B).

Example 3

CAR-T cells were co-incubated with KB cells, and by adding different concentrations of Folate-NBE-FITC, it was verified that CAR-T cells were efficiently activated by Folate-NBE-FITC with an EC50 equal to about 85pM (fig. 7-a); the T cell activation surface markers CD25 and CD69 also increased significantly with the addition of Folate-NBE-FITC (FIG. 7-B); cytokine release levels consistent with cytotoxic effects were significantly activated by Folate-NBE-FITC and exhibited dose-dependent effects (FIG. 7-C); in addition, it was clearly observed by microscopy that only the experimental group in which CAR-T cells were co-incubated with KB cells exhibited a clear T cell clumping effect, whereas none of the remaining control groups exhibited a clear phenomenon. This protocol demonstrates that Folate-NBE-FITC can mediate efficient activation of CAR-T cells and has a dose-dependent effect.

Example 4

From in vitro cellular experiments, it was demonstrated that CAR-T can be "switch-mode" regulated on the basis of cleavable switching compounds. CAR-T cells and target cells were co-incubated and a Folate-NBE-FITC-mediated CAR-T regulation study was performed as shown in the protocol (FIG. 8-A). The results show that Folate-NBE-FITC can effectively mediate the killing of CAR-T cells to target cells in 0-4 hours; after the bond breaking reaction is carried out in 4 hours, the 'closing' effect is successfully carried out on the CAR-T cells, so that the cytotoxic effect of the CAR-T cells is obviously inhibited; on the other hand, after the 21 st hour re-addition of Folate-NBE-FITC, CAR-T cells were successfully reactivated to restore normal cytotoxicity (FIG. 8-B). IL-2 and IFN- γ release levels were consistent with the results of cytotoxic effects (FIG. 8-C, D); on the other hand, during the experiment, the clustering effect of CAR-T cells was also found to be consistent with the addition-fragmentation-addition of Folate-NBE-FITC by direct microscopic observation of the experimental groups (FIG. 8-E). Taken together, this result demonstrates that the Folate-NBE-FITC molecule can effectively mediate the "switch" mode activity modulation of CAR-T.

Example 5

It was verified that activation of CAR-T cells in vivo could be effectively dependent on the addition of Folate-NBE-FITC and exhibited a dose-dependent effect by the mouse's fringe tumor model (FIG. 9).

From the subcutaneous tumor-bearing model in mice, it was demonstrated that CAR-T can be "switch-mode" regulated in vivo based on cleavable switch compounds. The specific experimental procedure is shown in FIG. 10-A. In a model experiment of subcutaneous tumor-bearing mice, it was observed that the addition of Folate-NBE-FITC effectively inhibited tumor growth relative to a control group (group 1) to which Folate-NBE-FITC was not added (group 2); after fragmentation of Folate-NBE-FITC by irradiation, tumor cell growth reached control levels at the last monitoring point on day 19 due to significant inhibition of CAR-T cell effects (group 3); in particular, in group 4, it was clearly observed that the activity of CAR-T cells was reactivated by re-supplementation with Folate-NBE-FITC after the disruption, and that the inhibitory effect on tumor cells was restored (FIG. 10-B). By measuring the tumor cell volume every other day, the size of the tumor-bearing volume was plotted against time, and it was observed that the trend changes between groups with the addition-fragmentation-addition of Folate-NBE-FITC were consistent with the animal imaging data (FIG. 10-C). On the other hand, it was confirmed by in vitro transdermal experiments that UV365nm ultraviolet light can penetrate the epidermis to cleave Folate-NBE-FITC at the cellular level, demonstrating that the above-mentioned regulation phenomenon is achieved based on the cleavage reaction (FIG. 10-D).

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the technical principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. A linker arm for controlling CAR-T activation/inhibition, comprising a targeting molecule capable of recognizing a target cell at one end and a moiety capable of recognizing a specific CAR-T cell and having bio-orthogonality at the other end, forming a specific orthogonal pair with an extracellular domain of a CAR-T cell, and coupling the targeting molecule capable of recognizing a target cell with a chemical group capable of being cleaved by bio-orthogonality, wherein the targeting molecule capable of recognizing a target cell is folic acid, the moiety capable of recognizing a specific CAR-T cell and having bio-orthogonality is fluorescein isothiocyanate, and the chemical group capable of being cleaved by bio-orthogonality is o-nitrobenzyl ester group.

2. A therapeutic agent comprising the linker arm of claim 1 for controlling CAR-T activation/inhibition.

3. Use of a linker arm according to claim 1 for controlling CAR-T activation/inhibition in the manufacture of a medicament for the treatment of a disease.

4. A method of controlling CAR-T activation/inhibition for non-disease treatment purposes by coupling a CAR-T cell and a target cell via the linker arm of claim 1, thereby activating the CAR-T cell; through a controllable fragmentation reaction, the CAR-T cell and a target cell are subjected to instant and effective fragmentation, and side effects of the CAR-T cell are further inhibited.