CN114426952A

CN114426952A - T cell potentiators for CAR T cell therapy of leukemia and methods of obtaining potentiated T cells

Info

Publication number: CN114426952A
Application number: CN202011176773.7A
Authority: CN
Inventors: 魏海明; 粘志刚; 郑小虎; 孙汭; 田志刚
Original assignee: University of Science and Technology of China USTC
Current assignee: University of Science and Technology of China USTC
Priority date: 2020-10-29
Filing date: 2020-10-29
Publication date: 2022-05-03
Also published as: WO2022088555A1

Abstract

The present invention relates to T cell potentiating agents for use in CAR T cell therapy against hematological tumors such as acute myeloid leukemia, and methods of using the same to enhance the effect of CAR T cell therapy. The methods include treating T cells used in CAR T therapy with a T cell potentiating agent during in vitro culture. The T cell synergist disclosed by the invention can enhance migration of CAR T/NK cells to bone marrow and improve tumor clearing effect of CAR T cell therapy.

Description

T cell potentiators for CAR T cell therapy of leukemia and methods of obtaining potentiated T cells

Technical Field

The present invention relates to Chimeric Antigen Receptor (CAR) T cell therapies against tumors, in particular to T cell potentiators for use in CAR T cell therapies against hematological tumors such as acute myeloid leukemia, and methods of using the same to enhance the effects of CAR T cell therapies.

Background

Acute Myeloid Leukemia (AML) is a tumor derived from bone marrow and most patients relapse, although chemotherapy can induce remission rates as high as 70%. ((Bishop,1997)), myeloid leukemia cells are an intramedullary recurrence when more than 5% of the cells in the bone marrow and an extramedullary recurrence (usually the CNS and testis) if they occur in the extramedullary fraction, with pediatric leukemias occurring in about half of the extramedullary recurrence and adult leukemias occurring in 95% of the total recurrence. Whether the leukemia stem cells in the bone marrow can be eliminated is a key factor for treating the success or failure of acute myelogenous leukemia.

The use of CD 19-targeted chimeric antigen receptor T cells for the treatment of Acute Lymphoblastic Leukemia (ALL) has been reported to be surprisingly effective (Maude et al, 2014). CAR T cell therapy is shown to have great potential for the treatment of hematological diseases, especially hematological tumors. To date, targets such as CD123, CD33, CLL1 have been clinically validated as potential targets for acute myeloid leukemia (Kenderian et al, 2015; Mardiros et al, 2013; Wang et al, 2018). However, cell therapies using CAR T cells designed to these sites did not achieve satisfactory results in treating AML, i.e., there were no reports of CAR T cell therapies achieving significant effects in treating AML.

As to how CAR T cells are difficult to act in AML, the inventors have come to the opinion after the study that important reasons affecting CAR T onset are that CAR T cells are difficult to enter tumor sites and that CAR T entering the body is difficult to persist in the body. Several clinical studies exist to date suggesting that migration of CAR T cells into the bone marrow is the basis for influencing the therapeutic effect of CAR T cells. It is reported that the more CAR T cells used for treatment migrate to the bone marrow, the better the treatment of the patient (ritchai et al, 2013; Wang et al, 2015). Therefore, increasing the ability of CAR T cells to migrate to the tumor site during therapy is one direction of research to improve the therapeutic efficacy of CAR T cells.

Another key factor determining the efficacy of CAR T cells is whether CAR T cells persist in vivo, and short term CAR T cell survival in vivo often results in poor therapeutic efficacy. Thus, there is a need for a method of improving survival of CAR T cells in vivo.

Summary of The Invention

The in vitro construction of CAR T cells involves the proliferative differentiation of T cells. In this process, the PI3K-Akt/mTOR signaling pathway plays a key role in T cell proliferation, survival, migration, and differentiation of effector/memory subsets. Among them, signals transmitted to T cells by CD3/CD28 antibody magnetic beads, IL2 cytokine, CAR structure and the like can cause the activation of PI3K-Akt/mTOR signaling pathway. Excessive activation of the PI3K-Akt/mTOR signaling pathway promotes the formation of short-lived end effector T cells and down-regulates the expression of chemokine receptors such as CD62L, CCR7 and CXCR4 (Arojo et al, 2018; Sinclair et al, 2008). However, it has been reported that over-expression of chemokine receptors in CAR T cells promotes migration of CAR T cells into tumors (Di Stasi et al, 2009; Moon et al, 2011).

Rapamycin was produced by streptomyces hygroscopicus, first isolated in 1972 by surland radard and colleagues from samples of streptomyces hygroscopicus in easter island. This compound was originally named rapamycin (Sirolimus) under the original name of rapaunous island. Sirolimus was originally developed as an antifungal agent. However, this use was abandoned due to the discovery of its ability to inhibit mTOR, its powerful immunosuppressive and antiproliferative properties. Approved by the U.S. food and drug administration for marketing at 9 months 1999, and sold by the company Peucedanum (formerly Hewlett-packard) under the trade name Rapamune.

Rapamycin is a macrolide compound that is used clinically as an immunosuppressant for the prevention of organ transplant rejection and for the treatment of a rare pulmonary disease known as lymphangioleiomyomatosis. It is used in the treatment concentration (10-30 mug. L)^-1) Has immunosuppressive function in human body, and is especially useful for preventing kidney transplant rejection. As to its mechanism of action, it is generally considered to reduce the leukocyte mediation of T cells and B cells by mTOR inhibitionSensitivity to IL 2(IL-2) and thus the ability to inhibit activation of T and B cells.

Previous studies by the inventors have found that expression of CXCR4 is reduced during the in vitro construction of CAR T cells, and more CAR T cells with a short lifespan are produced.

The inventors have conducted intensive studies and found, by transcriptome analysis, that the PI3K-AKT/mTOR signaling pathway is significantly activated in CAR T cells. It was finally found that during CAR T cell construction, by adding the classical inhibitor rapamycin of mTOR to the culture system, the expression of CXCR4 could be effectively upregulated and longer-lived CAR T cells were generated. The CAR T cells thus obtained can migrate more efficiently to the bone marrow and survive longer in vivo, resulting in better therapeutic efficacy for leukemia.

The present invention relates to a CAR T cell therapy for targeting tumors, preferably hematological tumors (leukemias), comprising the use of an inhibitor of the PI3K-AKT/mTOR signaling pathway as a T cell potentiator, thereby enhancing bone marrow migration and prolonging survival in vivo of CAR T/NK cells, in particular, the PI3K-AKT/mTOR signaling pathway inhibitor is rapamycin.

In one embodiment, over-activation of PI3K-Akt/mTOR during CAR T cell production in vitro can be attenuated, expression of CXCR4 is upregulated, and killing of CAR T on myeloid AML can be enhanced by addition of rapamycin at appropriate concentrations, e.g., 20nM, during culture. Furthermore, the anti-cancer activity of CAR T cells in AML treatment can be further enhanced by increasing the proportion of Tscm cells, down-regulating the expression of depletion markers, thereby increasing the in vivo survival time of potent CAR T cells.

The invention relates to the use of rapamycin as a T-cell potentiator in the preparation of a medicament for the treatment of hematological tumors by use in combination with CAR T-cell therapy, and to the use of a cell for use in said CAR T-cell therapy for the treatment, to pharmaceutical compositions containing such a cell. By using rapamycin, expression of CXCR4 on CAR T cells can be enhanced, the ability of CAR T cells to migrate to bone marrow is increased, elimination of tumor cells in bone marrow is accelerated, and the efficacy of CAR T cells is improved, resulting in more CAR T cells with longer life span.

The present invention specifically includes the following.

Use of an inhibitor of the PI3K-AKT/mTOR signaling pathway as a T cell potentiator for the treatment of CAR T cells or a population of cells containing CAR T cells, wherein,

the cell therapy is a chimeric antigen receptor T/NK cell therapy and the PI3K-AKT/mTOR signaling pathway inhibitor is, for example, LY294002 or rapamycin, preferably rapamycin.

2. Use according to claim 1, wherein the CAR T-cells or the population of cells containing CAR T-cells are preferably activated by a cytokine, wherein the cytokine treats the T-cells simultaneously with the PI3K-AKT/mTOR signaling pathway inhibitor, or before or after the PI3K-AKT/mTOR signaling pathway inhibitor treats the T-cells.

3. A method of potentiating a CAR T cell or a cell population containing a CAR T cell comprising culturing a T cell or a cell population containing a T cell, preferably activated by a cytokine, in a medium supplemented with a PI3K-AKT/mTOR signaling pathway inhibitor, wherein the cytokine is treated with the T cell simultaneously with, or before or after treatment of the T cell with a PI3K-AKT/mTOR signaling pathway inhibitor, e.g. LY294002 or rapamycin, preferably rapamycin.

4. The method according to item 3, the cytokines are IL-2, IL7 and IL15, preferably IL-2.

5. The method according to item 3 or 4, wherein the final concentration of rapamycin in the medium is 5, 10, 20, 30, 40nM, preferably 20nM, preferably the rapamycin treatment time is not less than 3 days, more preferably not less than 5 days.

6. A synergistic CAR T cell prepared by the method of any one of items 3-5.

7. The method of any of claims 3-5 or 6, wherein the CAR is a human EpCAM chimeric antigen receptor and has an amino acid sequence as set forth in SEQ ID No: 2, respectively.

8. Use of the use of

item

1 or 2, or the method of any one of items 3-5, or the CAR T cell of item 6, or the method of item 7 or the CAR T cell in the manufacture of a medicament for the treatment of a tumor, preferably leukemia.

9. A pharmaceutical composition comprising a CAR T cell obtained by the method of any of items 3-5, or the method of item 7.

Brief Description of Drawings

Figure 1 flow-through assay of expression of CXCR4 in initially isolated T cells and CAR T cells.

Figure 2 flow-through assay of phosphorylation of the Mtor signaling pathway in CAR T cells.

Figure 3 flow-through map of expression levels of CXCR4 of rapamycin treated CAR T cells.

FIG. 4 transwell migration experiments in which rapamycin treatment enhanced chemotaxis of CAR T cells in response to CXCL 12.

FIG. 5 flow assay of bone marrow migration of rapamycin treated CAR T cells.

FIG. 6 flow assay plot of CAR T cell subpopulation ratios and expression of depletion markers

Figure 7 clearance of myeloid AML by rapamycin treated EpCAM CAR T cells. (a) Fluorescence imaging detects changes in the tumor. (b) Tumor burden representative plot for fluorescence imaging in mice (n-8). (c) Total fluorescence value statistical plots. (d) And counting total fluorescence values of bone marrow parts. (e) Kaplan-Meier survival analysis. (f) Bone marrow cross-sectional histochemical staining for anti-human EpCAM antibody.

FIG. 8 rapamycin treatment enhanced the anti-tumor capacity of CD33CAR T cells. (a) Fluorescence imaging detects changes in the tumor. (b) Tumor burden representative of fluorescence imaging in mice (n-6). (c) Total fluorescence value statistical plots.

Detailed Description

Although rapamycin is used in the examples described below, other drugs which inhibit the PI3K-AKT/mTOR signaling pathway, such as LY294002, may be used. When such a drug is used, it is administered at a concentration equivalent to the effect of raising CXCR4 of T cells by administering rapamycin at 5-40 nM.

For the rapamycin concentrations used, the cells were incubated at a final concentration of 20nM in the examples described below, but may be at concentrations of 5, 10, 20, 30, 40nM or in the range between these.

For the timing of the addition of the PI3K-AKT/mTOR signaling pathway inhibitor to the cell culture, it can be added at any stage of the in vitro culture of the T cells or T cell containing population, either simultaneously with the addition of a cytokine such as IL-2 or after a period of IL-2 addition to the culture medium as described in the examples.

Cytokines that can be used in the present invention may be, for example, in addition to IL-2: IL7 and IL15, preferably IL-2, or IL-2 in combination with these.

Example 1 construction and preparation of EpCAM CAR T cells and CD33CAR T cells

In this example EpCAM CAR T cells and CD33CAR T cells were prepared, both cells being identical in terms of the procedures and reagents except for the different CAR chimeric sequences.

CAR chimeric sequence of EpCAM CAR T cells: the murine anti-human EpCAM scFv was ligated to the CD8 transmembrane region, 41bb co-stimulatory domain and CD3 zeta to obtain a murine anti-human EpCAM chimeric sequence (AE4 scFV-CD8-CD28-CD3 zeta, sequence shown in SEQ ID No: 1) and inserted into PCDH-MSCV-MCS-EF1-copGFP (available from Addgene).

CAR chimeric sequence of CD33CAR T cells: the humanized anti-human CD33 scFv was ligated to the CD8 transmembrane region, 41bb co-stimulatory domain and CD3 zeta to obtain a humanized anti-human CD33 chimeric sequence (SEQ ID NO: 3), which was inserted into PCDH-MSCV-MCS-EF1-copGFP (available from Addgene Co., Ltd.)

Next, 3 plasmids of the following table were transfected into 293T cells (purchased from Shanghai cell Bank of China academy of sciences) using PEI (Polyscience,23966) according to the protocol, and cell culture supernatants of 48h and 72h were collected to transduce the T cells, respectively.

Mononuclear cells were isolated from fresh human peripheral blood (from the blood center of Anhui province) by Ficoll density gradient centrifugation, and T cells were isolated using the CD 3T cell positive separation kit (Miltenyi, 30-097-043). The initially isolated T cells were split into two, one for flow assay and the other for construction of CAR T cells. Hereinafter, unless otherwise specified, the day on which the initial separation was performed was defined as day 0.

For the isolated T cells used to construct each set of CAR T cells, X-VIVO15 medium (lonza, BE02-060F) supplemented with 5% human AB serum (GEMINI,100-512), 2mmol/L glutamine at day 0 at 5X 10⁵The T cells were resuspended at a concentration of/ml and anti-CD 3/CD28 Dynabeads (thermo, 11161D) were added in a number ratio of 1: 1. IL-2 was added simultaneously with the addition of anti-CD 3/CD28 Dynabeads to give a final IL-2 concentration of 100U/ml, and IL-2 was supplied every two days.

After 24 hours of stimulation, concentrated lentivirus obtained from the supernatant of the cell culture broth collected above by centrifugation at 50000g, 4 ℃ for 2 hours was added to activated T cells at MOI 50 and polybrene (Sigma, H9268) at a final concentration of 8ng/ml according to the polybrene protocol, centrifuged at 720g, 32 ℃ for 1 hour, and replaced after 6-8 hours, and rapamycin (Sigma, V900930-1MG) at a final concentration of 20nM was added, and cultured in an incubator at 37 ℃ with rapamycin supplemented every two days, while a control group not treated with rapamycin was established. Cell culture was monitored daily during transduction and complete X-VIVO15 medium was added (to maintain cell concentration at 0.5-1X 10)⁶cells/mL. On day 4 post transduction, Dynabeads were removed. Activated T cells, EpCAM CAR T cells and CD33CAR T cells were harvested after day 5 for subsequent analysis and in vivo experiments, respectively.

Example 2 chemokine Down-Regulation, mTOR activation of CAR T cells after in vitro culture

And (3) determination groups: t cells initially isolated in example 1 (denoted as T), and EpCAM CAR T cells after 5 days in vitro culture in example 1 (denoted as CAR T).

Flow cytometric assay

The cells to be assayed were detected using a BD LSRII flow cytometer and analyzed using Flowjo V10.

In the assay of this example, anti-human antibody EpCAM from biolenged, (324208), anti-human CXCR4 from eBioscience (12-9999-42), anti-human mTOR from BD (583489), anti-human S6 from CST (14733S), anti-human CD62L from BD (555544), and anti-human CD45RO from BD (560607) were used.

The EpCAM T cell lines harvested in vitro were washed once in phosphate buffered saline supplemented with 2% fetal bovine serum and incubated with mouse serum for 30min, stained at 4 ℃ in the dark after blocking Fc receptors, washed twice with PBS and tested on the machine (fig. 1, 2, T cells from the same source were connected in a straight line to CAR T cells).

Specifically, for intracellular staining, the cell was immobilized and permeabilized with ebiscience-based immobilized transmembrane solution (Invitrogen, cat # 00-5521-00), then intracellular antibody was added in an amount recommended by the manufacturer's instructions, and the cell was stained in the dark for 1 hour, and the transmembrane solution and PBS were washed once and then subjected to flow cytometry (BD LSRII) detection.

The results show that expression of CXCR4 was significantly down-regulated in CAR T cells after 5 days in vitro culture compared to the original isolated T cells; phosphorylation of ribosomal S6 protein was significantly upregulated compared to mTOR in CAR T cells after 5 days in vitro culture. The ribosomal S6 protein is a protein downstream of mTORC1(mTOR complex 1). The ribosome S6 protein includes several subtypes, and p 70S 6K is phosphorylated and activated by upstream regulatory factor such as mammalian mTOR to form p-p 70S 6K.

The results suggest that after preparation as CAR T cells, the ability of T cells to migrate to the bone marrow is reduced and there is activation of the mTOR pathway, suggesting that this may be one of the reasons for poor therapeutic effect of CAR T cells on AML.

Example 3 Effect of rapamycin treatment on chemokines of EpCAM CAR T cells

And (3) determination groups: EpCAM CAR T cells cultured in vitro and treated with rapamycin starting at 24h (indicated as + Rapa), EpCAM CAR T cells cultured in vitro but not treated with rapamycin (indicated as-Rapa). Sample collection time point: days 3, 6, 9, 12.

Concentration screening: t cells were isolated as in example 1 and lentivirus transfected, CAR T cells were treated with 0nM,5nM,10nM,20nM,40nM rapamycin, respectively, and expressed CXCR4 flow-assayed 6 days after incubation. The results are shown in FIG. 3.

The results show that: 5nM,10nM,20nM, and 40nM can be used to treat CAR T cells, with 20nM being the optimal concentration for the most efficient upregulation of CXCR4 expression.

T cells were isolated as in example 1 and subjected to lentiviral transfection and rapamycin (20nM) treatment, resulting in rapamycin-treated CAR T cells (hereinafter also referred to as boosted CAR T cells) and CAR T cells not treated with rapamycin (control). Where cells were harvested on days 3, 6, 9, 12 of culture after lentiviral transfection, and CAR T cells not treated with rapamycin and treated with rapamycin were flow tested using CXCR4 antibody (as in example 1). Sample preparation and flow assay methods were the same as in example 1, and the results of flow assay of expression levels of CXCR4 from CAR T cells not treated with rapamycin and CAR T cells treated with rapamycin are shown in figure 3.

The results show that: the expression of CXCR4 (CXCR4 MFI) was significantly higher in the rapamycin treated group than in the untreated group at time points of days 3, 6, 9, 12, more than doubled at days 3, 6, 9, 12.

The results suggest that the ability of T cells to migrate to bone marrow during the preparation of CAR T cells was significantly reduced after T cells were activated by antibodies to CD3 and CD28 and infected with viruses, whereas by treatment with rapamycin, T cells with improved ability to migrate to bone marrow could be obtained, and even excellent synergistic T cells with higher ability to migrate to bone marrow than the initially isolated T cells could be obtained. Such T cells are presumed to be able to enter tumor sites more efficiently, and an increased therapeutic effect thereof can be expected.

Example 4T cell potentiation Transwell migration assay under chemotaxis of CXCL12

To further investigate the migratory capacity of the synergistic EpCAM CAR T cells obtained with the T cell potentiators of the present invention, the response of the synergistic CAR T cells to CXCL12 chemotaxis was examined using a transwell (Costar corporation) with a pore size of 5 μm.

And (3) determination groups: EpCAM CAR T cells isolated, cultured in vitro and treated with rapamycin (denoted as + Rapa), EpCAM CAR T cells not treated with rapamycin (denoted as-Rapa), as in example 1, were divided into a group (denoted as + CXCL12) to which CXCL12(SDF-1) was added in the lower chamber and a group (denoted as-CXCL 12) to which CXCL12(SDF-1) was not added, to give a total of 4 groups.

In the test, 500ul of RPMI 1640 complete medium containing 100ng/ml (CXCL12) SDF-1 (Aibixin, abs01114) is added into a 24-well plate; the upper chamber was then placed into the well, followed by 200ul of synergistic CAR T cells (1X 10) labeled with cellTrace bead (thermo, C34571) as described in the specification⁵) Add to the transwell upper chamber. Each group of cells was incubated at 37 ℃ for 4h with 5% CO 2.

The lower chamber cells were collected, centrifuged at 300g at 4 ℃ for 5min, the supernatant discarded and resuspended in 200ul of precooled PBS buffer, 1X 10 cells per sample⁵The CAR T cells not labeled with cellTrace violet of (1) were subjected to flow cytometry in the same manner as in example 1 (fig. 4).

The results show that: there was no significant difference in the ability of CAR T cells from the rapamycin-untreated group and rapamycin-treated group to migrate toward the lower chamber between the two groups to which the chemokine CXCL12(SDF-1) was not added; whereas between the two groups with CXCL12(SDF-1) added, there were more cells in the rapamycin treated group that were able to migrate to the lower chamber compared to CAR T cells in the non-rapamycin treated group (a, indicating p < 0.01).

CXCL12(SDF-1) is a ligand for CXCR 4. The above results suggest that CAR T cells potentiated by rapamycin treatment can respond better to chemotaxis of the cytokine CXCL12(SDF-1), which is a ligand of CXCR4, and then migrate, further demonstrating that the potentiated CAR T cells of the present invention have enhanced bone marrow migration ability and are expected to have increased therapeutic effects.

Example 5 migration tracking of synergistic CAR T cells in immunodeficient mice

To demonstrate in vivo bone marrow migration of the synergistic CAR T cells treated with the T cell potentiators of the invention, a migration follow-up of the synergistic EpCAM CAR T cells in immunodeficient mice was performed.

And (3) determination groups: EpCAM CAR T cells isolated, cultured in vitro, and treated with rapamycin (+ Rapa) and EpCAM CAR T cells not treated with rapamycin (-Rapa) as in example 1.

Immunodeficient female NCG mice (6-10 weeks, available from Jiejicaokang) were used and divided into two groups of 4 mice each, and 5X 10 mice were transfused into the tail vein⁶Or a CAR T cell not treated with rapamycin. Mice were harvested 7 days after infusion, peripheral blood and bone marrow were collected, and flow assays were performed as above after lysing red blood cells using RBC lysate (Biolegend, 420301) as described herein (fig. 5).

The results show that: as can be seen from figure 5, there was no significant difference (ns) in the number of non-rapamycin treated CAR T cells and rapamycin treated groups in Peripheral Blood (PB) in mice harvested on day 7 after CAR T cell transfusion. But in Bone Marrow (BM), the proportion of rapamycin-treated CAR T cells was (0.38), significantly higher than that of the group not treated with rapamycin (0.10), approaching 4-fold (×, p < 0.01); when the amount was counted, the number of rapamycin treated CAR T cells was 175043 (numerical value), which is significantly higher than the number 77790 of the group not treated with rapamycin. It was further confirmed by in vivo animal testing in immunodeficient mice that the bone marrow migratory capacity of CAR T cells potentiated by treatment with rapamycin was indeed enhanced.

Example 6 reduced expression of depletion markers in synergistic CAR T cells

T cells can be divided into four subpopulations of Tem, Tcm, Tscm, Teff by CD62L and CD45RO, where Tcm, Tscm represent a long-lived subpopulation, especially the Tscm subpopulation. The length of survival and decline in efficacy of CAR T cells in vivo can be measured by detecting the expression of their depletion markers. When the level of expression of the depletion marker is higher, it indicates that such CAR T cells have a short survival time in vivo and poor therapeutic activity. Thus in this example the synergistic EpCAM CAR T cell subsets Tem, Tcm, Tsccm, Teff were divided and the expression of the typical depletion markers PD-1 and Tim3 were examined.

Cell isolation, in vitro culture of CAR T cells was performed as in example 1, where both groups were tested 12 days after in vitro expansion. The proportion of CAR T cell subsets (Tem, Tcm, Tscm, Teff) was measured using flow assay, which were divided using CD45RO and CD62L (antibodies used see description of example 1). The proportion of CAR T cells positive for the expression of the exhaustion marker PD-1, Tim3 was measured separately by flow (figure 6, CAR T cells from the same source were connected by straight lines).

The results show that the proportion of the Tscm subpopulation was significantly increased in the rapamycin treated group compared to CAR T cells not treated with rapamycin. An increase in the Tsccm subpopulation indicates that more long-lived cells were generated in the synergized cells. Compared to CAR T cells not treated with rapamycin, expression of the CAR T depletion markers PD-1 and Tim3 was significantly reduced in the rapamycin treated group, with statistically poor differences in PD-1 (P ═ 0.0628) and statistical differences in Tim3 (indicates P < 0.05). The above results demonstrate that the CAR T cells obtained are of higher quality and longer life-span by treatment with the potentiator rapamycin of the invention.

Example 5 in vivo anti-tumor assay of enhanced EpCAM CAR T cells

First, immunodeficient mice were used to test for the anti-tumor capacity of the enhanced EpCAM CAR T cells in vivo.

And (3) determination groups: PBS (negative control group), EpCAM CAR T cells not treated with rapamycin, EpCAM CAR T cells treated with rapamycin.

Female NCG mice (6-10 weeks, purchased from synergestic) were used to establish tail vein transfusion leukemia xenograft models (Kenderian et al, 2015) and tumors were identified and grouped on day 5 to ensure consistent mean fluorescence intensity for each group. Then tail vein infusion of EpCAM CAR T (infusion volume: 1X 10)⁶Cells/time), tumor burden was detected weekly by intraperitoneal injection of 15mg/ml fluorescein potassium salt (Gold biotechnology, lock) for fluorescence imaging,the processing schedule and the test results are shown in fig. 7.

Specifically, it comprises tail vein transfusion of NCG mice with 1 × 10⁶HL60 (purchased from shanghai cell bank of chinese academy of sciences) stably expressing luciferase leukemia cells induced into tumors, and after determining the tumors on day 5, the mice were divided into three groups of 8 mice each, so that the average fluorescence remained consistent. 1X 10 respective tail vein transfusions⁶EpCAM CAR T and 1X 10 not treated with rapamycin⁶Rapamycin treated EpCAM CAR T in quantity, equal volume of PBS. Fluorescence imaging was then performed on days 5, 9, 16, and 23 to detect changes in the tumor. The processing schedule is shown in fig. 7 a. A representative plot of tumor burden (n-8) and a statistical plot of total fluorescence values for fluorescence imaging in mice are shown in fig. 7 b.

Further, statistics of total fluorescence values at the bone marrow sites (FIG. 7d) and (FIG. 7c) were performed on day 23 of injection induction by Kaplan-

Meier survival analysis (fig. 7 e). Bone marrow tissue of mice was fixed overnight in 4% paraformaldehyde solution, decalcified, and then subjected to tissue embedding and sectioning, followed by histochemical staining using anti-human EpCAM antibody (abcam, ab223582) and anti-human CD3 antibody (CST, #85061) (fig. 7 f).

The results show that, based on the tumor burden fluorescence plots and the corresponding total fluorescence statistics, it can be seen that the tumors in the rapamycin-treated mice were significantly lower at day 9 compared to the non-rapamycin-treated CAR T cells, and at day 23, almost no tumor was observed, and it can be seen that the synergistic CAR T cells of the present invention have a better elimination effect. Statistics of total fluorescence values show quantification of tumor residual amounts, and it can be seen that in mice injected with HL60, the fluorescence values at each time point for both CAR T cell groups were lower than the PBS group, with the total fluorescence values for the rapamycin treated group being lower than the rapamycin untreated group, and by day 23, the total fluorescence values in the rapamycin treated group were significantly lower.

The above results indicate that EpCAM CAR T cells treated with rapamycin are able to more effectively clear tumors in an iv leukemia xenograft model in vivo.

Further, statistics of total fluorescence at bone marrow sites (BM) showed no significant decrease in fluorescence at bone marrow sites in the group of CAR T cells not treated with rapamycin compared to the PBS group on day 23, whereas significant decrease in fluorescence at bone marrow sites in the group of CAR T cells treated with rapamycin suggested that tumor clearance at bone marrow sites was achieved. Bone marrow cross sectional histochemical staining with anti-human EpCAM antibody showed that there was almost no EpCAM positive AML in the bone marrow of the rapamycin treated EpCAM CAR T group, while there was still EpCAM positive tumor cell residue in the rapamycin untreated group.

The Kaplan-Meier survival analysis results showed 75% survival for the rapamycin treated EpCAM CAR T cell treated group, 100% survival for the rapamycin treated EpCAM CAR T cell treated group, 25% survival for the rapamycin treated EpCAM CAR T cell treated group, and 75% survival for the rapamycin treated EpCAM CAR T cell treated group on day 42, day 40. It is thus seen that rapamycin-treated EpCAM CAR T cells significantly prolonged the survival time of mice compared to PBS group, non-rapamycin-treated EpCAM CAR T cells (p <0.001 and p <0.05, respectively). The above results demonstrate that rapamycin treated EpCAM CAR T cells can better clear myeloid AML and more effectively treat AML.

Example 6 in vivo anti-tumor assay of synergistic CD33CAR T cells

Then, the in vivo antitumor ability of rapamycin-treated CD33CAR T cells was examined using immunodeficient mice in the same method and procedure as example 5.

And (3) determination groups: PBS (negative control group), CD33CAR T cells without rapamycin treatment, CD33CAR T cells with rapamycin treatment

1X 10 infusion of the NCG rat tail vein as in example 5⁶HL60, tumors were determined on day 5 and mice were divided into three groups of 6 mice each, ensuring consistent mean fluorescence intensity. Then transfusing 1X 10 tail vein respectively⁶CD33CAR T and 1X 10 without rapamycin treatment⁶Rapamycin treated CD33CAR T cells, equal volume of PBS. Then used for 5, 16, 23, 30 daysTumor changes were detected by fluorescence imaging with an animal imager (PerkinElmer). The processing schedule is shown in fig. 8 a. A representative plot of tumor burden (n-6) for fluorescence imaging in mice is shown in fig. 8b, and a statistical plot of total fluorescence values is shown in fig. 8 c.

The results show that, according to the tumor burden fluorescence map and the statistics of the corresponding total fluorescence value, compared with the CAR T cell group without rapamycin treatment, the tumor fluorescence in the rapamycin-treated mice is obviously lower on day 16, and almost no tumor can be observed on day 30, so that the synergistic CAR T cells are known to have better elimination effect. Statistics of total fluorescence values show quantification of tumor residual amounts, and it can be seen that in mice injected with HL60, the fluorescence values at each time point for both CAR T cell groups were lower than the PBS group, with the total fluorescence values for the rapamycin treated group being lower than the rapamycin untreated group, and by day 30, the total fluorescence values in the rapamycin treated group were close to 0.

The above results indicate that CD33CAR T cells treated with rapamycin are also able to clear tumors in an iv leukemia xenograft model more effectively in vivo than before treatment.

The above results demonstrate that by treating cells used in cell therapy for anti-tumor using rapamycin as a T cell potentiator, CAR T cells can be obtained that enhance the effect of anti-tumor, particularly AML, and that such a method can be applied to EpCAM targets, as well as to other targets for treating leukemia such as CD 33.

Therefore, the T cell potentiating agent for use in CAR T cell therapy against hematological tumors such as acute myeloid leukemia of the present invention, and the method for enhancing the effect of CAR T cell therapy using the same are effective, and the potentiating T cell obtained by such method can be used for the preparation of antitumor drugs based on various sites, with wide applicability.

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Claims

Use of an inhibitor of the PI3K-AKT/mTOR signaling pathway as a T cell potentiator for the treatment of CAR T cells or a population of cells containing CAR T cells, wherein,

the cell therapy is a chimeric antigen receptor T/NK cell therapy and the PI3K-AKT/mTOR signaling pathway inhibitor is, for example, LY294002 or rapamycin, preferably rapamycin.
2. Use according to claim 1, wherein the CAR T-cells or the cell population containing CAR T-cells are preferably activated by a cytokine, wherein the cytokine treats the T-cells simultaneously with the PI3K-AKT/mTOR signaling pathway inhibitor, or before or after the PI3K-AKT/mTOR signaling pathway inhibitor treats the T-cells.
3. A method of potentiating a CAR T cell or a cell population containing a CAR T cell comprising culturing a T cell or a cell population containing a T cell, preferably activated by a cytokine, in a medium supplemented with a PI3K-AKT/mTOR signaling pathway inhibitor, wherein the cytokine is treated with the T cell simultaneously with, or before or after treatment of the T cell with a PI3K-AKT/mTOR signaling pathway inhibitor, e.g. LY294002 or rapamycin, preferably rapamycin.
4. The method according to claim 3, wherein the cytokines are IL-2, IL7 and IL15, preferably IL-2.
5. The method according to claim 3 or 4, wherein the final concentration of rapamycin in the culture medium is 5, 10, 20, 30, 40nM, preferably 20nM, preferably the rapamycin treatment time is not less than 3 days, more preferably not less than 5 days.
6. A synergistic CAR T cell prepared by the method of any one of claims 3-5.
7. The method of any of claims 3-5 or the CAR T cell of claim 6, wherein the CAR is a human EpCAM chimeric antigen receptor having an amino acid sequence as set forth in SEQ ID No: 2, respectively.
8. Use according to claim 1 or 2, or the method of any one of claims 3 to 5, or the CAR T cell of claim 6, or the method of claim 7 or the use of a CAR T cell in the manufacture of a medicament for the treatment of a tumour, preferably leukaemia.
9. A pharmaceutical composition comprising a CAR T cell obtained by the method of any one of claims 3-5, or the method of claim 7.