CN116904400B

CN116904400B - Application of calicheamicin in optimization of in-vitro CAR/TCR-T cell product preparation process

Info

Publication number: CN116904400B
Application number: CN202311173465.2A
Authority: CN
Inventors: 胡以国; 孙媛媛; 王嘉欣
Original assignee: Chengdu Yibang Pharmaceutical Technology Co ltd
Current assignee: Chengdu Yibang Pharmaceutical Technology Co ltd
Priority date: 2023-09-12
Filing date: 2023-09-12
Publication date: 2023-12-01
Anticipated expiration: 2043-09-12
Also published as: CN116904400A

Abstract

The invention relates to the technical field of genetically modified T cell medicines, in particular to application of calicheamicin in optimization of in-vitro CAR/TCR-T cell product preparation process: genetically modified T cells including CAR-T cells and TCR-T cells are subjected to in vitro culture for a certain time before reinfusion, and a certain concentration of the colimycin is added into a culture system, so that the depletion process of the genetically modified T cells can be effectively slowed down, the expression level of cytokines can be improved, the expression level of immunosuppressive receptors can be inhibited, and CD4 can be improved ⁺ And CD8 ⁺ The ratio of the T cells and the central memory T cell group ratio, thereby improving the reinfusion survival rate and the tumor killing effect of the genetically modified T cells. The technical scheme can solve the technical problem that the treatment effect of the CAR-T/TCR-T cell therapy is not ideal due to factors such as T cell activity, T cell exhaustion and the like, and has ideal application and popularization prospects.

Description

Application of calicheamicin in optimization of in-vitro CAR/TCR-T cell product preparation process

Technical Field

The invention relates to the technical field of genetically modified T cell medicines, in particular to application of calicheamicin as an in-vitro T cell proliferation activation inhibitor, a genetically modified T cell preparation and a tumor treatment medicine, and more particularly relates to application of calicheamicin in-vitro CAR/TCR-T cell product preparation process optimization.

Background

In recent years, chimeric antigen receptor-modified T cell (Chimeric antigen receptor modified T cells, CAR-T) therapy or T cell receptor-modified T cell (T cell receptor modified T cells, TCR-T) therapy has become a popular means of tumor immunotherapy. The T cell functional state has an important influence on the treatment effect of the CAR-T/TCR-T cells, and most tumor patients are in tumor burden state for a long time or receive radiotherapy and chemotherapy, and the T cells in the body are in situations of inhibiting or tolerizing immunity and the like.

In addition to the problem of T cell activity in tumor patients, T cell depletion is also a concern in CAR-T/TCR-T cell therapy. T cell depletion refers to a progressive loss of T cell immune function in an in vitro culture environment where the body is in a tumor burden or virus infection state for a long period of time, which is manifested by a decrease in proliferation killing capacity and cytokine secretion level, while immunosuppressive receptor molecule expression is elevated. In the processes of in vitro stimulation activation, virus-mediated CAR modification and amplification of T cells, the T cells are continuously stimulated by intracellular activation signals, the depletion process is faster than that of normal T cells, and the rapid depletion of the CAR-T/TCR-T cells can lead to the in vivo amplification maintenance time to be shortened, and the killing capacity of tumor cells to be weakened, so that the patient has poor response to treatment.

In addition, conventional CAR-T/TCR-T cell preparation procedures also affect T cell CD4 ⁺ /CD8 ⁺ Proportion. T cells need to be stimulated, activated and expanded in vitro due to CD8 ⁺ T cells proliferate at a significantly faster rate than CD4 ⁺ T cells, such that the CD4/CD8 ratio is reduced, and CD4 ⁺ /CD8 ⁺ The proportion balance is important to the good anti-tumor effect of T cells, and research shows that CD8 ⁺ T cells, although having direct tumor killing, lack CD4 ⁺ T cells, which lead to CD8 ⁺ The rapid depletion of T cells and reduced function affect the expansion and anti-tumor effect of CAR-T/TCR-T cells after subsequent return to the body.

Because most of the clinically used CAR-T/TCR-T cells are derived from autologous T cell modification of tumor patients, the functions of the T cells are often poor, so that the effect of CAR-T/TCR-T cell therapy is poor. Therefore, how to reverse the T cell depletion state, ensure the T cell immunotherapy to play a longer-acting stable anti-tumor role in clinical treatment, and research the reactivation, the clonal proliferation, the improvement of the tumor infiltration capacity, the tumor cell killing capacity and the like of the T cells become field hot spots.

Disclosure of Invention

The invention aims to provide application of calicheamicin in preparation of genetically modified T cell preparations, so as to solve the technical problem of unsatisfactory treatment effect of CAR-T/TCR-T cell therapy caused by factors such as T cell activity, T cell exhaustion and the like.

In order to achieve the above purpose, the invention adopts the following technical scheme:

use of calicheamicin in the preparation of a genetically engineered T cell preparation.

Further, the genetically engineered T cell is a CAR-T cell or a TCR-T cell.

Further, the calicheamicin is added to an in vitro culture system of genetically engineered T cells.

Further, the working concentration of the calicheamicin in the in vitro culture system is 1.25 mu M-5 mu M.

Further, the in vitro culture system is lymphocyte culture medium containing colimycin, IL-7, IL-15, and double antibody of green streptomycin and FBS.

The technical scheme also provides application of the curomycin in preparing a tumor therapeutic drug, wherein the tumor therapeutic drug comprises genetically modified T cells subjected to in vitro culture, and the in vitro culture environment contains 1.25 mu M-5 mu M of the curomycin; the tumor is a hematological tumor or a solid tumor.

Further, the calicheamicin is used for slowing down the exhaustion of genetically engineered T cells and increasing CD4 ⁺ T cells and CD8 ⁺ T cell ratio and increase central memory T cell population ratio.

Genetically engineered T cells (e.g., CAR-T cells or TCR-T cells) may slow the progress of depletion of the genetically engineered T cells if a certain amount of calicheamicin is added during the culture phase in an in vitro culture phase prior to reinfusion of the organism. In particular in the proliferation rate of genetically engineered T cells is slowed, cytokine secretion is increased, the expression level of immunosuppressive receptors is inhibited, in vivo and in vivo The killing effect of external tumor is improved, the survival rate of cells after reinfusion is improved, etc. In addition, the treatment of the in vitro culture stage with the colimycin can also promote CD4 ⁺ T cells and CD8 ⁺ The proportion of T cells is made to be closer to the normal physiological level in the body, and the negative influence of imbalance of proportion of different subtype cells caused by the process of preparing genetically modified T cells is reduced. In addition, the treatment of the curcomycin in the in vitro culture stage can also improve the proportion of the central memory T cell group and further improve the treatment effect of the genetically modified T cells. Among them, genetically engineered T cells are a mature technology of the prior art, and have been widely used in clinical treatment, and are not described here in detail.

The technical scheme also provides application of the colimycin in preparing the depletion inhibitor of the genetically modified T cells.

Further, the calicheamicin is a proliferation inhibitor of genetically engineered T cells, a cytokine secretion promoter of the genetically engineered T cells, an inhibitor of an immunosuppressive receptor of the genetically engineered T cells, and a tumor killing effect promoter of the genetically engineered T cells.

The technical scheme also provides application of the colimycin in preparing an in-vitro T cell proliferation activation inhibitor.

The colimycin not only can be used for preparing the intermediate process of the preparation for medical application, but also can be used as a tool reagent in basic research, so that the depletion of T cells is avoided, the proliferation and activation of the T cells are slowed down, and the related action mechanism and the occurrence and development processes of diseases are further researched.

The principle of the technical scheme is as follows:

in the practical operation of CAR-T/TCR-T cell therapy, it is necessary to take T cells in the body (e.g., derived from human peripheral blood, isolate and collect all CD3 ⁺ T lymphocytes, i.e., whole T lymphocytes). Then, the T cells are cultured and genetically modified in vitro to obtain the CAR-T/TCR-T cells. The obtained CAR-T/TCR-T cells are subjected to in vitro culture to form cells with relatively stable states, and the cells are returned to organisms to realize anti-tumor effects (hematological tumors, solid tumors and the like). T cells stimulated activation in vitro, virusThe mediated modification and expansion of CAR and TCR continues to be stimulated by intracellular activation signals, which deplete faster than unmodified T cells, CD4 of CAR-T/TCR-T cells relative to normal T cells ⁺ /CD8 ⁺ The proportion balance is destroyed, which affects the action effect of the CAR-T/TCR-T cells, the CAR-T/TCR-T cells are returned into organisms, and the survival rate and the anti-tumor effect are greatly reduced. The technical problems are all caused by the self-specificity of the CAR-T/TCR-T cells, which exhibit different properties (e.g. increased depletion rate, CD 4) ⁺ cell/CD 8 ⁺ The proportion of cells decreased significantly). The inventors aimed at how to reduce the rate of CAR-T/TCR-T cell depletion and how to prevent CD4 ⁺ And CD8 ⁺ Extensive and intensive studies on the occurrence of T cell occurrence proportion disorder and the like are carried out, and the fact that the condition of the CAR-T/TCR-T cells can be effectively improved by adjusting the in-vitro culture condition before the CAR-T/TCR-T cells are back-transfused is found. More specifically, the application of a certain concentration of the colimycin in the in vitro culture stage can inhibit the proliferation rate of CAR-T/TCR-T cells, reduce the depletion rate of CAR-T/TCR-T cells and recover CD4 to a certain extent ⁺ And CD8 ⁺ T cell ratio, etc., thereby ensuring survival rate and antitumor activity of CAR-T/TCR-T cells infused back into the body.

Prior art (CN 113577086A) has reported that administration of colimycin in mice increases T cell proliferation activity and thus increases T cell fraction (CD 3) ⁺ T lymphocytes) and at the same time, to elevate CD4 ⁺ T cells and CD8 ⁺ The respective content of T cells (but has no effect on the ratio of the two cells). It can be speculated from the prior art that: calicheamicin is a T cell proliferation promoting substance. The CAR-T/TCR-T cells are genetically engineered T cells, and it is possible that the calicheamicin would also promote proliferation of the CAR-T/TCR-T cells. In the genetic modification process of the CAR-T/TCR-T cells, the CAR-T/TCR-T cells are subjected to a large amount of in vitro stimulation and activation, the proliferation speed is high, and the proliferation speeds of the CAR-T/TCR-T cells of different types of surface antigens are different, so that the CAR-T/TCR-T cells are fast in exhaustion and CD4 is brought ⁺ And CD8 ⁺ Adverse consequences of T cell dysregulation, therefore, inhibition of CAR-Proliferation rate of T/TCR-T cells, preventing cell depletion. With such considerations in mind, the skilled artisan does not prefer to apply the calicheamicin to the in vitro culture stage prior to CAR-T/TCR-T cell reinfusion. The inventors have unexpectedly found that after screening a large number of CAR-T/TCR-T cell in vitro culture additives: a concentration of calicheamicin not only does not promote massive proliferation of CAR-T/TCR-T cells in vitro, but also inhibits the proliferation rate of CAR-T/TCR-T cells to some extent (example 4), which is quite contrary to what the inventors speculated from the prior art. It was further determined that calicheamicin has a certain potential as an in vitro culture supplement to CAR-T/TCR-T cells and can be used as a substance to slow down CAR-T/TCR-T cell depletion.

T cell depletion, which is a progressive loss of T cell immune function, is also one of the problems to be solved in current CAR-T/TCR-T cell therapies, which is manifested by a decrease in both the killing capacity for the substance of interest and the level of cytokine secretion, while the expression of immunosuppressive receptor molecules increases. The excessive proliferation and activation of T cells (as would be the case, for example, in the preparation of CAR-T/TCR-T cells) results in an increased process of T cell depletion. The inventors have further conducted intensive studies on the effect of calicheamicin on T cell depletion to determine its availability. It was found that the proliferation rate of CAR-T/TCR-T cells could be effectively inhibited by applying 1.25 μm-5 μm of calicheamicin during the CAR-T/TCR-T cell in vitro culture phase (example 4); in vitro culture with the application of 1.25. Mu.M-2.5. Mu.M (preferably 2.5. Mu.M) of colimycin, the resulting CAR-T/TCR-T cells have a greater in vitro tumor killing effect (example 7); in vitro culture with the application of 1.25. Mu.M-2.5. Mu.M (preferably 2.5. Mu.M) of calicheamicin is effective in increasing the cytokine secretion level of CAR-T/TCR-T cells (example 8); in vitro culture with 1.25. Mu.M-5. Mu.M (preferably 2.5. Mu.M) of calicheamicin is effective to reduce the expression level of the immunosuppressive receptor molecules of CAR-T/TCR-T cells (example 9); 2.5 mu M of the curcomycin is applied by in vitro culture, and then the obtained CAR-T/TCR-T cells are returned to the animal body, so that stronger treatment effects of blood tumor and solid tumor can be obtained (examples 10-12); in vitro culture with 2.5. Mu.M of calicheamicin, the resulting CAR-T/TCR-T cells were returned to the animal to ensure higher CAR-T/TCR-T cell viability (examples 13, 14). The above experiments demonstrate that the administration of 1.25 μm-2.5 μm (preferably 2.5 μm) of calicheamicin during the in vitro culture phase of CAR-T/TCR-T cells can effectively inhibit proliferation and thereby avoid premature depletion of T cells, ensuring that CAR-T/TCR-T cells subsequently reinfused into the body are in a more active state. While the administration of 5 μm of colimycin, while effective in inhibiting the excessive proliferation of CAR-T/TCR-T cells, had no positive effect on cytokine secretion levels. The experimental data show that the colimycin can be used as an in vitro proliferation inhibitor, an activity maintaining reagent and a CAR-T/TCR-T cell failure inhibitor of genetically modified T cells, and further can be applied to the preparation of the CAR-T/TCR-T cells, so that the treatment effect of the CAR-T/TCR-T cell therapy is improved.

In addition to the inhibitory effect of calicheamicin on CAR-T/TCR-T cell failure, the inventors have also found that calicheamicin has the ability to elevate CD4 in CAR-T/TCR-T cells ⁺ And CD8 ⁺ Effect of the ratio (example 5), and thus maintain CD4 ⁺ And CD8 ⁺ Proportional balance, reduced CD4 due to CAR-T/TCR-T cell preparation process ⁺ And CD8 ⁺ Too low a ratio, which is as close as possible to CD4 in normal physiological conditions ⁺ And CD8 ⁺ Proportion. In addition, calicheamicin also has the effect of increasing the proportion of the central memory T cell population of CAR-T/TCR-T cells (example 6). T cells can be classified into primary T cells, central memory T cells, effector memory T cells and effector T cells according to their surface expression molecules, and in the conventional CAR-T/TCR-T cell therapy, it has been found that the central memory T cell population has a good proliferation effect and a long-lasting antitumor effect in vivo, and it has been found that the proportion of central memory T cells in tumor patients is lower and the proportion of effector memory T cells is higher than in healthy patients. Through the addition of the calicheamicin, the proportion of the central memory T cells can be improved in the preparation process of the CAR-T/TCR-T cells in vitro, and the in vivo tumor treatment effect of the CAR-T/TCR-T cells can be effectively improved. CD4 as described above ⁺ And CD8 ⁺ The ratio improving effect and the central memory T cell ratio improving effect are unexpected findings of the inventor in the process of researching the medicines capable of slowing down the T cell exhaustion, and the calicheamicin has more action effects and further improves the effect of the CAR-T/TCR-T cell therapy compared with other medicines capable of simply inhibiting the T cell exhaustion.

To sum up, the beneficial effects of this technical scheme lie in:

(1) The in vitro expansion speed of the CAR-T/TCR-T cells treated by the colimycin is reduced, and the overactivation, proliferation and depletion processes in the preparation process of the CAR-T/TCR-T cells can be delayed.

(2) The CAR-T/TCR-T cells treated with the calicheamicin can well maintain CD4 ⁺ /CD8 ⁺ The proportion balance can improve CD4 on the basis of original ⁺ /CD8 ⁺ The ratio has obvious help to the CAR-T/TCR-T cells prepared in vitro to exert more effective anti-tumor effect in vivo.

(3) The central memory T cell proportion of the CAR-T/TCR-T cells treated by the calicheamicin is increased, and the prior study finds that the central memory T cell population has good proliferation effect and long-acting anti-tumor effect in vivo, so that the proliferation capacity of the CAR-T/TCR-T cells treated by the calicheamicin in vivo is enhanced.

(4) The CAR-T/TCR-T cells treated by the calicheamicin have the advantages of enhanced in-vivo and in-vitro tumor killing capability, increased secretion level of cytokines, reduced expression of immunosuppressive receptor molecules, and increased number of CAR-T/TCR-T cells in a model mouse, and the effect that the calicheamicin can enhance the anti-tumor effect of the CAR-T/TCR-T cells, delay the depletion process of the CAR-T/TCR-T cells and improve the long-acting expansion capability of the CAR-T/TCR-T cells in vivo is shown.

(5) The optimal working concentration of the in vitro treatment of CAR-T/TCR-T cells with calicheamicin was determined to be 1.25-2.5 μm (preferably 2.5 μm), at which the effect of the calicheamicin exhibited very significant advantages.

Drawings

FIG. 1 is a schematic of the flow chart of the calicheamicin-treated in vitro CAR-T/TCR-T cell product of example 3.

FIG. 2 is an in vitro proliferation curve of CAR-T/TCR-T cells treated with different concentrations of calicheamicin of example 4 (CM: calicheamicin; A: CAR-T cells; B: TCR-T cells).

FIG. 3 is a plot of CD4+/CD8+ ratio at day 7 after treatment of CAR-T/TCR-T cells with different concentrations of calicheamicin of example 5 (CM: calicheamicin; A is a plot of results of flow cytometry analysis for different treated CAR-T; B is CD4 of different treated CAR-T) ⁺ /CD8 ⁺ A statistical plot of the scale; c is a graph of flow cytometry analysis results for TCR-T for different treatments; d is CD4 of TCR-T of different treatments ⁺ /CD8 ⁺ Statistical plot of the ratio).

FIG. 4 is a graph showing CCR7 and CD45RA expression at day 7 after CAR-T cell treatment with different concentrations of calicheamicin of example 6 (CM: calicheamicin; A is CD4 for different treatments) ⁺ A flow cytometry analysis result graph of CAR-T; b is a statistical plot for four types of T cells according to the results of A; c is CD8 for different treatments ⁺ A flow cytometry analysis result graph of CAR-T; d is a statistical plot for four types of T cells based on C results).

FIG. 5 shows CCR7 and CD45RA expression on day 7 after treatment of TCR-T cells with different concentrations of calicheamicin of example 6 (CM: calicheamicin; A is CD4 for different treatments) ⁺ Flow cytometry analysis results for TCR-T; b is a statistical plot for four types of T cells according to the results of A; c is CD8 for different treatments ⁺ Flow cytometry analysis results for TCR-T; d is a statistical plot for four types of T cells based on C results).

FIG. 6 shows tumor cell lysis (CM: colimycin; A: CAR-T cells; B: TCR-T cells) on day 7 after treatment of CAR-T/TCR-T cells with different concentrations of colimycin according to example 7.

FIG. 7 is a graph showing cytokine secretion levels of CAR-T cells treated with different concentrations of calicheamicin of example 8 (CM: calicheamicin; A is CD 4) ⁺ CAR-T cells; b is CD8 ⁺ CAR-T cells).

FIG. 8 shows cytokine secretion levels (CM:a calicheamicin; a is CD4 ⁺ TCR-T cells; b is CD8 ⁺ TCR-T cells).

FIG. 9 shows the expression levels of immunosuppressive receptor molecules of CAR-T/TCR-T cells treated with different concentrations of calicheamicin of example 9 (CM: calicheamicin; A: CAR-T cells; B: TCR-T cells).

FIG. 10 is a live imaging of a CAR-T treated mouse hematological tumor model treated with calicheamicin of example 10.

FIG. 11 is a live image of a CAR-T treated mouse subcutaneous tumor model treated with calicheamicin of example 11.

FIG. 12 is a live image of a mouse subcutaneous tumor model treated with the curcomycin-treated TCR-T of example 12.

FIG. 13 is a graph of T cell fraction in mice treated with calicheamicin of example 13 on day 21 (A is placebo T cells; B is CAR-T cells; C is CAR-T cells after treatment with calicheamicin).

FIG. 14 shows the T cell fraction of mice treated with calicheamicin of example 14 on day 21 (A is the control T cells; B is the TCR-T cells; C is the TCR-T cells after treatment with calicheamicin).

Detailed Description

The present invention will be described in further detail with reference to examples, but embodiments of the present invention are not limited thereto. Unless otherwise indicated, the technical means used in the following examples and experimental examples are conventional means well known to those skilled in the art, and the materials, reagents and the like used are all commercially available.

Example 1: medicine source and preparation

The calicheamicin belongs to 16-membered macrolide antibiotics, and has active groups of carboxyl, alkoxy, epoxy, keto and aldehyde groups and a pair of conjugated C=C, and has a molecular weight of about 884-982. The specifically used calicheamicin for subsequent experimental study is purchased from the Co-pharmaceutical industry Co., ltd (Shanghai, china) and has the specification of 200 mg/tablet, wherein the content of each tablet of the calicheamicin is 112.28 mg, and the effective component of the calicheamicin is calculated when the drug solution is prepared subsequently. When in preparation, a proper amount of the curcomycin medicine powder (used after grinding and crushing tablets) is weighed into a 1.5 mL centrifuge tube, and added with DMSO solution to be dissolved into 10 mM mother solution for standby.

Example 2: preparation of CAR-T/TCR-T cells

The CAR-T/TCR-T cells are all T cells which are conventional in the prior art and are genetically modified, and expression plasmids containing CAR or TCR genes are transferred into the T cells mainly through a transgenic technology, so that the T cells express CAR or TCR proteins, and the immunotherapy effect of the T cells is improved. The focus of the technical scheme is how to further improve the therapeutic effect of the CAR-T/TCR-T cells, and the construction mode of the CAR-T/TCR-T cells is specifically described below by taking specific CD19 CAR and NY-ESO1 TCR genes as examples. It should be noted that T cells expressing other types of CAR or TCR genes (genetically modified with CAR or TCR genes) can respond to specific concentrations of calicheamicin under in vitro culture conditions to achieve inhibition of cell proliferation in vitro and increase of CD4 ⁺ /CD8 ⁺ Proportion, proportion of central memory T cell population is raised. These genetically engineered T cells, after withdrawal, are infused back into the body with a significant increase in cellular activity and an increase in tumor therapeutic effect. In a specific embodiment, the CAR-T cell is a T cell expressing a CD19 CAR protein and the TCR-T cell is a T cell expressing an NY-ESO1 TCR protein.

(1) Construction of CD19 CAR and NY-ESO1 TCR expression lentiviral vector plasmids

The original lentiviral expression vector (empty vector) used was pLVX-IRES-mCherry (Takara doctor technologies Co., ltd., product number: 631237). The gene sequences of fusion proteins CD19 CAR and NY-ESO1 TCR are entrusted to be synthesized, and the two sequences are respectively inserted into an expression vector by utilizing EcoR1 and BamH1 cleavage sites to obtain pLVX-CD19 CAR-TSPAN32-IRES-mCherry and pLVX-NY-ESO1 TCR-TSPAN32-IRES-mCherry expression vectors. The two restriction sites EcoR1 and BanH1 are used for restriction enzyme digestion to identify whether the CD19 CAR and NY-ESO1 TCR sequences are connected or not, and the bands with the sizes of about 1600bp and 2600bp are respectively cut, which indicates that the two plasmids are constructed.

The chimeric antigen receptor CAR (CD 19 CAR) is constructed based on FMC63 antibody sequences, is a conventional chimeric antigen receptor of the prior art, and comprises the following parts in order from extracellular to intracellular: signal peptide (SEQ ID NO. 1), light chain variable region (mIglv, SEQ ID NO. 2), hinge region (Hinge chain, SEQ ID NO. 3), heavy chain variable region (mIghv, SEQ ID NO. 4), CD28 extracellular region (CD 28 Extra, SEQ ID NO. 5), CD28 transmembrane region (CD 28 TM, SEQ ID NO. 6), CD28 intracellular region (CD 28 cyto, SEQ ID NO. 7), CD3zeta intracellular region (CD 3zeta cyto, CD3zeta, SEQ ID NO. 8).

The T cell receptor TCR (NY-ESO 1 TCR) is constructed based on the NY-ESO1 antibody sequence and is a chimeric antigen receptor conventional in the prior art, and comprises the following parts in sequence from extracellular to intracellular: TCRα variable region (TCRVα, SEQ ID NO. 9), CD28 transmembrane region (CD 28 TM, SEQ ID NO. 10), CD28 intracellular region (CD 28 cyto, SEQ ID NO. 11), CD3zeta intracellular region (CD 3zeta cyto, CD3zeta, SEQ ID NO. 12), fusion protein linker (SEQ ID NO. 13), TCRβ variable region (TCRVβ, SEQ ID NO. 14), CD28 transmembrane region (CD 28 TM, SEQ ID NO. 15), 41BB intracellular region (41BB cyto,SEQ ID NO.16), CD3zeta intracellular region (CD 3zeta cyto, CD3zeta, SEQ ID NO. 17).

(2) Virus package

The virus packaging is carried out in a conventional manner in the prior art, and is specifically as follows:

293T cell culture: the culture medium is DMEM culture medium, and 10% FBS is added. When the cell grows until the fusion rate reaches 80-90%, the cell needs to be subjected to passage operation to expand the number of the cells and maintain the good growth state of the cells. At 37℃with 5% CO ₂ And 90% relative humidity in a cell incubator.

And (3) paving: 293T cells were plated in 10 cm dishes one night before virus inclusion to achieve a cell density of 80-90% when virus inclusion. Before virus inclusion, 1. 1 h, cell culture medium was changed to DMEM medium (1:4000 chloroquine addition) and placed in a cell incubator.

Plasmid addition: the composition and ratio of the mixture of the objective plasmid and the packaging plasmid (psPAX 2 and pMD2G packaging plasmids) are shown in Table 1. The mixture of Table 1 was mixed with 500. Mu.L of 2 XHBS and added dropwise to a culture dish of 293T cells.

Table 1: viral plasmids and packaging plasmid mixtures

Liquid replacement: after 12h, the culture medium is changed into a normal culture medium, and after 24h, the liquid is continuously changed.

Collecting viruses: the three virus supernatants were collected at 36h, 48h and 60h, respectively, and mixed well.

And (3) filtering: the supernatant was filtered with a 0.45 μm filter membrane to remove cellular impurities and the like.

Virus cryopreservation: freezing the packaged virus liquid in a refrigerator at-80 ℃.

(3) Isolated culture of primary human T cells

The technical scheme uses T cells for preparing the CAR-T/TCR-T, and the T cells can be collected in peripheral blood. In this example, T cells isolated from peripheral blood will be described. The technical scheme does not relate to a blood sampling process, only uses T cells separated from peripheral blood for preparing cell preparations, and does not relate to a surgical operation process.

Isolation of peripheral blood T cells: the obtained peripheral blood was diluted 1:1 with PBS, and an equal amount of lymphocyte separation liquid was added to a 15ml centrifuge tube, so that the separation liquid: blood: pbs=1:1:1, diluted blood was carefully added to the upper layer of the separation solution, centrifuged at 800g for 20min at room temperature, and both the centrifuge speed up and down were adjusted down to 2. After centrifugation, carefully sucking the cell layer of PBMC (human peripheral blood mononuclear cells), transferring to a new 15ml centrifuge tube, adding 3-5 times volume of PBS for dilution, centrifuging at 1500rpm for 5min, washing the cells once with PBS, incubating anti-CD3 magnetic beads for 20min, and separating CD3 with a magnetic separation column ⁺ T cells, cell counts, PBS wash cells 1-2 times. Wherein, CD3 ⁺ T lymphocytes represent whole T lymphocytes, including helper/inducer T lymphocytes (CD 3 ⁺ CD4 ⁺ ) Suppressor/cytotoxic T lymphocytes (CD 3 ⁺ CD8 ⁺ ) Etc. all types of T lymphocytes.

T cell in vitro stimulation culture: the T lymphocyte stimulating liquid is prepared, and the components and the proportions are shown in Table 2. Cells were resuspended in 12-well plates, 2 mL/well, with the prepared stimulation solution. Cell density was observed under the microscope, the stimulation solution was changed every day, stimulation was continued for three days, and after the third day the medium was changed to one containing only IL-2.

Table 2: t lymphocyte stimulating liquid

(4) T cell viral infection

The stimulated T cells were plated in six well plates at day 3 at a cell density of 2X 10 ⁶ Each well was prepared with a virus-infected solution and the reagents are shown in Table 3. Six-well plate centrifugation (1000 g,37 ℃,90 min), then placing in a cell incubator for 8-10h, and changing to normal medium (containing IL-2 in the medium). After 48h of T cell infection, the cells are subjected to fluorescence detection to detect whether transfection is successful. Thus, the construction of CAR-T/TCR-T cells was completed.

Table 3: viral infection solution component

Example 3: treatment of CAR-T/TCR-T cells with calicheamicin in vitro

The prepared CAR-T/TCR-T cells were used in a 1.0X10 scale ⁶ Each well was seeded in 24-well plates (i.e., day 5) and CAR-T/TCR-T cells were cultured in vitro with a DMSO negative control, a concentration gradient of calicheamicin of 1.25. Mu.M, 2.5. Mu.M, and 5. Mu.M, with simultaneous addition of recombinant human IL-7 (10 ng/ml) and IL-15 (5 ng/ml) to the medium. The medium was lymphocyte medium containing 10% FBS and 1% Streptomyces lividans (see Table 2, without IL2, anti-CD3, anti-CD 28), three duplicate wells were made per group, CAR-T/TCR-T cells were expanded in vitro to day 12, and CAR-T/TCR-T cells were treated in vitro with calicheamicin for day 7, after which the CAR-T/TCR-T cell phenotype and function were examined in an in vitro experiment. Relay in the above mannerAnd (3) continuously culturing until the 14 th day, namely, treating the CAR-T/TCR-T cells in vitro by using the calicheamicin, removing the calicheamicin, the IL-7 and the IL-15, taking the CAR-T/TCR-T cells, reinjecting tail veins of the CAR-T/TCR-T cells into a tumor model mouse, and observing the killing effect and in-vivo effect maintenance condition of the CAR-T/TCR-T cells on the tumor cells in the mouse (the schematic diagram is shown in figure 1).

Example 4: calicheamicin inhibits CAR-T/TCR-T cell proliferation in vitro

To demonstrate the inhibition of CAR-T/TCR-T cell proliferation by calicheamicin in vitro, this example examined CAR-T/TCR-T cells proliferation in vitro following administration of different calicheamicin drug concentrations. CD3 in peripheral blood of healthy volunteers on day 0 ⁺ T cells were isolated and purified at 1.0X10 ⁶ After 3 days of in vitro stimulated culture with CD3 antibody (600 ng/ml), CD28 antibody (300 ng/ml) and recombinant human IL-2 (10 ng/ml), each well was inoculated into 24-well plates and lentiviruses were infected to prepare CAR-T and TCR-T cells, respectively (see example 2 for specific procedures). CD3 antibody (600 ng/ml), CD28 antibody (300 ng/ml) and recombinant human IL-2 (10 ng/ml) were replaced with recombinant human IL-7 (10 ng/ml) and IL-15 (5 ng/ml), the concentration gradient of calicheamicin was set to 1.25. Mu.M, 2.5. Mu.M and 5. Mu.M with DMSO as negative control, three wells were made per group, CAR-T/TCR-T cells were expanded in vitro, cell counts were performed on days 3, 7, 11 and 15 of in vitro culture, and CAR-T/TCR-T cell proliferation curves were plotted, respectively.

As a result of the experiment, referring to fig. 2, it is known from the result of the experiment that the colimycin has an inhibitory effect on the proliferation of CAR-T/TCR-T cells in vitro, wherein the inhibitory effect of the 5 μm group is most remarkable (P < 0.0001).

Prior art CN113577086A (application of isovaleryl spiramycin compounds or compositions thereof in preparing medicines for treating immune disorder) mentions that administration of the curomycin to mice can significantly promote increase of total T cells (CD 3 positive cells) in the mice, wherein both CD4 and CD8 positive cells are increased (see example 4 of patent CN113577086A and figure 11: CD3 in peripheral blood) ⁺ Bar graph of cell scale). It can be seen that in vivo administration of calicheamicin was not genetically engineeredThe effect of T cells is to promote proliferation. This trend of action is greatly different from the present solution, and according to the report of the prior art, the trend of action of the calicheamicin is that the effect of promoting proliferation is generated on T cells, and the genetically modified CAR-T/TCR-T cells which are T cells are theoretically subject to the effect of promoting proliferation from the calicheamicin. However, the inventors have unexpectedly found that the application of different concentrations of calicheamicin to genetically engineered CAR-T/TCR-T cells in vitro produced an inhibition of proliferation of CAR-T/TCR-T cells, resulting in unexpected technical results.

Indeed, the CAR-T/TCR-T cell therapy method requires genetic engineering and in vitro culture of T cells from an organism, during which we do not want the proliferation activity of T cells to be too strong, otherwise, T cells continue to be stimulated by intracellular activation signals during in vitro stimulation activation, virus-mediated CAR modification and expansion, and the depletion process is faster than normal T cells, and the rapid depletion of CAR-T/TCR-T cells will lead to a shorter in vivo expansion maintenance time, i.e. the activity of CAR-T/TCR-T cells returned to the patient is poor, and the killing ability of tumor cells is reduced, thus making the patient respond poorly to the therapy. Therefore, properly inhibiting the in vitro proliferative capacity of CAR-T/TCR-T cells is very helpful to boost the effect of CAR-T/TCR-T therapy.

The inhibition effect of the colimycin on the CAR-T/TCR-T cells is found, the colimycin can be used as an in vitro T cell inhibitor, particularly an in vitro CAR-T/TCR-T cell inhibitor, can be applied to CAR-T/TCR-T cell therapy, can be used in early T cell genetic modification and CAR-T/TCR-T cell in-vitro culture stages, can inhibit the premature activation and the premature exhaustion of the CAR-T/TCR-T cells, can be removed when the CAR-T/TCR-T cells are returned to a patient or other organisms, can eliminate the proliferation inhibition state of the CAR-T/TCR-T cells, and can fully exert the tumor treatment effect in vivo.

Example 5: calicheamicin increases CD4 in CAR-T/TCR-T cell in vitro culture ⁺ /CD8 ⁺ Proportion of

To demonstrate that calicheamicin was used in vitro culture of CAR-T/TCR-T cellsCD4 ⁺ /CD8 ⁺ Ratio maintenance, in this example, CD4 on day 7 after administration of different concentrations of calicheamicin drug to CAR-T/TCR-T cells was detected using flow cytometry ⁺ And CD8 ⁺ T cell ratio. The prepared CAR-T/TCR-T cells were grown at 1.0X10 on day 0 ⁶ The individual wells were seeded in 24-well plates with a DMSO negative control, and a concentration gradient of calicheamicin of 1.25. Mu.M, 2.5. Mu.M and 5. Mu.M, with simultaneous addition of recombinant human IL-7 (10 ng/ml) and IL-15 (5 ng/ml), and three wells were made per group. Detection of CD4 in CAR-T/TCR-T cells by flow cytometry on day 7 of in vitro culture ⁺ And CD8 ⁺ Proportion, statistical analysis was performed for mapping.

Experimental results show that the 1.25 mu M and 2.5 mu M groups of the colimycin can improve CD4 in vitro culture of CAR-T/TCR-T cells ^＋ /CD8 ^＋ The ratio, in which the ratio increase was most pronounced for the 2.5 μm group (< P < 0.001), whereas no significant difference was seen for the 5 μm group compared to the control group (see fig. 3). Thus, the addition of proper calicheamicin during the in vitro culture of CAR-T/TCR-T cells can not only slow down the proliferation trend of cells and prevent cell decay, but also properly promote CD4 ^＋ /CD8 ^＋ The ratio, which has not been reported in the prior art. In the current conventional CAR-T/TCR-T cell preparation procedure, T cells need to be stimulated, activated and expanded in vitro due to CD8 ⁺ T cells proliferate at a significantly faster rate than CD4 ⁺ T cells, such that CD4 ⁺ /CD8 ⁺ Reduced ratio, while CD4 ⁺ /CD8 ⁺ The proportion balance is important to the good anti-tumor effect of T cells, and research shows that CD8 ⁺ T cells, although having direct tumor killing, lack CD4 ⁺ T cells, which lead to CD8 ⁺ The rapid depletion of T cells and reduced function affect the expansion and antitumor effect of CAR-T/TCR-T cells in vivo. In this technical scheme we can see that when CAR-T/TCR-T cells are cultured in vitro, CD4 is obtained if no calicheamicin is added (DMSO is used instead) ⁺ /CD8 ⁺ The ratio is as low as about 0.2-0.3, and the addition of 1.25 μm of colimycin can make CD4 ⁺ /CD8 ⁺ The proportion is increased to about 0.4, and 2.5 mu M of cocoa is addedLimycin, which can cause CD4 ⁺ /CD8 ⁺ The proportion is increased to about 0.6, and a proper amount of the colimycin is added, so that the proportion is increased by about 2 times, the lifting amplitude is very large, and the CD4 is maintained ⁺ /CD8 ⁺ The ratio to achieve an improvement in the therapeutic effect of CAR-T/TCR-T cells was also unexpected by the inventors prior to experimental studies. And, if the amount of the calicheamicin is continuously increased to 5 μm, the above-mentioned elevating effect is lost.

Prior art CN113577086A (application of isovaleryl spiramycin compounds or compositions thereof in preparing medicines for treating immune disorder) mentions that administration of the curomycin to mice can significantly promote increase of both CD4 and CD8 positive cells in mice (see example 4 of patent CN113577086A and figure 8: CD4 in peripheral blood) ⁺ /CD3 ⁺ And CD8 ⁺ /CD3 ⁺ Bar graph of cell scale). Experimental data from the blank and the calicheamicin-applied group of the figure were analyzed, CD4 of the blank ⁺ /CD8 ⁺ The ratio is about 45%/30%; CD4 of the calicheamicin-applied group ⁺ /CD8 ⁺ The ratio is about 50%/35%; there is no very significant difference between the two sets of data. It can be seen that the application of in vivo calicheamicin to normal, non-genetically engineered T cell CD4 ⁺ /CD8 ⁺ The ratio has no significant effect. And, in vivo CD4 ⁺ /CD8 ⁺ The higher ratio is beneficial to the efficacy of the T cells. As one of ordinary skill in the art can infer, there is no significant effect of calicheamicin on the modulation of T cell composition, and on CD4 of genetically engineered CAR-T/TCR-T cells ⁺ /CD8 ⁺ The ratio is also likely to have no effect. However, the actual situation is not as inferred that CD4 could be exponentially promoted if the appropriate concentration of calicheamicin was applied to CAR-T/TCR-T cells in vitro ⁺ /CD8 ⁺ The ratio is increased and unexpected technical effects are obtained. During in vitro culture and genetic engineering, CD8 is responsible for ⁺ Cell proliferation rate is high leading to CD4 ⁺ /CD8 ⁺ The down regulation of the ratio was severe (CD 4 of normal T cells in vivo according to the experimental data of CN113577086A ⁺ /CD8 ⁺ The ratio was maintained around 1.5, and there was a significant difference from the DMSO experimental group of fig. 3), affecting the activity and therapeutic effect of the cells. In the process of culturing the CAR-T/TCR-T cells in vitro, proper application of the calicheamicin can inhibit the proportion decreasing trend and recover CD4 to a certain extent ⁺ /CD8 ⁺ In proportions, unexpected technical effects are obtained.

Example 6: calicheamicin for improving T in CAR-T/TCR-T cell in-vitro culture process _CM (Central memory T cell population) ratio

To demonstrate that calicheamicin increases the proportion of TCM (central memory T cell population) in the in vitro culture of CAR-T/TCR-T cells, this example uses a flow cytometer to detect CD4 on day 7 after administration of different calicheamicin drug concentrations to CAR-T/TCR-T cells, respectively ⁺ And CD8 ⁺ T in T cells _N (naive T cells, CCR 7) ⁺ CD45RA ⁺ ）、T _CM (Central memory T cells, CCR 7) ^- CD45RA ⁺ ）、T _EM (effector memory T cells, CCR 7) ^- CD45RA ^- ) And T _E (effector T cells, CCR 7) ^- CD45RA ⁺ ). The prepared CAR-T/TCR-T was used at 1.0X10 on day 0 ⁶ The individual wells were seeded in 24-well plates with a DMSO negative control, and a concentration gradient of calicheamicin of 1.25. Mu.M, 2.5. Mu.M and 5. Mu.M, with simultaneous addition of recombinant human IL-7 (10 ng/ml) and IL-15 (5 ng/ml), and three wells were made per group. Detection of CD4 by flow cytometry on day 7 of in vitro culture ⁺ And CD8 ⁺ Expression of CCR7 and CD45RA in CAR-T/TCR-T cells, wherein T _N Is CCR7 ⁺ CD45RA ⁺ 、T _CM Is CCR7 ⁺ CD45RA ^- 、T _EM Is CCR7 ^- CD45RA ^- 、T _E Is CCR7 ^- CD45RA ⁺ And carrying out statistical analysis and mapping on the obtained streaming data.

Experimental results show that 1.25 mu M and 2.5 mu M of the colimycin can improve CD4 ⁺ And CD8 ⁺ T in CAR-T/TCR-T cell in vitro culture _CM Proportion of 2.5. Mu.M group T _CM The ratio increase was most pronounced (< 0.0001) whereas the 5 μm group was not evident compared to the control group Differences (fig. 4 and 5). T cells can be classified into initial T cells, central memory T cells, effector memory T cells and effector T cells according to their surface expression molecules, and in the past CAR-T/TCR-T cell therapy, it was found that the central memory T cell population has a good proliferation effect and a long-lasting antitumor effect in vivo, and studies have found that the proportion of central memory T cells in T cells of tumor patients is lower compared to healthy persons, whereas the proportion of effector memory T cells is higher, if the proportion of central memory T cells is increased in the CAR-T/TCR-T cell preparation process in vitro, the in vivo tumor therapeutic effect of CAR-T/TCR-T cells will be improved.

The application of the calicheamicin in the in vitro culture process of the CAR-T/TCR-T cells can effectively improve the proportion of the central memory T cells, which is not reported in the prior art, and the treatment effect of the CAR-T/TCR-T cells is improved by the calicheamicin in various modes.

Example 7: calicheamicin for enhancing CAR-T/TCR-T cell in-vitro tumor killing capability

To demonstrate that calicheamicin enhances the in vitro tumor killing ability of CAR-T/TCR-T cells, this example utilizes in vitro tumor cell and CAR-T/TCR-T cell co-culture experiments to examine the in vitro killing ability of CAR-T/TCR-T cells on day 7 after administration of different calicheamicin drug concentrations to tumor cells. The prepared CAR-T/TCR-T was used at 1.0X10 on day 0 ⁶ Inoculating each cell/hole into 24-hole plate, taking DMSO as negative control, setting the concentration gradient of the colimycin to 1.25 mu M, 2.5 mu M and 5 mu M, simultaneously adding recombinant human IL-7 (10 ng/ml) and IL-15 (5 ng/ml) to in vitro amplification culture CAR-T cells for seventh day, co-culturing Raji tumor cell lines expressing CD19 antigen with CAR-T cells treated by different concentrations of the colimycin drug (after withdrawal of the colimycin) according to an effective target ratio of 1:1, namely respectively paving the Raji cells and the CAR-T cells in 48-hole plate for 2.0X10 × ⁵ Detecting the number of Raji cells after 48 hours; co-culturing A375 tumor cell line expressing NY-ESO1 antigen with TCR-T cells treated with different concentrations of calicheamicin at an effective target ratio of 1:1, respectively, by spreading A375 cells and TCR-T cells in 48-well plate at a ratio of 2.0X10 ⁵ Detecting the number of A375 cells after 48 hours, and calculating TCR-T cells of different groupsTumor lysis rate of (2).

The experimental results show that the colimycin can enhance the in vitro tumor killing capacity of the CAR-T/TCR-T cells, wherein the 2.5 mu M group effect is most obvious (P < 0.01) (figure 6).

Example 8: calicheamicin enhances cytokine secretion levels by CAR-T/TCR-T cells

To demonstrate the effect of calicheamicin on cytokine secretion during CAR-T/TCR-T cell in vitro tumor killing, this example uses a flow cytometer to detect intracellular expression levels of CAR-T/TCR-T cell interferon gamma (IFNG), tumor Necrosis Factor Alpha (TNFA), granzyme B (GZMB) and perforin 1 (PRF 1) after 48h of CAR-T/TCR-T cell and tumor cell co-culture experiments. Co-culturing Raji tumor cell lines with CAR-T cells treated by different concentrations of the colimycin according to an effective target ratio of 1:1, namely respectively paving Raji cells and CAR-T cells in a 48-well plate by 2.0X10 ⁵ A plurality of; co-culturing A375 tumor cell line with TCR-T cells treated with different concentrations of colimycin at an effective target ratio of 1:1, respectively, by spreading A375 cells and TCR-T cells in 48-well plates at 2.0X10 s ⁵ CD4 detection by 48h flow cytometry ⁺ And CD8 ⁺ Expression levels of INFG, TNFA, GZMB and PRF1 in CAR-T/TCR-T cells.

The experimental results found that 1.25. Mu.M and 2.5. Mu.M of calicheamicin increased cytokine secretion levels in CAR-T/TCR-T cells, with the 2.5. Mu.M increase being most pronounced, whereas the 5. Mu.M group was not significantly different from the control group (FIGS. 7 and 8).

Example 9: calicheamicin down-regulates the expression level of CAR-T/TCR-T cell immunosuppressive receptor molecules

To demonstrate the effect of calicheamicin on the expression level of immunosuppressive receptor molecules during CAR-T/TCR-T cell in vitro tumor killing, this example uses a flow cytometer to detect the expression levels of CAR-T/TCR-T cells PD1, LAG3, TIM3 after 48h of CAR-T/TCR-T cell and tumor cell co-culture experiments. Co-culturing Raji tumor cell lines with CAR-T cells treated by different concentrations of the colimycin according to an effective target ratio of 1:1, namely respectively paving Raji cells and CAR-T cells in a 48-well plate by 2.0X10 ⁵ A plurality of; co-culturing A375 tumor cell line with TCR-T cells treated with different concentrations of colimycin at an effective target ratio of 1:1, respectively, by spreading Raji cells and TCR-T cells in 48-well plates at a ratio of 2.0X10 ⁵ In addition, 48 h flow cytometry detected the expression levels of PD1, LAG3, TIM3 in CAR-T/TCR-T cells.

The experimental results found that 1.25. Mu.M, 2.5. Mu.M and 5. Mu.M of calicheamicin down-regulated the expression level of the immunosuppressive receptor molecules of CAR-T/TCR-T cells to varying degrees, with the 2.5. Mu.M group being the most pronounced effect (FIG. 9).

Example 10: calicheamicin enhances the in vivo therapeutic effect of CAR-T cells on hematological tumors

To demonstrate that the calicheamicin enhances the in vivo therapeutic effect of CAR-T cells on hematological tumors, this example utilizes NSG mouse hematological tumor models to examine the in vivo killing ability of CAR-T/TCR-T cells on day 7 after administration of different calicheamicin drug concentrations to hematological tumor cells. CD3 in peripheral blood of healthy volunteers on day 0 ⁺ T cells were isolated and purified at 1.0X10 ⁶ After 3 days of in vitro stimulation culture with CD3 antibody (600 ng/ml), CD28 antibody (300 ng/ml) and recombinant human IL-2 (10 ng/ml) each well was inoculated in 24-well plates, CD 19-targeted CAR lentiviruses were infected, CAR-T cells were given a treatment with calicheamicin on day 5 (working concentration of 2.5 μm, see fig. 1), CAR-T cells were expanded in vitro to day 14 (calicheamicin treatment time total of 9 days), and the treated CAR-T cells were taken for in vivo experiments (CM treated CAR-T group). CAR-T cells without calicheamicin were used as negative controls (DMSO was used instead of calicheamicin, CAR-T group) and unmodified T cells were used as blank controls (NC-T group). Wherein, as a negative control, the CAR-T cell treatment protocol without calicheamicin treatment was the same as in fig. 1, except that DMSO was used instead of calicheamicin; the in vitro culture procedure for unmodified T cells was the same as in fig. 1, except that no virus infection was performed. Raji-luc cells (expressing luciferase) were grown at 1.0X10 ⁶ The mice were divided into 3 groups of 5 mice each, and 5.0X10 cells were used on day 7 after Raji-luc cell injection ⁶ Each/only separatelyThe unmodified T cells, CAR-T cells and calicheamicin-treated CAR-T cells were reinfused via the tail vein and tumor cells were examined for in vivo growth using a biopsy imager on days 14 and 21.

Experimental results show that the proliferation speed of tumor cells in the mice of the unmodified T cell group is high, only one mouse remains to survive on the 21 st day, and the rest of mice die, the CAR-T cell group can inhibit tumor growth but is not completely cleared, the inhibition effect of the tumor cells in the mice of the CAR-T cell group treated by the calicheamicin is obviously superior to that of the CAR-T cell group, no fluorescent signals of the tumor cells are detected on the 14 th and 21 st days, and the effect of the calicheamicin on enhancing the in vivo killing effect of the CAR-T cells on hematological tumors is shown, and the in vivo treatment effect of the CAR-T is improved (figure 10).

Example 11: calicheamicin enhances the in vivo therapeutic effect of CAR-T cells on solid tumors

To demonstrate that calicheamicin enhances the in vivo therapeutic effect of CAR-T cells on solid tumors, this example utilizes NSG mouse subcutaneous tumor model to detect the in vivo killing ability of CAR-T cells on solid tumor cells on day 7 after administration of different calicheamicin drug concentrations. CD3 in peripheral blood of healthy volunteers on day 0 ⁺ T cells were isolated and purified at 1.0X10 ⁶ After 3 days of in vitro stimulation culture with CD3 antibody (600 ng/ml), CD28 antibody (300 ng/ml) and recombinant human IL-2 (10 ng/ml) each well was inoculated in 24-well plates, CD 19-targeted CAR lentiviruses were infected, CAR-T cells were given a calicheamicin treatment (working concentration of 2.5 μm) on day 5, CAR-T cells were expanded in vitro to day 14 (calicheamicin treatment time total of 9 days), and the treated CAR-T cells were taken for in vivo experiments (CM treated CAR-T group). The CAR-T group and NC-T group were the same as in example 10. Stable A375-CD19-luc strain (expressing both CD19 antigen and luciferase) was grown at 1.0X10 ⁶ Subcutaneous inoculation of the mice on the right upper back of NSG mice, detection of in vivo tumor cell distribution by in vivo imager on day 7 after inoculation, dividing mice into 3 groups of 5 mice each, and administering 5.0X10 at day 7 after injection of A375-CD19-luc cells ⁶ Infusion of unmodified T cells, CAR-T cells and calicheamicin-treated CAR-T cells via tail vein alone, respectively, with live use on day 14 and day 21The in vivo imager detects the in vivo growth of tumor cells.

Experimental results show that the proliferation speed of tumor cells in the mice of the unmodified T cell group is very high, the CAR-T cell group can inhibit tumor growth, but the tumor mass volume of the mice of the CAR-T cell group treated by the calicheamicin is increased compared with that of the mice of the CAR-T cell group on the 21 st day, the inhibition effect of the tumor cells in the mice of the CAR-T cell group is obviously superior to that of the mice of the CAR-T cell group, the tumor cell volume of the mice of the calicheamicin on the 21 st day is obviously reduced, and the in vivo killing effect of the CAR-T cells on solid tumors is shown to be enhanced, and the in vivo treatment effect of the CAR-T is improved (figure 11).

Example 12: calicheamicin enhances the in vivo therapeutic effect of TCR-T cells on solid tumors

To demonstrate that the effect of calicheamicin on enhancing the in vivo therapeutic effect of TCR-T cells on solid tumors, this example uses the NSG mouse subcutaneous tumor model to examine the in vivo killing ability of TCR-T cells on solid tumor cells on day 7 after administration of different calicheamicin drug concentrations. CD3 in peripheral blood of healthy volunteers on day 0 ⁺ T cells were isolated and purified at 1.0X10 ⁶ After 3 days of in vitro stimulation culture with CD3 antibody (600 ng/ml), CD28 antibody (300 ng/ml) and recombinant human IL-2 (10 ng/ml), each well was inoculated in 24-well plates, infected with TCR lentivirus targeting NY-ESO1, TCR-T cells were given a treatment with calicheamicin (working concentration of 2.5. Mu.M) on day 5, TCR-T cells were expanded in vitro to day 14 (treatment time with calicheamicin total of 9 days), and the treated TCR-T cells were taken for in vivo experiments (CM treated CAR-T group). TCR-T cells without calicheamicin were used as negative control (DMSO was used instead), and unmodified T cells were used as blank control. Wherein, as a negative control, the TCR-T cell treatment protocol without calicheamicin treatment was the same as in figure 1, except that DMSO was used instead of calicheamicin; the in vitro culture procedure for unmodified T cells was the same as in fig. 1, except that no virus infection was performed. A375-luc stable transformant (expressing NY-ESO1 antigen and luciferase) was grown at 1.0X10 ⁶ Subcutaneous inoculation of mice on the right upper back of NSG mice, detection of in vivo tumor cell distribution by in vivo imager on day 7 after inoculation, dividing mice into 3 groups of 5 mice each, and administration of 5.0X10 s on day 7 after injection of A375-luc cells ⁶ Individual/just divideThe in vivo growth of tumor cells was examined by in vivo imaging on days 14 and 21 by reinfusion of unmodified T cells, TCR-T cells and calicheamicin-treated TCR-T cells via the tail vein.

Experimental results show that the proliferation speed of tumor cells in the mice of the unmodified T cell group is very high, the TCR-T cell group can inhibit the tumor growth, but the tumor mass volume on the 21 st day is increased compared with that on the 14 th day, the in vivo tumor cell inhibition effect of the mice of the TCR-T cell group treated by the calicheamicin is obviously superior to that of the TCR-T cell group, the tumor cell volume on the 21 st day is obviously reduced, and the in vivo killing effect of the TCR-T cells on solid tumors is shown to be enhanced by the calicheamicin, and the in vivo treatment effect of the TCR-T is improved (figure 12).

Example 13: colamycin improves survival in CAR-T cell reinfusion

To demonstrate that the effect of calicheamicin on CAR-T cell survival in vivo was improved by calicheamicin, this example utilized NSG mouse subcutaneous tumor model to detect the effect of calicheamicin on CAR-T cell survival in vivo. Stable A375-CD19-luc strain (expressing both CD19 antigen and luciferase) was grown at 1.0X10 ⁶ Subcutaneously inoculating on the right upper back of NSG mice, detecting in vivo distribution of tumor cells by using in vivo imager at 7 days after inoculation, dividing mice into 3 groups of 5 mice each, and 5.0X10 ⁶ Detection of human CD3 in tumor, spleen and bone marrow in 3 groups of mice on day 21 by flow cytometry ⁺ T cell ratio.

Experimental results found that the proportion of T cells in small tumors, spleen and bone marrow was significantly increased in the calicheamicin-treated CAR-T cell group compared to the unmodified T cell group and the normal CAR-T cell group, indicating that calicheamicin can enhance the survival rate of CAR-T cells in vivo after reinfusion (fig. 13).

Example 14: colamycin improves survival in TCR-T cell reinfusion

To demonstrate that the effect of calicheamicin on the in vivo survival of TCR-T cells was improved by the use of NSG mouse subcutaneous tumor model in this example. Stabilizing A375-lucStrains (simultaneously expressing NY-ESO1 antigen and luciferase) at 1.0X10 ⁶ Subcutaneously inoculating on the right upper back of NSG mice, detecting in vivo distribution of tumor cells by using in vivo imager at 7 days after inoculation, dividing mice into 3 groups of 5 mice each, and 5.0X10 ⁶ Method for detecting human CD3 in tumor, spleen and bone marrow of 3 groups of mice by flow cytometry on day 21 ⁺ T cell ratio.

The experimental results found that the proportion of T cells in the small tumor, spleen and bone marrow was significantly increased in the group of TCR-T cells treated with calicheamicin compared to the group of unmodified T cells and the group of normal TCR-T cells, indicating that calicheamicin can enhance survival in vivo after TCR-T cell reinfusion (figure 14).

The foregoing is merely exemplary of the present application, and specific technical solutions and/or features that are well known in the art have not been described in detail herein. It should be noted that, for those skilled in the art, several variations and modifications can be made without departing from the technical solution of the present application, and these should also be regarded as the protection scope of the present application, which does not affect the effect of the implementation of the present application and the practical applicability of the patent. The protection scope of the present application is subject to the content of the claims, and the description of the specific embodiments and the like in the specification can be used for explaining the content of the claims.

Claims

1. Use of a calicheamicin for the preparation of an inhibitor of depletion of genetically engineered T cells, wherein the genetically engineered T cells are CAR-T cells or TCR-T cells;

The method comprises the steps of adding the calicheamicin into an in vitro culture system of genetically engineered T cells; the in vitro culture system is lymphocyte culture medium containing colimycin, IL-7, IL-15, green streptomycin diabody and FBS; the working concentration of the colimycin in the in vitro culture system is 1.25 mu M-5 mu M.

2. The application of the calicheamicin in preparing a tumor therapeutic drug is characterized in that the tumor therapeutic drug comprises genetically engineered T cells which are cultured in vitro, wherein the genetically engineered T cells are CAR-T cells or TCR-T cells; the in vitro culture environment contains a lymphocyte culture medium of colimycin, IL-7, IL-15, double antibody of green streptomycin and FBS; the working concentration of the colimycin is 1.25 mu M-5 mu M; the tumor is a hematological tumor or a solid tumor.

3. The use of a calicheamicin according to claim 2 for the preparation of a medicament for the treatment of tumors, wherein said calicheamicin is used for slowing down the depletion of genetically engineered T cells and for increasing CD4 ⁺ T cells and CD8 ⁺ T cell ratio and increase central memory T cell population ratio.

4. The application of the colimycin in preparing an in vitro T cell proliferation activation inhibitor is characterized in that the colimycin is added into an in vitro culture system of genetically modified T cells; the in vitro culture system is lymphocyte culture medium containing colimycin, IL-7, IL-15, green streptomycin diabody and FBS; the working concentration of the colimycin in an in vitro culture system is 1.25 mu M-5 mu M; the genetically engineered T cells are CAR-T cells or TCR-T cells.