CN117866906A - Application of FOXR1 inhibitor in preparation of medicines for treating tumors - Google Patents

Abstract

The invention discloses application of FOXR1 inhibitor in preparing medicaments for treating tumors, and discovers that the FOXR1 is CD8 for the first time ⁺ The main negative regulatory factor of the T anti-tumor reaction, and further proves that inhibiting the expression of FOXR1 in T cells can obviously improve the in-vivo and in-vitro killing capacity of CAR-T cells on tumor cells through cell experiments and animal experiments, so that the anti-tumor treatment effect is improved.

Description

Application of FOXR1 inhibitor in preparation of medicines for treating tumors

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to application of a FOXR1 inhibitor in preparation of a medicine for treating tumors.

Background

Tumor immunotherapy is one of cancer treatments aimed at stimulating the human immune system to recognize and attack cancer cells. It refers to a range of therapeutic approaches including immune checkpoint blockade, engineered T cells, cytokines, co-stimulatory receptor agonists, vaccines and other therapies. Compared with traditional tumor therapy, tumor immunotherapy is known for its good therapeutic effect, good specificity and few side effects. In tumor immunotherapy, CD8 ⁺ T cells are being widely studied by a large number of researchers as a promising approach to the treatment of cancer. CD8 ⁺ T cells can recognize tumor-associated antigens and specifically kill cancer cells, CD8 ⁺ The number and function of T determines to a large extent the efficacy and prognosis of tumor immunotherapy. During acute stimulation, primary CD8 ⁺ T cells differentiate into effector CD8 after stimulation by antigen ⁺ T cells. Subsequently, effect CD8 ⁺ T cells eliminate various antigens by producing cytotoxic molecules such as perforin and granzyme B, and secreting effector cytokines such as interleukin IL-2, interferon (IFN) -gamma, and Tumor Necrosis Factor (TNF) -alpha, etc. However, in long-term chronic stimulatory antigens and inflammatory factors, CD8 ⁺ T cells gradually exhibit decreased secretion of toxic molecules and cytokines, decreased proliferation capacity, overexpression of inhibitory receptors, and metabolic and epigenetic changes, leading to failure of antigen clearance, i.e. T cell depletion. T cell depletion is a process in which T cells gradually lose function due to prolonged exposure to antigen, resulting in a reduced immune response. This is a result of a combination of mechanisms, including up-regulation of inhibitory receptors such as PD-1, TIM-3 and LAG-3, which prevent T cells from effectively attacking target cells. To some extent, T cell depletion hampers the development of tumor immunotherapy, especially CAR-T depletion in tumor microenvironments, severely limiting the efficacy of CAR-T therapy.

Overall, understanding T cell depletion and developing strategies to overcome it is an important area of research in immunology. With the development of CRISPR technology, researchers now have powerful methods to explore key regulators of T cell depletion in tumor microenvironments. Previous studies have shown that whole genome CRISPR screening in vitro using primary T cells is feasible. However, it is notable that the in vitro environment does not fully replicate the complexity of the in vivo tumor microenvironment, resulting in the loss of some critical information. Thus, to better identify the resulting CD8 in the tumor microenvironment ⁺ Regulatory factor of T cell depletion, the invention relates to CD8 in tumor microenvironment in early experimental research ⁺ T cells were subjected to whole genome CRISPR screening. Based on this, the present invention found and clarified for the first time that FOXR1 is CD8 ⁺ The main negative control factor of the T anti-tumor reaction can inhibit the expression of FOXR1 in T cells, improve the expression of cytokines and toxic molecules such as IL-2, TNF-alpha, IFN-gamma, granzyme B, perforin and the like, and relieve CD8 in tumor microenvironment ⁺ The depletion of T cells improves the in-vitro and in-vivo killing capacity of CAR-T cells on tumor cells, thereby improving the anti-tumor treatment effect, which indicates that FOXR1 is a potential target point of tumor immunotherapy.

Disclosure of Invention

The invention aims to provide a novel tumor immunotherapy target FOXR1 and application of a FOXR1 inhibitor in preparing medicines for treating tumors.

The above object of the present invention is achieved by the following technical solutions:

in a first aspect of the invention, a recombinant T cell is provided.

Further, the recombinant T cell does not contain the FOXR1 gene, or the biological function of the FOXR1 gene product of the recombinant T cell is inhibited. Further, the recombinant T cell is obtained by knocking out the FOXR1 gene of the target T cell or inhibiting the FOXR1 gene expression of the target T cell. Further, the target T cell is a CAR-T cell or CD8 ⁺ T cells. Further, the CAR-T cells are CD19 CAR-T cells.

In the present invention, the information of the gene FOXR1 is as follows: gene ID for Gene FOXR1 is 283150, details of which are available in the NCBI database (https:// www.ncbi.nlm.nih.gov/Gene /) according to the Gene ID described above.

In the present invention, the CAR-T cell refers to a Chimeric Antigen Receptor (CAR) T cell engineered to recognize and bind to an antigen of interest expressed by a cell, such as an antigen expressed by a disease cell, such as an antigen expressed by a cancer cell. T cells expressing the CAR polypeptide are referred to as CAR T cells. CARs have the ability to redirect T cell specificity and reactivity to a selected target in a non-MHC restricted manner. non-MHC-restricted antigen recognition confers the ability of CAR-T cells to recognize antigen independent of antigen processing, thereby bypassing the primary mechanism of tumor escape. In a specific embodiment of the invention, the CD19 CAR-T and CAR 19T refer to chimeric antigen receptor T cells that target the human CD19 antigen.

In the invention, the in-vivo and in-vitro killing capacity of the recombinant T cells on tumor cells is obviously improved. The knockout of FOXR1 gene or the inhibition of FOXR1 gene expression in T cells can improve the expression of IL-2, TNF-alpha, IFN-gamma and other effector molecules and granzyme B, perforin and other toxic molecules, and relieve CD8 in tumor microenvironment ⁺ And (3) the T cells are exhausted, so that the killing capacity of the recombinant T cells to tumor cells is improved. In some embodiments, the recombinant T cells can be derived from a parent T cell (e.g., an unedited wild-type T cell) obtained from a suitable source (e.g., one or more mammalian donors). In other embodiments, the parent T cell is a primary T cell (e.g., untransformed and terminally differentiated T cells) obtained from one or more human donors. Alternatively, the parent T cells may be differentiated from precursor T cells obtained from one or more suitable donors or stem cells that may be cultured in vitro, such as hematopoietic stem cells or induced pluripotent stem cells (ipscs). In certain embodiments, the T cells may be derived from one or more suitable mammals, such as one or more human donors. T cells may be obtained from a variety of sources, including but not limited to: peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymusTissue, tissue at the site of infection, ascites, pleural effusion, spleen tissue, tumors. In certain embodiments, T cells can be obtained from a unit of blood collected from a subject using a number of techniques known to those skilled in the art, such as density gradient centrifugation (e.g., FICOLL separation).

In a second aspect, the invention provides a method for preparing a recombinant T cell according to the first aspect of the invention.

Further, the preparation method comprises the following steps: knocking out the FOXR1 gene of the target T cell or inhibiting the FOXR1 gene expression of the target T cell to obtain the recombinant T cell of the first aspect of the invention. Further, agents that knock out the FOXR1 gene or inhibit FOXR1 gene expression include: interfering molecules that specifically interfere with FOXR1 gene expression, CRISPR gene editing reagents for FOXR1 genes, homologous recombination reagents for FOXR1 genes, and/or site-directed mutagenesis reagents for FOXR1 genes; the homologous recombination reagent aiming at the FOXR1 gene or the site-directed mutagenesis reagent aiming at the FOXR1 gene can carry out the functional loss mutation on the FOXR 1. Further, the interfering molecule comprises shRNA, siRNA, miRNA and/or antisense nucleic acid; the CRISPR gene editing reagent comprises an RNP complex. Further, the sequence of the shRNA is shown as SEQ ID NO 1-2, SEQ ID NO 3-4, SEQ ID NO 5-6, SEQ ID NO 7-8 or SEQ ID NO 9-10; the RNP complex comprises crRNA, tracrRNA and Cas9 proteins targeting FOXR1, and the crRNA sequence is shown as SEQ ID NO. 11.

In the present invention, any agent capable of knocking out the FOXR1 gene or inhibiting the expression of the FOXR1 gene is within the scope of the present invention, including but not limited to: shRNA, siRNA, miRNA, antisense nucleic acids, CRISPR gene editing reagents, small molecule compounds, peptides, peptidomimetics, matrix analogs, aptamers, antibodies, dsRNA, and the like.

In some embodiments, the FOXR1 sequence information provided according to the present invention may be prepared by methods known in the art to obtain interfering molecules as described above, and in the present invention, the methods for preparing interfering molecules as described above are not particularly limited, including but not limited to: chemical synthesis, in vitro transcription, and the like. The interfering molecules may be delivered into the cells of interest (e.g., T cells) by use of an appropriate transfection reagent, or may also be delivered into the cells of interest using a variety of techniques known in the art. In some embodiments, the agent that knocks out the FOXR1 gene or inhibits expression of the FOXR1 gene further comprises a small molecule compound directed against FOXR 1. Those skilled in the art can use conventional screening methods in the art to screen such small molecule compounds, and such small molecule compounds that are able to knock out the FOXR1 gene or inhibit the expression of the FOXR1 gene obtained by screening are also included in the scope of the present invention. In some embodiments, the FOXR1 gene in T cells can be knocked out using a CRISPR/Cas (e.g., cas9, cas12a, cas13, cas 14) gene editing system. Common methods of knocking out the FOXR1 gene include: designing crrnas, complementing a Spacer (Spacer) sequence with a DNA target sequence, synthesizing the crrnas, mixing the crrnas with the tracrRNA, base pairing the crrnas with the tracrRNA through a repeat region (Repeats) of the crrnas to form guide RNAs (grnas), uniformly mixing the grnas with Cas9 proteins to form ribonucleoprotein bodies (RNPs), introducing the RNPs into cells by means of electrotransfection or the like, recognizing the DNA target sequence through the Spacer of the crrnas, recognizing the PAM sequence through the Cas9 proteins, and shearing the DNA to realize target gene knockout. In addition, other gene editing techniques (e.g., TALEN, ZFN, RNAi) can be used in the present invention as well. In some embodiments, the FOXR1 gene in T cells may be specifically targeted to be expressed defective or deleted using a method of homologous recombination. The Cre and loxp methods can also be used to selectively knock out the FOXR1 gene in the genome of T cells, reducing or inactivating its expression in T cells. In some embodiments, a site-directed mutagenesis approach may be employed to specifically mutate the FOXR1 gene in T cells, and FOXR1 genes disrupted by such mutation may contain one or more mutations (e.g., insertions, deletions, nucleotide substitutions, etc.) relative to the wild-type counterpart to substantially reduce or completely eliminate the activity of the encoded gene product. The one or more mutations may be located in a non-coding region, e.g., a promoter region, a regulatory region that regulates transcription or translation; or an intron region. Alternatively, the one or more mutations may be located in a coding region (e.g., in an exon). In some cases, the disrupted FOXR1 gene does not express or expresses the encoded protein at a greatly reduced level. In other cases, the disrupted FOXR1 gene expresses the encoded protein in a mutated form, which has no significant reduction in functionality or activity. In some embodiments, T cells comprising the disrupted FOXR1 gene do not express a detectable level of protein encoded by the gene (e.g., by antibody or flow cytometry). T cells that do not express detectable levels of protein may be referred to as T cells that knock out the FOXR1 gene.

A third aspect of the invention provides any one of the following products:

(1) A population of recombinant T cells comprising the recombinant T cells of the first aspect of the invention;

(2) An activated recombinant T cell obtained by activating the recombinant T cell of the first aspect of the invention;

(3) An activated recombinant T cell population comprising the activated recombinant T cells;

(4) A pharmaceutical composition comprising a recombinant T cell, the population of recombinant T cells, the activated recombinant T cell and/or the activated population of recombinant T cells according to the first aspect of the invention;

(5) A pharmaceutical formulation comprising the pharmaceutical composition;

(6) An in vitro method of enhancing the anti-tumor effect of T cells, the method comprising: knocking out the FOXR1 gene of the target T cell or inhibiting the FOXR1 gene expression of the target T cell;

(7) A method of screening for potential substances capable of promoting T cell anti-tumor effects, the method comprising the steps of: (1) treating the expression system expressing FOXR1 with a candidate substance; (2) detecting the expression or activity of FOXR1 in the system, wherein the candidate substance is a potential substance capable of promoting T cell anti-tumor effect if the candidate substance statistically significantly down-regulates the expression or activity of FOXR 1.

In some embodiments, the step (1) comprises: adding a candidate substance to the expression system in a test set; and/or, the step (2) comprises: detecting expression or activity of FOXR1 in the system; and comparing with a control group, wherein the control group is an expression system without the addition of the candidate substance; if the candidate agent statistically significantly down-regulates the expression or activity of FOXR1, the candidate agent is a potential agent that promotes the efficacy of T cell anti-tumor immunotherapy. In some embodiments, the candidate substance includes, but is not limited to: regulatory molecules designed for FOXR1, fragments or variants thereof, genes encoding same, or upstream and downstream molecules or signaling pathways thereof, or constructs thereof (e.g., shRNA, siRNA, gene editing agents, expression vectors, recombinant viral or non-viral constructs, etc.), chemical small molecules (e.g., specific inhibitors or antagonists), interactive molecules, etc. In some embodiments, the system is selected from: cell systems (e.g., FOXR1 expressing cells or cell cultures), subcellular (culture) systems, solution systems, tissue systems, organ systems, or animal systems. In some embodiments, the method further comprises: further cell experiments and/or animal experiments were performed to verify that the potential substances obtained were further selected and determined from candidate substances useful for promoting the therapeutic efficacy of T cell anti-tumor immunotherapy. In some embodiments, the activation of the recombinant T cells of the first aspect of the invention may be performed using methods well known in the art, including, but not limited to: antigen presenting therapy (APC), antibody Dependent Cellular Cytotoxicity (ADCC), antigen-antibody complexes (Ag-Ab), and Bioreactors (BR), etc., any recombinant T cell capable of activating the recombinant T cell of the first aspect of the invention, and thus the resulting activated recombinant T cell, is within the scope of the invention.

In some embodiments, the pharmaceutical compositions of the present invention may further comprise a pharmaceutically acceptable carrier and/or adjuvant. The pharmaceutical formulation according to the present invention may have any one of the formulations selected from the group consisting of: tablets, pills, powders, granules, capsules, suspensions, solutions, emulsions, syrups, sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations and suppositories. Furthermore, one or more administrations may be performed. Routes of administration include, but are not limited to: intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intranasal, intrapulmonary, intrarectal, and the like. In particular embodiments, the pharmaceutical formulations provided herein can be formulated into a variety of dosage forms as desired, and the dosage to be beneficial to the patient can be determined by the clinician based on the type, age, weight, general disease condition, mode of administration, etc. of the subject patient.

Furthermore, the present invention provides a method of treating a tumor in a subject in need thereof, the method comprising: administering to a subject in need thereof a therapeutically effective amount of a recombinant T cell according to the first aspect of the invention, a recombinant T cell population according to the third aspect of the invention, activated recombinant T cells, activated recombinant T cell population, pharmaceutical composition and/or pharmaceutical formulation.

Further, the tumors include any tumor known in the art at present and new types of tumors that may be found in the future, including but not limited to: solid tumors, non-solid tumors, including but not limited to: lung cancer, melanoma, ovarian cancer, cervical cancer, bladder cancer, breast cancer, head and neck cancer, pancreatic cancer, liver cancer, stomach cancer, colorectal cancer, renal cancer, neuroblastoma, osteosarcoma, hodgkin's lymphoma, chondrosarcoma, and the like. The non-solid tumors include, but are not limited to: glioma, non-hodgkin lymphoma, multiple myeloma, leukemia (e.g., acute lymphoblastic leukemia, acute myelogenous leukemia, chronic lymphoblastic leukemia, chronic myelogenous leukemia), and the like.

A fourth aspect of the invention provides the use of any one of the following:

(1) The application of the substance for knocking out the FOXR1 gene or the substance for inhibiting the expression of the FOXR1 gene in the preparation of a reagent for promoting the curative effect of T cell anti-tumor immunotherapy;

(2) Use of a FOXR1 gene knockout substance or a FOXR1 gene expression inhibiting substance in the preparation of a reagent for promoting CAR-T cell anti-tumor immunotherapy efficacy;

(3) The application of the substance for knocking out the FOXR1 gene or the substance for inhibiting the expression of the FOXR1 gene in preparing the T cell immune tumor related medicament;

(4) Use of a FOXR1 gene knockout substance or a FOXR1 gene expression inhibiting substance in the preparation of a reagent for reducing T cell depletion;

(5) Use of a FOXR1 gene knockout substance or a FOXR1 gene expression inhibiting substance in the preparation of a reagent for enhancing T cell activity;

(6) Use of a FOXR1 gene knockout substance or a FOXR1 gene expression inhibiting substance in the preparation of recombinant T-cells for the treatment of tumors;

(7) Use of a FOXR1 gene knockout substance or a FOXR1 gene expression inhibiting substance in the preparation of a medicament for the treatment of a tumor.

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

the invention discovers that FOXR1 is CD8 for the first time ⁺ The main negative regulation factor of the T anti-tumor reaction and further proves that inhibiting the expression of FOXR1 in T cells can relieve CD8 in tumor microenvironment through cell experiments and animal experiments ⁺ The invention provides a new therapeutic target and therapeutic strategy for the technical field of tumor immunotherapy, and has important scientific significance and clinical application value.

Drawings

FIG. 1 illustrates FOXR1 inhibition of mouse CD8 for further screening ⁺ T cell function, wherein, panel a: structure of CAR19 viral plasmid, panel B: qPCR detects the knockdown efficiency of FOXR1 in EL4 lymphoma cells, panel C: qPCR assay of IL-2 expressed in EL4 lymphoma cells after knockdown of FOXR1, D plot: primary CD8 transfected with shFOXR1 retrovirus or control retrovirus ⁺ Flow cytometry analysis and quantification of IL-2 and IFN- γ in T cells, < 0.05, < 0.01, < 0.001, < P;

FIG. 2 illustrates FOXR1 inhibition of mouse CD8 for further screening ⁺ T cell function, wherein, panel a: for detecting FOXR1 inMouse CD8 ⁺ Pattern diagram of antitumor function in T, B: flow cytometry analysis and quantification of IL-2, IFN- γ and TNF- α in primary OT 1T cells transfected with shFOXR1 retrovirus or control retrovirus 9 days after adoptive feedback, < 0.05, < 0.01, < 0.001;

FIG. 3 is a FOXR1 deletion promoting human CD8 ⁺ T cell function, wherein, panel a: western blot analysis showed FOXR1 protein levels in Jurkat E6-1 cells transfected with shFOXR1 lentivirus or control lentivirus, panel B: FOXR1 expressed mRNA levels in Jurkat E6-1 cells transfected with shFOXR1 lentivirus or control lentivirus, C panels: mRNA expression levels of IL-2 (n=3) in Jurkat E6-1 cells transfected with shFOXR1 lentivirus or control lentivirus after 8 hours of treatment with phorbol 12-tetradecanoate 13-acetate (PMA) (50 ng/mL) and ionomycin (I) (1 μg/mL), D plot: cas9RNP complex electrotransformation of human primary CD8 ⁺ Fluorescence microscopy and flow cytometry analysis of T cells; qPCR and western blot analysis of FOXR1 expression levels in human primary cd8+ T cells transfected with FOXR1-KO-Cas9RNP complex;

FIG. 4 is a FOXR1 deletion promoting human CD8 ⁺ T cell function, wherein, panel a: perforin MFI (left effector phase CD 8) ⁺ T, right depletion phase CD8 ⁺ T), B and C: FOXR1 KO human primary CD8 at effector phase (B panel) or depleting phase (C panel) after 6 hours of treatment with CD3 and CD28 antibody activated beads ⁺ T cells and WT CD8 ⁺ qPCR analysis of IL-2, TNF, IFNG, GZMB mRNA expression in T and flow cytometry analysis of IL-2, TNF-alpha, IFN-gamma and granzyme B, perforin in cell supernatants after stimulation of 24 h,: P<0.01，***P<0.001，***P<0.0001；

FIG. 5 shows a human CD8 ⁺ Knockout of FOXR1 in T cells constructs CAR 19T and schematic representation of CAR 19T adoptive feedback;

figure 6 is that the deletion of FOXR1 increases the antitumor efficacy of CAR-T cells, wherein, panel a: flow detection of lentivirus-infected primary CD8 overexpressing CAR 19T ⁺ Percentage of CAR 19T cells 3 days after T, panel B: non-radioactive cytotoxicity of CAR 19T co-cultured with Raji, K562-hcd19 and Hepg2-hcd19Sex analysis (n=3), panel C: CBA analysis CAR 19T with tumor cells co-culture supernatants IFN- γ, IL-2, IL-4, IL-6, IL-10 (n=3), D plot: raji cells (5×10) ⁵ ) 6 days after inoculation, by 5X 10 ⁵ Tumor volume (mm) of B-NDG mice treated with human CAR 19T cells (WT and FOXR1 KO) ³ ) One-way anova was compared to multiple, untreated experimental animals (n=4). CAR T treated experimental mice (n=11), E plot: tumor-bearing B-NDG mice receiving CAR-19T reinfusion survived until 1500 mm ³ The statistical data is a log rank Mantel-Cox test, and the results are expressed as mean ± standard deviation, P < 0.05, P < 0.01, P < 0.001, P < 0.0001, ns, not significant.

Detailed Description

The invention is further illustrated below in conjunction with specific examples, which are provided solely to illustrate the invention and are not to be construed as limiting the invention. One of ordinary skill in the art can appreciate that: many changes, modifications, substitutions and variations may be made to the embodiments without departing from the spirit and principles of the invention, the scope of which is defined by the claims and their equivalents. The reagents and materials used in the present invention are readily available to those of ordinary skill in the art, and are commercially available unless otherwise indicated, and the present invention is not directed to specific conditions for the experimental procedure, and the detection is usually carried out under conventional conditions or under conditions suggested by the manufacturer.

Example FOXR1 inhibitor for use in the preparation of a medicament for the treatment of tumors

1. Experimental materials

(1) Experimental animal

C57BL/6 mice were from Beijing visual River laboratory (Beijing, china). Rosa26-Cas9 knockout mice were purchased from Nanjing Seiko Biotech Inc. Rag1 KO mice were purchased from Shanghai, south model biotechnology, inc. OT-1 mice were crossed with Cas9 mice and then bred indoors. B-NDG (NOD-Parkdcsccid IL2rgtm 1/Bcgen) mice were purchased from the Beijing animal center of the Baiocin diagram. All mice were maintained under specific pathogen-free conditions. All mouse experiments were performed under supervision of the Institutional Animal Care and Use Committee (IACUC) at the national academy of medicine basic medical institute. All studies were performed on animals of 6 to 8 weeks of age.

(2) Cell lines

293T cell line, jurkat E6-1 lymphoma cell line, EL4 lymphoma cell line and human Raji lymphoma cell line were obtained from the Beijing institute of medicine and cell resource center (PCRC). B16-OVA and MC38-OVA cell lines and Platinum-E (Plat-E) cell lines were derived from the Beijing institute of basic medicine, proc. MC38-OVA and B16-OVA cell lines were maintained in RPMI-1640 (Corning) supplemented with 10% FBS (Gibco) at 37℃with 5% CO ₂ Is maintained. 293T and Plat-E cells in DMEM (Corning) supplemented with 10% FBS serum, 100U/mL penicillin (Solarbio) and 100 mg/mL streptomycin (Solarbio) at 37℃with 5% CO ₂ Is maintained. EL4 cells were incubated in DMEM (Corning) supplemented with 10% horse serum at 37℃with 5% CO ₂ Is maintained. STR assays were performed on all cell lines and were determined to be mycoplasma free.

(3) Reagent consumable

The main reagent consumables used in this example are shown in Table 1 below.

Table 1 major reagent consumables

2. Experimental method

(1) Isolation and culture of primary T cells

Spleen and inguinal lymph nodes were collected from Cas9OT1 mice and placed in pre-chill PBS (Sangon Biotech) containing 2% FBS (Gibco). The minced spleen and inguinal lymph node tissue was then gently triturated into single cells by a 70 mM filter. After centrifugation, the erythrocytes are lysed by using a Soxhobao erythrocyte lysis buffer solution, incubated on ice for 15 minutes, mixed uniformly by reversing every 3 minutes, then the cell lysis solution is neutralized by using PBS, centrifuged for 500 g and 5 minutes, resuspended by using PBS, the resuspended cells are subjected to a filter screen of 40 μm, resuspended by using PBS again,10. Mu.L of cells were diluted 10-fold and counted, the remaining cells were centrifuged again, the total number of cells was counted while centrifugation was completed, and after centrifugation was completed, 1X 10 cells were washed with easy Sep ™ buffer (STEMCELL Cat # 20144) ⁸ The cells were resuspended at a density of/mL. Mouse Naive CD8 using easy Sep ™ ⁺ T cell isolation kit (STEMCELL Cat # 19858) for enriching Naive CD8 ⁺ T cells, after sorting, a fraction of the cells were stained with anti-mouse CD8 antibody and CD8 was verified by flow cytometry ⁺ Purity of T cells, which were then resuspended in a suspension containing GlutaMax, 10% FBS (Gibco), 1% nonessential amino acids (Gibco), 100U penicillin/streptomycin, 55. Mu.M beta-mercaptoethanol (Gibco), 10 ng/mL mouse IL-2 (R)&D Systems) in RPMI 1640 medium. The cells were mixed at 1X 10 ⁶ Concentration of individual/mL 72 h activated cells were cultured on plates pre-coated with 5. Mu.g/mL anti-mouse CD3 (Biolegend) and 1. Mu.g/mL anti-mouse CD28 antibody (Biolegend), followed by adherent cells being blown off the plate and cultured in suspension. Cells were passaged at 1:2 every two days and maintained at 1X 10 ⁶ Individual cells/mL.

Isolation of human primary T cells: human active CD8 by Lymphoprep ™ gradient centrifugation (Stemcell Technologies) and easy Sep ™ ⁺ T cell isolation kit II (Stemcell Technologies) isolation of human CD8 from peripheral blood of healthy volunteers ⁺ T lymphocytes. According to the instructions, T cells were stimulated with ImmunoCult ™ human CD3/CD 28T cell activator (Stemcell Technologies) for 72 hours. Isolated T cells were cultured in ImmunoCurt ™ -XF T cell expansion medium (Stemcell Technologies) supplemented with 10 ng/mL recombinant human IL-2 (Stemcell Technologies). Cells were passaged every two days at 1:2 and maintained at a culture density of one million cells/mL.

(2) Tumor model

MC38-OVA cells resuspended in PBS were injected 5X 10 subcutaneously on the ventral side of each Rag1 KO mouse ⁵ /only. Each B-NDG mouse was inoculated subcutaneously with 5X 10 in a 1:1 mixture of PBS and Matrigel (Corning, standard formulation) ⁵ Raji/r. The mice body weight and tumor size were monitored every 3 days since tumor cell bearing. Tumor size was measured as (length. Times. Width)Degree)/2. To tumor with 1500 mm ³ Mice of (2) were regarded as the end point of the experiment and euthanized.

(3) Tumor invasive CD8 ⁺ Isolation of T cells

On day 9 after T cell reinfusion, mice were euthanized to harvest T cells in the tumor. Tumors were cut into small pieces, 1 h digested with type I and type IV collagenases, 0.5 mg/mL, and 200 IU/mL DNase I at 37℃and the tumor mass was gently crushed into individual cells by a 70 mM filter. Tumor infiltrating immune cells were isolated by density gradient centrifugation using Percoll (GE Healthcare). Enrichment of CD8 using EasySep ™ mouse CD8a Positive selection kit II (Stemcell Technologies) ⁺ T cells.

(4) Measurement of cytokine production

Mouse T cells were treated with 10. Mu.g/mL Brefeldin A (BFA), 50 ng/mL phorbol-ester (MCE) and 500 ng/mL ionomycin (MCE) at 37℃for 4 h and labeled with Zombie Violet ™ Fixable Viability Kit (bioleged) at room temperature for 20 minutes in the absence of light. Then, the cells were washed once with 1.5 mL of Biolegend's cell staining buffer (catalog No. 420201), the cell surface antibody mixture was added, and incubated at room temperature for 20 minutes in the absence of light. Next, the cells were washed twice and fixed for 20 minutes at room temperature in dark using cytofast ™ Fix/Perm Buffer (Biolegend), then the fixed cells were washed 2 times with wash Buffer and intracellular cytokines were stained for 20 minutes at room temperature in dark. The cells were then washed twice and filtered through a 40 μm filter and subjected to flow analysis using a BD LSR Fortessa machine (BD). Data analysis was performed using FlowJo software. All experiments were performed in at least two biological replicates. Antibodies were purchased from Biolegend and used according to the reagent instructions.

After stimulation of 24 h with CD3 and CD28 antibody activated magnetic beads by human T cells WT and KO FOXR1 CAR 19T, supernatants were collected and assayed for IL-2, TNF- α, interferon- γ, granzyme B and perforin expression using the biolegend LEGENDplex (catalog number: 741186) following the protocol described herein.

（5）FOXR1 KO

(1) shRNA mediated FOXR1 KO

Freshly isolated mouse primary CD8 ⁺ T cell isolation followed by 24 h activation with 5 μg/mL CD3 and 1 μg/mL CD28 pre-coated in well plates, infection with freshly coated shFOXR1 (shRNA targeting FOXR 1) retrovirus, mixing the virus supernatant with mouse T cell medium at a 1:2 volume ratio and assisted transfection with 8 μg/mL polybrene and centrifugation at 32℃for 60 min at 1100 g. After 4 hours, the virus-containing medium was replaced with fresh medium, and the next day 3.5. Mu.g/mL puromycin was added to the medium for selection, 48 h, and the knock-out effect was confirmed by Q-PCR detection Western blot analysis. Wherein, the sense sequence and antisense sequence information of shFOXR1 are shown in the following table 2.

TABLE 2 shFOXR1 sequence information

In the subsequent experiments, mouse shFOXR1-2 was selected in a murine cell experiment or a mouse-related animal experiment, and human shFOXR1-1 was selected in a human cell experiment.

(2) Cas9RNP mediated FOXR1 KO

Freshly isolated human primary CD8 ⁺ T activation of CD8 in ImmunoCurt ™ -XF T cell expansion Medium (Stemcell Technologies) supplemented with T cells TransAct (Miltenyi) and 10 ng/mL human IL-2 ⁺ T cells for 24 hours. The following day, cas9-RNP complex was constructed: mu.L of 100. Mu.M gene-specific Alt-R-crRNA (sgFOXR 1, IDT) and 1.5. Mu.L of 100. Mu.M Alt-R-tracrRNA (IDT, 1075928) were mixed in a nuclease-free double-stranded buffer, wherein the Alt-R-crRNA had a sequence of 5'-AAGGCAAACCGCTTCCGAG-3' (SEQ ID NO: 11), PCR was heated to 95℃for 5 minutes, and then cooled to 25℃at a rate of-0.1℃per minute. Then 3 μl of these annealed crRNAtracrRNA complexes were incubated with 1 μl of 61 μΜ Cas9 nuclease V3 (IDT, 1081058) at room temperature for 30 min in the dark to form ribonucleoprotein complexes, then 24 h activated human primary CD8 was activated ⁺ T cells, 500 g, were centrifuged for 5 min and after centrifugation, 1X 10 ⁶ Individual cells were resuspended at 20 μlP3 electrotransport buffer. Then 20 μl of cells were mixed with 3 μl of Cas9RNP complex and 1.5 μl of 100 μΜ electrotransport enhancer (IDT, 1075916). Next, the mixture was transferred to an electrocuvette in a 16 well X-cell test tube strip (Lonza) using the 4D Nucleofector System (Lonza) electrometer EH115 procedure. Immediately after electrotransformation, 100 μl of pre-heated medium was added to the tube and incubated at 37 ℃ for 20 minutes. The cells were then incubated at 1X 10 ⁶ The density of individual cells/mL was transferred to fresh medium containing 10 ng/mL human IL-2, after 2 hours, human T cell activating reagent (Miltenyi) was added again, and after 8 hours ATTO 550TM was detected by flow cytometry to examine transduction efficiency. The next day, if necessary, the viral infection described above is added. Gene knockout effects were tested using Western blot analysis.

(6) Construction of CAR-T cells and adoptive feedback

To construct human CD8 ⁺ CAR-T cells, according to the reagent instructions, using EasySep ™ human CD8 ⁺ T cell isolation kit II (Stemcell Technologies) for isolating CD8 from peripheral blood of healthy volunteers ⁺ T lymphocytes, which are activated by T cell TransAct for 24 hours, are transduced by CAR19 lentivirus under 20 to 25 (MOI) infection, wherein the CAR19 viral plasmid is biologically constructed by Yun Zhou, and has a structure shown in figure 1A, and is obtained by sequentially connecting in series a CD8 leader peptide, a CD19-scfv sequence, a CD8 hinge region, a CD8 transmembrane domain, a CD28 co-stimulatory domain and a CD3-zeta signaling domain, wherein the corresponding amino acid sequences are shown in SEQ ID NO 12-17, and the corresponding nucleotide sequences are shown in SEQ ID NO 18-23. After 3 days, to determine the transduction efficiency of anti-CD 19 CAR, cells were stained with FITC-labeled human CD19 (20-291) protein, his-Tag DMF-filled antibody (bpuse) and detected by flow cytometry. All CD8 was cultured during the whole culture using ImmunoCurt ™ -XF T cell expansion Medium (Stemcell Technologies) supplemented with 10 ng/mL recombinant human IL-2 ⁺ T cells. Cells were passaged 1:2 every two days and maintained at 1X 10 ⁶ Individual cells/mL, CAR-T cells were expanded for 6-7 days until numbers were sufficient, 500 g,5 minutes, centrifuged, washed in PBS, counted,2×10 ⁶ CAR-T cells were resuspended in PBS and transferred back through the tail vein of the mice to B-NDG mice subcutaneously vaccinated with Raji. Mice were monitored for body weight, tumor volume size every 3 days since tumor cell inoculation. The construction of the CAR-T cells specifically comprises the following steps: peripheral Blood Mononuclear Cell (PBMC) separation and CD3 separation ⁺ T lymphocytes (also sorted with CD4, CD8 magnetic beads), infected cells, and expanded culture cells.

(7) In vitro killing assay of CAR-T cells

CAR-T cells, raji, K562-hcd19, hepG2 cells were collected separately and washed twice with PBS, trypan blue staining was used to detect cell viability above 90%, raji cells and K562-Hcd cells were co-cultured with CAR 19T cells of CAR19 and FOXR1-KO of WT (the CAR 19T cells of FOXR1-KO were obtained by the FOXR1 KO method described in the previous step (5) on the basis of T cells obtained by the FOXR1 KO method described in the previous step (5)), further co-cultured in 96 well plates according to E: T5:1, after 16 hours, cell culture medium supernatants were collected, and Cytox 96 was used ^® Non-radioactive cytotoxicity assay (Promega) and human Th1/Th2 cytokine cell assay (CBA) kit II (BD) were used to detect killing and cytokine secretion.

(8) In vitro T cell depletion assay

To induce mouse T cell depletion in vitro, after activation of 72 h of naive T cells with CD3-28, T cells were cultured using an orifice plate pre-coated with 5 μg/mL of anti-mouse CD3 antibody for chronic stimulation. Cells were passaged every two days into new plates pre-coated with CD3 antibodies and T cells were collected on days 0, 2, 4, 6 and 8 for Q-PCR analysis of T cell depletion. In contrast, acutely stimulated cells were cultured in suspension after 72 h activation, maintained with 10 ng/mL IL-2, passaged 1:2 every two days, and cell density was maintained at one million cells per mL.

To induce human T cell depletion in vitro, human CD8 was stimulated continuously with fresh CD3/CD28 activating magnetic beads (Thermo Fisher Scientific) in a 3:1 magnetic bead to cell ratio in a 48 well plate ⁺ T（2×10 ⁵ cells/mL), one cycle every 3 days, for 4 cycles. On day 12, T cells were stained and analyzed in parallel for expression of T cell depletion related markers. T cells were collected on days 0, 3, 6, 9, 12 and observed for depletion changes.

(9) Reverse transcription and real-time quantitative polymerase chain reaction

Total RNA was obtained using RNAfast200 kit (Shanghai Fei Biotechnology Co., ltd.) or TRIzol reagent (Invitrogen) using ReverTra-Ace ^® qPCR RT Master Mix (Toyobo, FSQ-301) cDNA was prepared and SYBR was used ^® Green Real-time PCR Master Mix (Toyobo, QPK-201) performed qPCR on Quantum studio 7 Flex machine (Thermo Fisher Scientific). Primer pairs for quantitative real-time PCR were designed and synthesized using a primer design method conventional in the art (primer sequences were designed using primer blast from NCBI website, primers were synthesized at the division of biological engineering (Shanghai)).

(10) Western blot

Cells were lysed using RIPA lysate (Thermo Fisher Scientific) supplemented with PMSF and CT to collect proteins, cellular protein extracts were separated on 10% SDS-PAGE gels, proteins were transferred onto nitrocellulose blotting membranes (cytova), membranes were blocked with Tris buffer/Tween 20 (TBST) containing 5% skim milk (Cell Signaling Technology) for 1 hour at room temperature, membranes were rinsed with TBST for 10 minutes, then incubated with the required primary antibody overnight at 4 ℃, membrane re-washed three times a day for 10 minutes each time, then HRP-coupled secondary antibody incubation membranes diluted 1:2000 for 1 hour at room temperature, membrane washed three times again with TBST, and immunoblots were detected using chromogenic solution (Millipore).

(11) Statistical analysis

Statistical analysis was performed using GraphpadPrism software. Data are expressed as mean ± Standard Deviation (SD). The Student's t test was used to evaluate the significance of differences in the mean between the two groups. Kaplan-Meier survival data was analyzed using the log rank (Mantel-Cox) test. Primary tumor growth over time was analyzed using a one-factor anova. All p-values were bilateral and were evaluated for statistical significance at the 0.05 level.

3. Experimental results

(1) Inhibition of FOXR1 promotes mouse CD8 ⁺ Antitumor function of T cells

To better focus on effector genes, this example intersected the first 100 genes enriched in two in vivo whole genome screens in the previous study and was performed in primary mouse CD8 ⁺ Dynamic changes in these top-ranked genes were detected in the T cell in vitro depletion model. We have primarily focused on a group of genes that are most significantly altered during chronic stimulation of primary T cells, suggesting their possible involvement in CD8 ⁺ T cell depletion process. Subsequently, we validated these molecules in the EL4 cell line and primary mouse CD8 ⁺ Function in T cells we found that decreasing FOXR1 expression was effective in increasing IL-2 expression in EL4 cell lines (fig. 1B and 1C). Similarly, inhibition of FOXR1 effectively enhanced expression of IL-2 and TNF- α in primary mouse T cells (FIG. 1D). To further explore FOXR1 tumor infiltration CD8 in vivo ⁺ Role in T we adoptive transfer of FOXR 1-reduced OT1 cells to Rag1 carrying MC38-OVA tumors ^-/- Mice (fig. 2A). After 9 days we found that knocking down FOXR1 increased tumor-invasive CD8 ⁺ IL-2, TNF-a and IFN-gamma expression of T (FIG. 2B).

(2) Knockout of FOXR1 promotes human CD8 ⁺ Function of T cells

To investigate whether the function of FOXR1 is conserved among different species, we subsequently knocked down FOXR1 in the human lymphoma cell line Jurkat E6-1, which was found to be effective in increasing IL-2 expression (fig. 3A, 3B and 3C). Furthermore, we utilized Cas9RNP system to knock out human primary CD8 ⁺ Expression of FOXR1 in T (fig. 3D). And increase effector cells and deplete CD8 ⁺ Expression of perforin, IL-2, TNF, IFNG and GZMB by T cells (FIGS. 4A, 4B and 4C), which indicates that FOXR1 functions in CD8 of different species ⁺ Is conserved in the T state.

(3) Knockout of FOXR1 promotes human CAR19 CD8 ⁺ Antitumor function of T cells

Whereas knockout of FOXR1 can enhance human primary CD8 ⁺ T cell function we then investigated whether FOXR1 can promote CAR-T cell function. We obtained Naive CD8 from peripheral blood of healthy volunteers ⁺ T and electro-knocking out FOXR1 from Cas9RNP complex 24 hours after activation we obtained the original CD8 in humans ⁺ T, FOXR1 was knocked out and transfected with CAR-19 lentivirus to construct FOXR1 KO CAR-19T, the experimental technical scheme of CAR-19T is shown in FIG. 5, and the constructed FOXR1 KO CAR-19T is shown in FIG. 6A. Co-culturing KO FOXR1 CAR-19T in vitro with tumor cells expressing human CD19 antigen, we found that KO FOXR1 was effective in enhancing the killing capacity of CAR-19T cells (FIG. 6B), enhancing the expression of IL-2, IL-4, IL-6, IFN-gamma by CAR-19T cells, and simultaneously reducing the expression of IL-10 in CAR-19T cells (FIG. 6C). In addition, to study the anti-tumor effect of FOXR1 KO CAR-T cells in vivo, we infused FOXR1 KO CAR-19T back into Raji lymphoma-inoculated B-NDG mice subcutaneously through the tail vein, found that FOXR1 KO CAR-19T could better inhibit tumor growth and prolong survival time of the carrying mice (fig. 6D and 6E), and as a result, it was shown that knockout of FOXR1 could significantly promote anti-tumor function of CAR-19T, FOXR1 was a potential clinical therapeutic target.

Claims (10)

1. A recombinant T cell, wherein the recombinant T cell does not contain a FOXR1 gene or the biological function of the FOXR1 gene product of the recombinant T cell is inhibited.

2. The recombinant T cell of claim 1, wherein the recombinant T cell is a target T cell obtained by knocking out the FOXR1 gene of the target T cell or by inhibiting the FOXR1 gene expression of the target T cell.

3. The recombinant T cell of claim 2, wherein the target T cell is a CAR-T cell or CD8 ⁺ T cells.

4. The recombinant T cell of claim 3, wherein the CAR-T cell is a CD19 CAR-T cell.

5. A method of producing a recombinant T cell according to any one of claims 1 to 4, comprising: knocking out the FOXR1 gene of the target T cell or inhibiting the FOXR1 gene expression of the target T cell to obtain the recombinant T cell of any one of claims 1 to 4.

6. The method of claim 5, wherein knocking out the FOXR1 gene or inhibiting the expression of the FOXR1 gene comprises: interfering molecules that specifically interfere with FOXR1 gene expression, CRISPR gene editing reagents for FOXR1 genes, homologous recombination reagents for FOXR1 genes, and/or site-directed mutagenesis reagents for FOXR1 genes;

the homologous recombination reagent aiming at the FOXR1 gene or the site-directed mutagenesis reagent aiming at the FOXR1 gene can carry out the functional loss mutation on the FOXR 1.

7. The method of claim 6, wherein the interfering molecule comprises shRNA, siRNA, miRNA and/or antisense nucleic acid;

the CRISPR gene editing reagent comprises an RNP complex.

8. The preparation method of claim 7, wherein the shRNA has a sequence shown in SEQ ID NO 1-2, SEQ ID NO 3-4, SEQ ID NO 5-6, SEQ ID NO 7-8 or SEQ ID NO 9-10;

the RNP complex comprises crRNA, tracrRNA and Cas9 proteins targeting FOXR1, and the crRNA sequence is shown as SEQ ID NO. 11.

9. A product of any one of the following:

(1) A population of recombinant T cells, wherein the population of recombinant T cells comprises the recombinant T cells of any one of claims 1-4;

(2) An activated recombinant T cell, wherein the activated recombinant T cell is obtained by activating the recombinant T cell of any one of claims 1-4;

(3) An activated recombinant T cell population, wherein the activated recombinant T cell population comprises the activated recombinant T cells;

(4) A pharmaceutical composition comprising the recombinant T cell, the population of recombinant T cells, the activated recombinant T cell, and/or the activated population of recombinant T cells of any one of claims 1-4;

(5) A pharmaceutical formulation, characterized in that the pharmaceutical formulation comprises the pharmaceutical composition;

(6) A method of screening for potential substances capable of promoting T cell anti-tumor effects, said method comprising the steps of:

(1) treating the expression system expressing FOXR1 with a candidate substance;

(2) detecting the expression or activity of FOXR1 in the system, wherein the candidate substance is a potential substance capable of promoting T cell anti-tumor effect if the candidate substance statistically significantly down-regulates the expression or activity of FOXR 1.

10. Use of any one of the following:

(1) The application of the substance for knocking out the FOXR1 gene or the substance for inhibiting the expression of the FOXR1 gene in the preparation of a reagent for promoting the curative effect of T cell anti-tumor immunotherapy;

(2) Use of a FOXR1 gene knockout substance or a FOXR1 gene expression inhibiting substance in the preparation of a reagent for promoting CAR-T cell anti-tumor immunotherapy efficacy;

(3) The application of the substance for knocking out the FOXR1 gene or the substance for inhibiting the expression of the FOXR1 gene in preparing the T cell immune tumor related medicament;

(4) Use of a FOXR1 gene knockout substance or a FOXR1 gene expression inhibiting substance in the preparation of a reagent for reducing T cell depletion;

(5) Use of a FOXR1 gene knockout substance or a FOXR1 gene expression inhibiting substance in the preparation of a reagent for enhancing T cell activity;

(6) Use of a FOXR1 gene knockout substance or a FOXR1 gene expression inhibiting substance in the preparation of recombinant T-cells for the treatment of tumors;

(7) Use of a FOXR1 gene knockout substance or a FOXR1 gene expression inhibiting substance in the preparation of a medicament for the treatment of a tumor.