WO2024092508A1

WO2024092508A1 - Use of mesenchymal stem cell

Info

Publication number: WO2024092508A1
Application number: PCT/CN2022/128989
Authority: WO
Inventors: 钟晓松; 顾爱琴
Original assignee: 卡瑞济(北京)生命科技有限公司
Priority date: 2022-11-01
Filing date: 2022-11-01
Publication date: 2024-05-10

Abstract

Provided are a method of a mesenchymal stem cell, e.g., a placenta-derived mesenchymal stem cell, for enhancing the treatment of a tumor by a CAR-T cell and/or treating a tumor in combination with a CAR-T cell, corresponding use, and a corresponding composition. The mesenchymal stem cell enhances the proliferation ability and persistence of a CAR-T cell in vivo, and regulates and controls the differentiation of the CAR-T cell, so as to allow the CAR-T cell to be in a low-differentiation state and increase the differentiation of the CAR-T cell into a memory cell subtype.

Description

Uses of Mesenchymal Stem Cells

Technical Field

The present invention relates to the field of cell therapy, and in particular to the use of mesenchymal stem cells for enhancing immunotherapy and corresponding pharmaceutical compositions.

Background technique

Chimeric antigen receptor (CAR) is a synthetic molecule that specifically recognizes antigens expressed on the surface of tumor cells to guide immune effector cells (e.g., T cells, NK cells) genetically engineered to express CAR to eliminate tumors (see Sampson JH et al., EGFRvIII mCAR-modified T-cell therapy cures mice with established intracerebral glioma and generates host immunity against tumor-antigen loss. Clinical cancer research: an official journal of the American Association for Cancer Research. 2014; 20(4): 972-984). For example, chimeric antigen receptor T cells (CAR-T) directly target surface antigens of tumor cells through chimeric antigen receptor (CAR) molecules on T cells, thereby achieving the purpose of recognizing and killing tumors, wherein the N-terminus of the chimeric antigen receptor contains an extracellular domain that recognizes the antigen. When the antigen-positive cells targeted by the CAR-T cells exist, the CAR-T cells can recognize and kill these antigen-positive cells. CAR-T cells have obvious advantages in the treatment of malignant tumors. More and more reports have shown that CAR-T cells are very effective in treating refractory hematological tumors (see Sadelain M et al., Therapeutic T cell engineering. Nature. 2017 May 24; 545(7655): 423-431.; June CH and Sadelain M. Chimeric Antigen Receptor Therapy. N Engl J Med. 2018 Jul 5; 379(1): 64-73.; Brudno JN and Kochenderfer JN. Chimeric antigen receptor T-cell thera pies for lymphoma. Nat Rev Clin Oncol. 2018 Jan; 15(1):31-46.; Brudno JN et al., T Cells Genetically Modified to Express an Anti-B-Cell Maturation Antigen Chimeric Antigen Receptor Causes Remissions of Poor-Prognosis Relapsed Multiple Myeloma. J Clin Oncol. 2018 Aug 1; 36(22):2267-2280.). Although the complete remission rate is high, most patients will still relapse; some of these patients are antigen-negative and some are antigen-low (see: Sadelain M et al., Therapeutic T cell engineering. Nature. 2017 May 24; 545(7655): 423-431.; June CH and Sadelain M. Chimeric Antigen Receptor Therapy. N Engl J Med. 2018 Jul 5; 379(1): 64-73.; Brudno JN et al., T Cells Genetically Modified to Express an Anti-B-Cell Maturation Antigen Chimeric Antigen Receptor Cause Remissions of Poor-Prognosis Relapsed Multiple Myeloma.J Clin Oncol.2018 Aug 1；36(22):2267-2280.；Orlando EJ et al., Genetic mechanisms of target antigen loss in CAR19 therapy of acute lymphob Lastic leukemia.Nat Med.2018 Oct；24(10):1504-1506.；Sotillo E et al.，Convergence of Acquired Mutations and Alternative Splicing of CD19 Enables Resistance to CART-19 Immunotherapy.Cancer Discov.2015Dec；5(12):1282-95.；Gardner R et al.，Acquisition of a CD19-negative myeloid Phenotype allows immune escape of MLL-rearranged B-ALL from CD19 CAR-T-cell therapy. Blood. 2016 May 19;127(20):2406-10.; Orlando EJ, et al., Genetic mechanisms of target antigen loss in CAR19 therapy of acute lymphoblastic leukemia. Nat Med. 2018 Oct;24(10):1504-1506.). Especially in the treatment of solid tumors, the efficacy is very limited (see: An Z et al., Antitumor activity of the third generation EphA2 CAR-T cells against glioblastoma is associated with interferon gamma induced PD-L1. Oncoimmunology. 2021 Aug 16; 10(1): 1960728.; Xu C et al., IL-13Rα2humanized scFv-based CAR-T cells exhibit therapeutic activity against glioblastoma. Mol Ther Oncolytics. 2022 Jan 10; 24: 443-451.), and there is still a long way to go.

If the results of CAR-T therapy do not meet expectations, one of the most important reasons may be that the related CAR-T cells have poor expansion and limited persistence. The in vivo expansion ability and persistence of CAR-T cells are related to the strength of CAR molecular signals, cytokine selection, stimulation domain, and T cell phenotype.

In addition, preclinical and clinical studies have shown that the function of CAR-T cells is also affected by the differentiation of CAR-T cells. Infusion of CAR-T cells with naive-like (TN) and central memory (TCM) phenotypes is associated with T cell persistence in vivo and superior antitumor efficacy (see: Wilkie S et al., Selective expansion of chimeric antigen receptor-targeted T-cells with potent effector function using interleukin-4. J Biol Chem. 2010 Aug 13; 285(33): 25538-44.; Shum T et al., Constitutive Signaling from an Engineered IL7 Receptor Promotes Durable Tumor Elimination by Tumor-Redirected T Cells. Cancer Discov. 2017 Nov; 7(11): 1238-1247.; Sukumaran S et al., Enhancing the Potency and Specificity of Engineered T Cells for Cancer Treatment.Cancer Discov.2018 Aug ；8(8):972-987.；Liu X et al., A Chimeric Switch Receptor Targeting PD1 Augments the Efficacy of Second-Generation CAR T Cells in Advanced Solid Tumors.Cancer Res.2016 Mar 15；76(6):1578-90.；Torres Chavez A et al., Expanding CAR T cells in human platelet lysate renders T cells with in vivo longevity.J Immunother Cancer.2019 Nov 28；7(1):330.）. Therefore, regulating the differentiation of CAR-T cells and keeping them in a low-differentiation state will greatly enhance their anti-tumor effect.

Mesenchymal stem cells (MSC) are multipotent stromal cells found in a variety of tissues such as bone marrow, fat, peripheral blood, umbilical cord, placenta and other tissues. It is the most promising allogeneic cell therapy in the clinic. Currently, mesenchymal stem cells from bone marrow and placenta are of the greatest interest in this field. Stem cells in the placenta are relatively primitive, have low immune rejection, strong proliferation and differentiation capabilities, are easy to collect and separate, and more importantly, are not accompanied by ethical barriers.

Mesenchymal stem cells have inherent immunomodulatory properties, nutritional capacity, high self-renewal ability and strong differentiation potential in vitro. Mesenchymal stem cells affect the function of most immune effector cells through direct contact with immune cells and paracrine activity, and can be easily designed to enhance their immunomodulatory function (see: Song N et al., Mesenchymal Stem Cell Immunomodulation: Mechanisms and Therapeutic Potential. Trends Pharmacol Sci. 2020 Sep; 41(9): 653-664.). It has been reported that MSCs inhibit the function of T cells by inhibiting the activation and proliferation of T cells (see: Ghannam S et al., Mesenchymal stem cells inhibit human Th17 cell differentiation and function and induce a T regulatory cell phenotype. J Immunol. 2010 Jul 1; 185(1): 302-12.), and it was found that MSCs inhibited the differentiation of naive CD4+T cells into Th17 cells and inhibited the expression of IL-17 and IL22. , IFN-γ and TNF-α production (see: Ghannam S et al., Mesenchymal stem cells inhibit human Th17 cell differentiation and function and induce a T regulatory cell phenotype. J Immunol. 2010 Jul 1; 185(1): 302-12.), induce T regulatory cell phenotype or T cell allergy (see: Glennie S et al., Bone marrow mesenchymal stem cells induce division arrest anergy of activated T cells. Blood. 2005 Apr 1; 105(7): 2821-7.). There are few reports on the effects of mesenchymal stem cells on CAR-T function. It has been reported that bone marrow mesenchymal stem cells suppress T cell responses but do not affect the activity of CD19 CAR-T cells (see: Zanetti SR et al., Bone marrow MSCs from pediatric patients with B-ALL highly immunosuppress T-cell responses but do not compromise CD19-CAR T-cell activity. J Immunother Cancer. 2020 Aug; 8(2): e001419.). Another study found that IL7-IL12 engineered mesenchymal stem cells improved CAR-T cell attack against colorectal cancer cells (see: Hombach AA et al., IL7-IL12 Engineered Mesenchymal Stem Cells (MSCs) Improve A CAR T Cell Attack Against Colorectal Cancer Cells. Cells. 2020 Apr 3; 9(4): 873.). Currently, the effect of MSC on CAR-T function is still controversial. Whether mesenchymal stem cells promote or inhibit CAR-T function requires more research.

How to improve the effectiveness of CAR-T cell therapy for tumors is the primary goal of researchers in this field.

Summary of the invention

The present invention has discovered for the first time the effect pattern of mesenchymal stem cells on CAR-T function, and for the first time provided the use of mesenchymal stem cells as CAR-T function enhancers. Specifically, the present invention co-cultured mesenchymal stem cells induced and cultured from the placenta of healthy donors and pregnant women with CAR-T cells of different targets, thereby confirming the effect of mesenchymal stem cells on the killing function of CAR-T cells. Furthermore, the present invention has confirmed for the first time that this effect is manifested through the persistence and differentiation of CAR-T cells.

In one aspect, the present invention relates to the use of mesenchymal stem cells for enhancing the efficacy of immunotherapy, wherein the surface of the mesenchymal stem cells does not express or substantially does not express the antigens targeted by the immunotherapy. In some embodiments, immunotherapy is cell therapy, that is, a therapy that administers therapeutic cells, preferably, therapeutic cells are immune effector cells. In some more specific embodiments, therapeutic cells are nonspecific immune effector cells. In some more specific embodiments, therapeutic cells are specific immune effector cells, preferably, immune effector cells are T cells; preferably, immune effector cells are modified immune effector cells; more preferably, immune effector cells are CAR-T cells. In some embodiments, human T cells are CD8 ⁺ T cells. In other specific embodiments, therapeutic cells are TIL, CAR-NK, TCR-T, CAR-DC, DC loaded with antigens, B cells, etc.

In some embodiments, the disease targeted by the immunotherapy is a tumor. In some more specific embodiments, the tumor is a hematological tumor. In some more specific embodiments, the tumor is a solid tumor. In some more specific embodiments, the immunotherapy is directed against one or more tumor-associated antigens (TAA) expressed by the tumor, wherein the MSC surface does not express or substantially does not express the tumor-associated antigen molecule. In a preferred embodiment, the tumor-associated antigen is IL-13Rα2, EphA2, CD19 and/or EGFRvIII.

In some embodiments, mesenchymal stem cells are derived from bone marrow, fat, peripheral blood, umbilical cord and/or placenta. In some embodiments, mesenchymal stem cells are derived from umbilical cord and/or placenta. In some embodiments, mesenchymal stem cells are derived from bone marrow, fat and/or placenta. In some embodiments, mesenchymal stem cells are derived from bone marrow and/or placenta. In some embodiments, mesenchymal stem cells are derived from placenta.

In some embodiments, the mesenchymal stem cells express CD105. In some embodiments, the mesenchymal stem cells do not express CD45. In some preferred embodiments, the mesenchymal stem cells are CD105 ⁺ CD45 ^- . In some preferred embodiments, the mesenchymal stem cells have the ability to be induced to differentiate into osteoblasts. In some preferred embodiments, the mesenchymal stem cells have the ability to be induced to differentiate into adipocytes. In some preferred embodiments, the mesenchymal stem cells have the ability to be induced to differentiate into chondrocytes.

In some embodiments, the mesenchymal stem cells are co-cultured with the immune effector cells in vitro. In some embodiments, the mesenchymal stem cells are co-administered with the immune effector cells into a subject, wherein the mesenchymal stem cells are administered prior to the immune effector cells, or administered simultaneously with the immune effector cells, or administered after the immune effector cells are administered.

In some embodiments, the ratio of the number of immune effector cells to the mesenchymal stem cells is 100:1, or 90:1, or 80:1, or 70:1, or 60:1, or 50:1, or 40:1, or 30:1, or 20:1, or 10:1, or 5:1, or 4:1, or 2:1, or 1:1, or 1:2, or 1:4, or 1:5, or 1:10, or a ratio therebetween.

In some specific embodiments, mesenchymal stem cells promote the immune effector cells (e.g., preferably, CAR-T cells) to secrete cytokines to kill tumor cells. In other preferred embodiments, mesenchymal stem cells enhance the direct killing effect of immune effector cells (e.g., preferably, CAR-T cells) on target cells. In some specific embodiments, mesenchymal stem cells promote the proliferation and amplification of immune effector cells (e.g., preferably, CAR-T cells), that is, the number increases. In some specific embodiments, mesenchymal stem cells promote the persistence of therapeutic cells (e.g., preferably, CAR-T cells), that is, the time of survival/existence in vivo is prolonged. In some specific embodiments, mesenchymal stem cells enhance the ability of immune effector cells (e.g., preferably, CAR-T cells) to reach the tumor area. In some specific embodiments, the efficacy enhancer promotes immune effector cells (e.g., preferably, CAR-T cells) to penetrate the body barrier (e.g., blood-brain barrier) so that more immune effector cells (e.g., preferably, CAR-T cells) reach the tumor (e.g., glioma) area, that is, increase the number of immune effector cells (e.g., preferably, CAR-T cells) that penetrate the body barrier (e.g., blood-brain barrier) In some specific embodiments, mesenchymal stem cells regulate the differentiation of immune effector cells (e.g., preferably, CAR-T cells) to maintain a low differentiation state. In some specific embodiments, mesenchymal stem cells promote immune effector cells (e.g., preferably, CAR-T cells) to differentiate into more memory cell subtypes. In some preferred embodiments, the enhancement is achieved by increasing the persistence of immune effector cells and/or maintaining a low differentiation state and/or more differentiation into memory effector cell subtypes.

In some specific embodiments, mesenchymal stem cells enhance cell therapy by at least one or more of the following:

(1) Enhance the proliferation ability of immune effector cells in vivo;

(2) increase the in vivo persistence of single immune effector cells;

(3) increasing the amount of therapeutic cytokines (e.g., interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), IL-4, IL-6, IL-10, and/or IL-17A) secreted by immune effector cells;

(4) Enhanced killing of cells carrying target antigens;

(5) Inhibit the differentiation of immune effector cells (e.g., CAR-T cells) to keep them in a low-differentiation state;

(6) Increase the formation of memory T cells;

(7) Increased target antigen neutralization/inhibition;

(8) Delaying lesion progression/reducing lesion size;

(9) reversing inhibitory factors/environments/conditions against immunotherapy in the treated subjects;

(10) Improving the survival rate and/or prolonging the survival period of the subjects receiving treatment (i.e., extending life span).

Preferably, the immune effector cell is modified. Preferably, the immune effector cell is a T cell. More preferably, the immune effector cell is a CAR-T cell.

(1) Enhance the proliferation ability of immune effector cells in vivo;

(2) increase the in vivo persistence of single immune effector cells;

(3) Enhanced killing of cells carrying target antigens;

(4) Inhibit the differentiation of immune effector cells (e.g., CAR-T cells) to keep them in a low-differentiation state;

(5) Increase the formation of memory T cells;

(6) Delaying lesion progression/reducing lesion size;

(7) Improving the survival rate and/or prolonging the survival period of the subjects receiving treatment (i.e., extending life span).

(1) Enhance the proliferation capacity of immune effector cells in vivo or increase the persistence of single immune effector cells in vivo;

(2) Inhibit the differentiation of CAR-T cells, keeping them in a low-differentiation state;

(3) increase the formation of memory T cells; and

(4) Improve the survival rate and/or prolong the survival of the subjects receiving treatment.

Thus, in another aspect, the present invention provides the use of mesenchymal stem cells for enhancing the in vivo proliferation ability of immune effector cells after administration and/or increasing the in vivo persistence of single immune effector cells after administration. Preferably, the immune effector cells are modified. Preferably, the immune effector cells are T cells. More preferably, the immune effector cells are CAR-T cells.

The present invention also provides the use of mesenchymal stem cells for regulating the differentiation of immune effector cells in vivo after administration. Preferably, the immune effector cells are modified. Preferably, the immune effector cells are T cells. More preferably, the immune effector cells are CAR-T cells.

The present invention also provides the use of mesenchymal stem cells for promoting the differentiation of immune effector cells into memory cell subtypes in vivo after administration. Preferably, the immune effector cells are modified. Preferably, the immune effector cells are T cells. More preferably, the immune effector cells are CAR-T cells.

In one aspect, the present invention relates to the use of mesenchymal stem cells in the preparation of anti-tumor drugs, wherein the drug also contains other cells. In a specific embodiment, the other cells are immune effector cells, and the surface of the mesenchymal stem cells does not express or substantially does not express the antigen specifically targeted by the CAR-T cells.

In some more specific embodiments, the tumor is a hematological tumor.In some more specific embodiments, the tumor is a solid tumor.

In some specific embodiments, immune effector cells are T cells. In some specific embodiments, immune effector cells are modified, preferably, transduced and express chimeric antigen receptor (CAR) T cells, i.e., CAR-T cells.

In some specific embodiments, the specific immune effector cells are directed against one or more tumor-associated antigens expressed by the tumor. In a preferred embodiment, the target TAA directed against by the specific immune effector cells is IL-13Rα2, EphA2, CD19 or EGFRvIII.

In some embodiments, the mesenchymal stem cells are of placental origin. In some specific embodiments, the placental-derived mesenchymal stem cells express CD105 and do not express CD45.

In some embodiments, the mesenchymal stem cells are co-cultured in vitro with therapeutic cells used in cell therapy. In some embodiments, the mesenchymal stem cells are co-administered with therapeutic cells used in cell therapy into a subject, wherein the mesenchymal stem cells are administered prior to, or simultaneously with, or after the therapeutic cells are administered.

In some embodiments, the number ratio of the therapeutic cells to the mesenchymal stem cells is 100:1, or 90:1, or 80:1, or 70:1, or 60:1, or 50:1, or 40:1, or 30:1, or 20:1, or 10:1, or 5:1, or 4:1, or 2:1, or 1:1, or 1:2, or 1:4, or 1:5, or 1:10, or a ratio therebetween.

In one aspect, the present invention relates to the use of mesenchymal stem cells in combination with other cells for preparing anti-tumor drugs, wherein the surface of the mesenchymal stem cells does not substantially express antigens specifically targeted by other cells, and the cancer cells express antigens specifically targeted by other cells.

In some embodiments, other cells are immune effector cells. In some embodiments, immune effector cells are T cells. In some specific embodiments, immune effector cells are modified, preferably, transduced and express chimeric antigen receptor (CAR) T cells, i.e., CAR-T cells.

In some embodiments, the mesenchymal stem cells are of placental origin. In some specific embodiments, the mesenchymal stem cells express CD105 and do not express CD45.

In another aspect, the present invention provides a composition comprising mesenchymal stem cells, preferably placental-derived mesenchymal stem cells. The composition is used as an immunotherapy efficacy enhancer. In a specific embodiment, the immunotherapy is directed against hematological tumors. In another specific embodiment, the immunotherapy is directed against solid tumors. In a preferred embodiment, the target molecule of the immunotherapy is IL-13Rα2, EphA2, CD19 or EGFRvIII.

In some specific embodiments, the composition promotes therapeutic cells (e.g., preferably, CAR-T cells) to secrete cytokines to kill tumor cells. In other specific embodiments, the composition enhances the direct killing effect of therapeutic cells (e.g., preferably, CAR-T cells) on target cells. In some specific embodiments, the composition promotes the proliferation of therapeutic cells (e.g., preferably, CAR-T cells), that is, the number increases. In some preferred embodiments, the composition enhances the ability of therapeutic cells (e.g., preferably, CAR-T cells) to reach the tumor area. In some specific embodiments, the efficacy enhancer promotes therapeutic cells (e.g., preferably, CAR-T cells) to penetrate the body barrier (e.g., blood-brain barrier) so that more therapeutic cells (e.g., preferably, CAR-T cells) reach the tumor (e.g., glioma) area, that is, increases the number of therapeutic cells (e.g., preferably, CAR-T cells) that penetrate the body barrier (e.g., blood-brain barrier). In some specific embodiments, mesenchymal stem cells regulate the differentiation of immune effector cells (e.g., preferably, CAR-T cells) to maintain a low differentiation state. In some specific embodiments, mesenchymal stem cells promote immune effector cells (e.g., preferably, CAR-T cells) to differentiate more into memory cell subtypes. In some preferred embodiments, the enhancement is achieved by increasing the persistence of immune effector cells and/or remaining in a poorly differentiated state and/or more differentiation into memory effector cell subtypes.

In yet another aspect, the present invention provides the use of mesenchymal stem cells for preparing one or more of the following:

(1) Agents that enhance the in vivo proliferation capacity of immune effector cells and/or the in vivo persistence of single immune effector cells;

(2) immune effector cell differentiation inhibitors that keep immune effector cells in a low-differentiation state;

(3) Agents that increase the formation of memory immune effector cell subtypes;

(4) An agent for enhancing the efficacy of cell (preferably immune effector cell) therapy;

Wherein, preferably, the immune effector cell is modified. Preferably, the immune effector cell is a T cell. More preferably, the immune effector cell is a CAR-T cell.

In another aspect, the present invention provides the use of mesenchymal stem cells in combination with immune effector cells for preparing a drug for treating cancer, wherein, preferably, the immune effector cells are modified. Preferably, the immune effector cells are T cells. More preferably, the immune effector cells are CAR-T cells.

In another aspect, the present invention provides a pharmaceutical composition comprising the aforementioned mesenchymal stem cells (e.g., placental-derived mesenchymal stem cells) or compositions of the present invention, and further comprising a suitable pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier makes the pharmaceutical composition suitable for administration by the intended route, for example, but not limited to, intravenous injection or local injection of tumors, etc. In some embodiments, the pharmaceutical composition of the present invention further comprises other cells (e.g., preferably, therapeutic cells, more preferably, CAR-T cells).

In another aspect, the present invention provides a method comprising administering immune effector cells to a subject in need thereof, and the cells have been co-cultured with mesenchymal stem cells in vitro/ex vivo, and/or co-administered with mesenchymal stem cells in vivo. Preferably, the immune effector cells are modified. Preferably, the immune effector cells are T cells. More preferably, the immune effector cells are CAR-T cells. In one embodiment, the subject has a condition described herein, e.g., the subject has cancer, e.g., the subject has a cancer that expresses a target antigen described herein. In one embodiment, the subject is a human.

In another aspect, the present invention relates to a method for treating a subject with a disease associated with the expression of a cancer-associated antigen as described herein, comprising administering immune effector cells to a subject in need thereof, and the cells have been co-cultured with mesenchymal stem cells in vitro/ex vivo, and/or co-administered with mesenchymal stem cells in vivo. Preferably, the immune effector cells are modified. Preferably, the immune effector cells are T cells. More preferably, the immune effector cells are CAR-T cells.

In another aspect, the present invention provides a method for treating a subject with a disease associated with the expression of a tumor antigen, comprising administering an immune effector cell to a subject in need thereof, and the cell has been co-cultured with mesenchymal stem cells in vitro/ex vivo, and/or co-administered with mesenchymal stem cells in vivo. Preferably, the immune effector cell is modified. Preferably, the immune effector cell is a T cell. More preferably, the immune effector cell is a CAR-T cell.

In another aspect, the present invention provides a method for enhancing the in vivo proliferation ability of immune effector cells after administration and/or increasing the in vivo persistence of a single immune effector cell after administration, the method comprising co-culturing the immune effector cells with mesenchymal stem cells in vitro/ex vivo, and/or co-administering with mesenchymal stem cells to the body. Therefore, in a specific embodiment, the present invention provides a method for enhancing the in vivo proliferation ability of immune effector cells after administration in vitro/ex vivo and/or increasing the in vivo persistence of a single immune effector cell after administration, the method comprising co-culturing the immune effector cells with mesenchymal stem cells in vitro/ex vivo. In another specific embodiment, the present invention provides a method for enhancing the in vivo proliferation ability of immune effector cells after administration in vivo and/or increasing the in vivo persistence of a single immune effector cell after administration, the method comprising co-administering the immune effector cells with mesenchymal stem cells to the body. Preferably, the immune effector cells are modified. Preferably, the immune effector cells are T cells. More preferably, the immune effector cells are CAR-T cells.

The present invention also provides a method for regulating the differentiation of immune effector cells in vivo after administration, the method comprising co-culturing the immune effector cells with mesenchymal stem cells in vitro/ex vivo, and/or co-administering them with mesenchymal stem cells in vivo. Therefore, in a specific embodiment, the present invention provides a method for regulating the differentiation of immune effector cells in vivo after administration in vitro/ex vivo, the method comprising co-culturing the immune effector cells with mesenchymal stem cells in vitro/ex vivo. In another specific embodiment, the present invention provides a method for regulating the differentiation of immune effector cells in vivo after administration in vivo, the method comprising co-administering the immune effector cells with mesenchymal stem cells in vivo. Preferably, the immune effector cells are modified. Preferably, the immune effector cells are T cells. More preferably, the immune effector cells are CAR-T cells.

The present invention also provides a method for promoting the differentiation of immune effector cells to memory cell subtypes in vivo after administration, the method comprising co-culturing the immune effector cells with mesenchymal stem cells in vitro/ex vivo, and/or co-administering them with mesenchymal stem cells in vivo. Therefore, in a specific embodiment, the present invention provides a method for promoting the differentiation of immune effector cells to memory cell subtypes in vivo after administration in vitro/ex vivo, the method comprising co-culturing the immune effector cells with mesenchymal stem cells in vitro/ex vivo. In another specific embodiment, the present invention provides a method for promoting the differentiation of immune effector cells to memory cell subtypes in vivo after administration, the method comprising co-administering the immune effector cells with mesenchymal stem cells in vivo. Preferably, the immune effector cells are modified. Preferably, the immune effector cells are T cells. More preferably, the immune effector cells are CAR-T cells.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1-4: Characterization of placental mesenchymal stem cells from healthy natural delivery pregnant women. Figure 1 shows the microscopic morphology of placental-derived mesenchymal stem cells. Figure 2 shows a flow chart for counting the proliferation of mesenchymal stem cells in vitro (top), and the calculation of the proliferation of mesenchymal stem cells according to the population doubling of six passages in vitro (bottom). Figure 3 shows the immunophenotype of the third generation of MSCs amplified by flow cytometry analysis, with the vertical axis reflecting the number of cells and the horizontal axis representing the staining intensity. The left figure shows that the cell population CD45-specific staining (by anti-CD45mAb) is basically negative; the right figure shows the cell population CD105-specific staining (by anti-CD105mAb), the dark curve represents the isotype-matched negative control mAb staining, and the light color represents the mAb-specific stained cells. It can be seen that the vast majority of cells can be clearly stained with CD105 antibodies. Figure 4 shows the osteogenic differentiation ability of placental-derived mesenchymal stem cells by Alizarin red staining. After inducing osteogenic differentiation, obvious clumps and nodular calcium deposits can be stained. MSC, mesenchymal stem cells, N=3.

Figure 5: Detection of tumor surface antigen expression in MSC by flow cytometry. (A) Negative control; (B, C, D) Detection of the expression of uPAR, IL13Rα2 and CD19 on the surface of MSC, respectively. MSC, mesenchymal stem cell.

Figures 6-10: Preparation of CAR-T cells and the effect of mesenchymal stem cells on in vitro CAR-T function. Figure 6 shows a schematic diagram of the structures of three CAR molecules that specifically target CD19, IL13Rα2 and uPAR. Figure 7 shows a flowchart of the preparation of CAR-T cells together with in vitro functional studies by time nodes. Figure 8 shows the effect of MSC on the proliferation of CD19-IL15 CAR-T cells detected by flow cytometry. CAR-T cells were first co-cultured with mesenchymal stem cells at a ratio of 10:1 for 24 hours, and then collected and co-cultured with/without NALM-6 cells at a ratio of 1:1 for 3 days for flow cytometric analysis. CAR-T cells that were not co-cultured with MSC under the same conditions (including co-culture with/without NALM-6 cells) were used as the control group. Figure 9 shows the effect of MSC on the activation of CD19-IL15 CAR-T cells detected by flow cytometry. CAR-T cells were co-cultured with MSC in advance and co-cultured with/without NALM-6 for 6 hours. Figure 10 shows the effect of MSC on cytokine release of CD19-IL15 CAR-T cells detected by CBA method. CAR-T cells were co-cultured with MSC for 24 hours, and then co-cultured with NALM-6 (A) and Raji (B) cells overnight, with an E:T ratio of 1:1, and the co-culture supernatant was collected for cytokine detection. For each cytokine, the 5 bars from left to right represent the target tumor cell group alone, the CD19-IL15 CAR-T cell group alone, the CD19-IL15 CAR-T cell + target tumor cell group, the CD19-IL15 CAR-T cell + target tumor cell group co-cultured with MSC, and the CD19-IL15 CAR-T cell group co-cultured with MSC. Data are shown as mean ± SEM. "*" represents p < 0.05; "**" represents p < 0.01; "***" represents p < 0.001; MSC: mesenchymal stem cells, N = 3. CAR-T cells N = 3.

Figures 11-13: Effects of MSCs on the anti-tumor function of CD19-IL15 CAR-T cells in vitro. Figure 11 shows the effect of MSCs on CD19-IL15 CAR-T cells against NALM-6-GL cell lines by flow cytometry analysis. CAR-T cells were first co-cultured with MSCs at a ratio of 10:1 and then co-cultured with NALM-6-GL at a ratio of 1:1. Flow cytometry result analysis graphs and statistical bar graphs are provided. Figure 12 shows the effect of MSCs on CD19-IL15 CAR-T cells against Raji-GL cell lines by flow cytometry analysis. CAR-T cells were co-cultured with mesenchymal stem cells at a ratio of 10:1 in advance and then co-cultured with Raji-GL at a ratio of 1:1. Flow cytometry result analysis graphs and statistical bar graphs are provided. Figure 13 shows the effect of MSCs on CD19-IL15 CAR-T cells against NALM-6-GL cells (A) and against Raji-GL cells (B) by detecting luciferase activity. Among them, CAR-T cells were first co-cultured with MSCs at a ratio of 10:1, and then collected and co-cultured with NALM-GL or Raji-GL at three ratios of 1:1, 2:1, and 5:1. "*" represents p < 0.05; "**" represents p < 0.01; "***" represents p < 0.001; GL: eGFP-Luc; MSC: mesenchymal stem cells, N = 3; E:T ratio is effector-target ratio; CAR-T cells N = 3.

Figures 14-16: Effect of MSC on the function of IL13Rα2 CAR-T cells against U87 cells. Figure 14 shows the activation of CD107a analyzed by flow cytometry. CAR-T cells were first co-cultured with mesenchymal stem cells at a ratio of 10:1, and then co-cultured with/without U87-GL cell line for 6 hours. Figure 15 shows the release of cytokines detected by ELISA. CAR-T cells were co-cultured with MSC for 24 hours, then collected and co-cultured with U87-GL cells overnight at an E:T ratio of 1:1, and the supernatant was obtained for cytokine detection. For each cytokine, the 5 bars from left to right represent the U87 cell group alone, the IL13Rα2 CAR-T cell group alone, the IL13Rα2 CAR-T cell + U87 cell group, the IL13Rα2 CAR-T cell + U87 cell group co-cultured with MSC, and the IL13Rα2 CAR-T cell group co-cultured with MSC. Figure 16 shows the anti-tumor effect analyzed by flow cytometry. CAR-T cells were co-cultured with MSC for 24 hours and then collected and co-cultured with U87-GL cells overnight, with an E:T ratio of 1:1. Flow cytometry result analysis chart and statistical bar graph are provided. "*" represents p<0.05; "**" represents p<0.01; "***" represents p<0.001. GL: eGFP-Luc; MSC: mesenchymal stem cells, N=3; CAR-T cells N=3.

Figures 17-21: Effects of MSC on the function of uPAR CAR-T cells against H460-GL cells. Figure 17 shows the activation of CAR-T cell CD107a. Figure 18 shows the detection of cytokine release. Figure 19 shows the in vitro anti-tumor effect of an E:T ratio of 1:1, and provides a flow cytometry result analysis chart and a statistical bar chart. Figure 20 shows the anti-tumor effect of uPAR CAR-T cells at different E:T ratios by detecting luciferase activity. Figure 21 shows that uPAR CAR-T cells exhibit killing function against MSC. GL: eGFP-Luc; MSC: mesenchymal stem cells, N = 3; E:T ratio is the effector-target ratio; CAR-T cells N = 3.

Figures 22-25: Effect of MSC on differentiation of CD19-IL15 CAR-T cells. Figure 22 shows flow cytometric analysis of differentiation of TCM phenotype detected by flow cytometry. CAR-T cells were first co-cultured with mesenchymal stem cells for 24 hours, then collected and co-cultured with/without NALM-6 cells. Figure 23 shows a bar graph of statistical results of the same assay. Figure 24 shows the expression of transcription factor TCF-7 detected by qRT-PCR. Figure 25 shows the release of cytokine IL2 detected by ELISA. CAR-T cells were cultured in medium without IL2 for 3 days, co-cultured with MSC for 24 hours, and then cultured with NALM-6 (A) or Raji cells (B) in the same medium without IL2 overnight, and the co-culture supernatant was obtained for IL2 detection. "*" represents p < 0.05; "**" represents p < 0.01; "***" represents p < 0.001. MSC: mesenchymal stem cells, N = 3; CAR-T cells N = 3.

Figures 26-29: Effect of MSC on the anti-tumor effect of CD19-IL15 CAR-T cells in the NALM-6-GL xenograft mouse model. Figure 26 shows a flow chart of the in vivo experiment, with time nodes indicated. 1×10 ⁶ NALM-6-GL cells were injected into NOD-SCID mice (N=12) via the tail vein, and 1×10 ⁷ CAR-T cells were injected via the tail vein 24 hours later, including CAR-T cells co-cultured with/without MSC. The mice were randomly divided into 3 groups, with 4 mice in each group. Figure 27 shows the tumor fluorescence imaging of mice, and Figure 28 shows the relative quantitative results of fluorescence for each mouse, among which the survival period of the tumor-bearing mice in the control group of CAR-T cells was extremely short, and only the relevant data for the first two weeks were obtained, which is also reflected in the survival curve of the mice shown in Figure 29. "*" represents p<0.05;"**" represents p<0.01;"***" represents p<0.001.

Detailed ways

As used herein, the singular forms "a", "an", "the" and "said" may include more than one or one referent unless the context clearly dictates otherwise. As used herein, "about" should be understood to mean within the range of -5% to +5% of the referenced number. In addition, all numerical ranges herein are understood to include all integers or fractions within the range. The compositions disclosed herein may be free of any elements not specifically disclosed herein. Therefore, disclosures of embodiments using the term "comprising" or "including" include disclosures of embodiments "consisting essentially of the specified components" and "consisting of the specified components."

As used herein, the term "mesenchymal stem cells", also known in the art as "mesenchymal stem cells (MSC)", refers to a group of multipotent stromal cells derived from the mesoderm, with certain differentiation potential and can differentiate into a variety of cell types. They are mainly derived from and exist in the bone marrow, and also include multipotent cells widely derived from other "non-bone marrow" tissues, such as: placenta, umbilical cord blood, adipose tissue, adult muscle, corneal stroma, deciduous tooth pulp, etc.

Mesenchymal stem cells suitable for the present invention

The isolated mesenchymal stem cell population described herein can be produced by the following method: digesting tissues (e.g., but not limited to, bone marrow, placenta, adipose, umbilical cord, peripheral blood, etc.) containing mesenchymal stem cells with tissue-destroying enzymes to obtain a mesenchymal stem cell population containing mesenchymal stem cells, and isolating or substantially isolating a plurality of mesenchymal stem cells from the remainder. All or any portion of the tissue can be digested to obtain the isolated mesenchymal stem cells described herein.

When cultivated in primary culture or in cell culture, the mesenchymal stem cells used as described herein adhere to a tissue culture substrate, for example, a tissue culture vessel surface (e.g., a tissue culture plastic). The mesenchymal stem cells in culture present a general fibroblast-like star-shaped appearance, in which a plurality of cytoplasmic processes extend from a central cell body. However, since the mesenchymal stem cells show these processes more than the fibroblast number, the mesenchymal stem cells are distinguishable in morphology from the fibroblasts cultivated under the same conditions. In morphology, the mesenchymal stem cells are also distinguishable from hematopoietic stem cells, which generally present a more rounded or pebble morphology when cultivated.

The isolated mesenchymal stem cells (e.g., isolated multipotent mesenchymal stem cells or isolated mesenchymal stem cell populations) useful in the technical solutions disclosed herein are adherent cells (e.g., adherent to tissue culture plastic) that have the characteristics of multipotent cells or stem cells and express a variety of markers that can be used to identify and/or isolate the cells or cell populations containing the stem cells.

In some embodiments, the separated mesenchymal stem cells are CD45 ^- and CD105 ⁺ , as detected by flow cytometry. In some embodiments, the mesenchymal stem cells are CD73 ⁺ , CD90 ⁺ and CD105 ⁺ , and CD14, CD34 and CD45 expression levels are extremely low/not expressed. In certain embodiments, the separated mesenchymal stem cells have the potential to differentiate into neural phenotype cells, osteogenic phenotype cells and/or chondrogenic phenotype cells. In a specific embodiment, the mesenchymal stem cells express about 95% of CD105 on the surface, substantially do not express CD45, and can differentiate into osteoblasts after being exposed to osteogenic induction medium for 3-4 weeks.

The isolated mesenchymal stem cell populations described above may generally contain about, at least, or no more than 1×10 ⁵ , 5×10 ⁵ , 1×10 ⁶ , 5×10 ⁶ , 1×10 ⁷ , 5×10 ⁷ , 1×10 ⁸ , 5×10 ⁸ , 1×10 ⁹ , 5×10 ⁹ , 1×10 ¹⁰ , 5×10 ¹⁰ , 1×10 ¹¹ or more isolated mesenchymal stem cells. The isolated mesenchymal stem cell populations useful in the methods of treatment described herein contain at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% viable isolated mesenchymal stem cells, e.g., as determined by, e.g., trypan blue exclusion.

For any of the above mesenchymal stem cells or mesenchymal stem cell populations, the cells or mesenchymal stem cell populations are or can include cells that have been passaged for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18 or 20 generations or more, or expanded for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38 or 40 population doublings or more.

In a preferred embodiment, the mesenchymal stem cells of the present invention do not express or substantially do not express the antigen molecules specifically targeted by cell therapy. Unless otherwise specified, when the mesenchymal stem cells are referred to herein as "not expressing" and "substantially not expressing" a certain antigen, they have the same meaning, meaning that in a certain specified mesenchymal stem cell population, only a very small number of cells, for example, less than 20% of cells, less than 19% of cells, less than 18% of cells, less than 17% of cells, less than 16% of cells, less than 15% of cells, less than 14% of cells, less than 13% of cells, less than 12% of cells, less than 11% of cells, less than 10% of cells, Less than 9% of cells, less than 8% of cells, less than 7% of cells, less than 6% of cells, less than 5% of cells, less than 4% of cells, less than 3% of cells, less than 2% of cells or less than 1% of cells express the antigen to a detectable degree; or, although the antigen is expressed on more than a few cells, the expression is extremely low, so that compared with the molecules normally expressed by the cells, or compared with the expression on cells that normally express the antigen molecule, the expression is negligible, or even below the detection limit of conventional methods. For example, in certain embodiments, the separated mesenchymal stem cells do not express or substantially do not express antigens specifically targeted by immune effector cells (e.g., CAR-T cells). In some embodiments, the separated mesenchymal stem cells do not express or substantially do not express CD19.

Placental mesenchymal stem cells suitable for the present invention

The isolated placental stem cell population described herein can be produced by the following method: digesting placental tissue with a tissue-destroying enzyme to obtain a placental cell population containing placental stem cells, and isolating or substantially isolating a plurality of placental stem cells from the remainder of the placental cells. All or any portion of the placenta can be digested to obtain the isolated placental stem cells described herein. In specific embodiments, for example, the placental tissue can be a whole placenta (e.g., including the umbilical cord), amnion, chorion, a combination of amnion and chorion, or any combination of the above. In other specific embodiments, the tissue-destroying enzyme is trypsin or collagenase. In various embodiments, the isolated placental stem cells contained in the cell population obtained from placental digestion are at least 50%, 60%, 70%, 80%, 90%, 95%, 99% or at least 99.5% of the placental cell population.

Among them, mesenchymal stem cells derived from the placenta (also referred to as placental-derived mesenchymal stem cells, placental-derived mesenchymal stem cells, placental mesenchymal stem cells, and the above terms are used interchangeably herein) can be fetal or maternal in origin (i.e., can have a maternal or fetal genotype). A placental stem cell population or a cell population comprising placental stem cells can include placental stem cells that are only of fetal or maternal origin, or can include a mixed population of placental stem cells of fetal and maternal origin.

The isolated mesenchymal stem cells and/or placental cell populations described herein (e.g., two or more isolated placental stem cells) include placental stem cells obtained directly from the placenta or any part thereof (e.g., chorion, placental villous leaves, etc.) and cell populations containing placental cells. Isolated placental cell populations also include (i.e., two or more) isolated placental stem cell populations in culture and cell populations in containers (e.g., bags).

Immune effector cells suitable for the present invention

The term "immune effector cell" refers to cells that have differentiated (i.e., not hematopoietic stem cells) from the human body and are capable of regulating or influencing the form or realization of an immune response, such as B cells, dendritic cells, natural killer cells, and T cells. It is known in the art to collect naturally occurring immune effector cells in vivo, optionally and preferably after a certain procedure of treatment in vitro, to convert them into therapeutic products and apply them to patients, i.e., immune effector cell therapy. These cell therapy products are an important part of immunotherapy, a new pillar of cancer treatment, which effectively uses the patient's own immune system to attack tumors. Immune effector cell therapy can also be designed to promote the immune system to stop attacking itself (in the presence of autoimmune diseases).

CAR-T cell therapy is the most studied immune effector cell therapy in recent years. The term "CAR-T" or "CAR T" refers to a T lymphocyte transduced with and expressing a chimeric antigen receptor (CAR). The chimeric antigen receptor refers to one or more sets of polypeptides that, when expressed on immune effector cells, provide the cells with specificity for a target antigen and have intracellular signal generation. The target antigen is expressed by a target cell (usually a cancer cell). Typically, CAR includes at least one extracellular binding region, a transmembrane region, and an intracellular signal region. Exemplary CAR construction methods and/or CAR-T cell transduction methods are described in, for example, Chinese Patent Publication No. CN114014941A. In some specific embodiments, the CAR-T cell is constructed according to the first generation CAR-T technology. In some specific embodiments, the CAR-T cell is constructed according to the second generation CAR-T technology. In some specific embodiments, the CAR-T cell is constructed according to the third generation CAR-T technology. In some specific embodiments, the CAR-T cell is constructed according to the fourth generation CAR-T technology. In some specific embodiments, the CAR molecule comprises an extracellular specific target antigen binding domain, preferably, the target antigen binding domain is a scFv. Preferably, the target antigen binding domain is a natural interaction molecule of the target antigen, such as, but not limited to, its ligand (when the target antigen is a receptor) or its soluble receptor fragment (when the target antigen is a ligand). In some embodiments, the target antigen is selected from one or more of the following: CD19, CD123, CD22, CD30, CD171, CS-1 (also known as CD2 subset 1, CRACC, SLAMF7, CD319, and 19A24), C-type lectin-like molecule-1 (CLL-1 or CLECL1), CD33, epidermal growth factor receptor variant III (EGFRvIII), ganglioside G2 (GD2), ganglioside GD3 (aNeu5Ac (2-8) aNeu5Ac (2-3) bDGalp (1-4) bDGlcp (1-1) Cer), TNF receptor family member B cell maturation (BCMA), Tn antigen ((Tn Ag) or (GalNAcα-Ser/Thr)), prostate specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), Fms-like tyrosine kinase 3 (FLT3), tumor-associated glycoprotein 72 (TAG72), CD38, CD44v6, carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EPCAM), B7H3 (CD276), KIT (CD117), interleukin 13 receptor subunit α-2 (IL-13Rα2 or CD 213A2), mesothelin, interleukin 11 receptor alpha (IL-11Ra), prostate stem cell antigen (PSCA), testisin or PRSS21, vascular endothelial growth factor receptor 2 (VEGFR2), Lewis (Y) antigen, CD24, platelet-derived growth factor receptor beta (PDGFR-β), stage-specific embryonic antigen-4 (SSEA-4), CD20, folate receptor alpha, receptor tyrosine protein kinase ERBB2 (Her2/neu), cell surface-associated mucin 1 (MUC1), epidermal growth factor receptor (EGFR) , neural cell adhesion molecule (NCAM), prostase, prostatic acid phosphatase (PAP), mutated elongation factor 2 (ELF2M), ephrin B2, fibroblast activation protein alpha (FAP), insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX), proteasome (Prosome, Macropain) subunit, beta type, 9 (LMP2), glycoprotein 100 (gp100), oncogene fusion protein composed of breakpoint cluster region (BCR) and Alelson murine leukemia virus oncogene homolog 1 (AB1) (bcr- abl), tyrosinase, ephrin type A receptor 2 (EphA2), fucosyl GM1, sialyl Lewis adhesion molecule (sLe), ganglioside GM3 (aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer), transglutaminase 5 (TGS5), high molecular weight melanoma-associated antigen (HMWMAA), o-acetyl GD2 ganglioside (OAcGD2), folate receptor β, tumor endothelial marker 1 (TEM1/CD248), tumor endothelial marker 7-related (T7-related EM7R), Claudin6 (CLDN6), thyroid stimulating hormone receptor (TSHR), G protein-coupled receptor class C group 5, member D (GPRC5D), X chromosome open reading frame 61 (CXORF61), CD97, CD179a, anaplastic lymphoma kinase (ALK), polysialic acid, placenta-specific 1 (PLAC1), hexose portion of globoH glycoceramide (GloboH), breast differentiation antigen (NY-BR-1), uroplakin 2 (UPK2), hepatitis A HAVCR1, adrenergic receptor β3 (ADRB3), pannexin 3 (PANX3), G protein-coupled receptor 20 (GPR20), lymphocyte antigen 6 complex, locus K9 (LY6K), olfactory receptor 51E2 (OR51E2), TCRγ alternate reading frame protein (TARP), nephroblastoma protein (WT1), cancer/testis antigen 1 (NY-ESO-1), cancer/testis antigen 2 (LAGE-1A), melanoma associated antigen 1 (MAGE-A1), ETS-prone ETV6-AML, sperm protein 17 (SPA17), X-antigen family, member 1A (XAGE1), angiopoietin binding cell surface receptor 2 (Tie2), melanoma cancer testis antigen-1 (MAD-CT-1), melanoma cancer testis antigen-2 (MAD-CT-2), FOS-related antigen 1, tumor protein p53 (p53), p53 mutant, prostein, survivin, telomerase, prostate cancer tumor antigen-1 (PCTA-1 or galectin 8), T cell Recognized melanoma antigen 1 (MelanA or MART1), rat sarcoma (Ras) mutant, human telomerase reverse transcriptase (hTERT), sarcoma translocation breakpoints, melanoma inhibitor of apoptosis (ML-IAP), ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene), N-acetylglucosaminyltransferase V (NA17), paired box protein Pax-3 (PAX3), androgen receptor, cyclin B1, V-myc avian myelocytic virus oncogene neuroblastoma-derived homolog (MYCN) , Ras homolog family member C (RhoC), tyrosinase-related protein 2 (TRP-2), cytochrome P450 1B1 (CYP1B1), CCCTC binding factor (zinc finger protein)-like (brother of regulator of BORIS or imprinting sites), squamous cell carcinoma antigen recognized by T cells 3 (SART3), paired box protein Pax-5 (PAX5), proacrosin binding protein sp32 (OY-TES1), lymphocyte-specific protein tyrosine kinase (LCK), A kinase anchoring protein 4 (AKAP-4), Synovial sarcoma, SSX2, RAGE-1, RU1, RU2, legumain, HPV E6, HPV E7, intestinal carboxylesterase, mutated heat shock protein 70-2, CD79a, CD79b, CD72, leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), IgA receptor Fc fragment (FCAR or CD89), leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2), CD300 molecule-like family member f (CD300LF), C-type lectin domain family 12 member A (CLEC12A), bone marrow stromal cell antigen 2 (BST2), mucin-like hormone receptor-like 2 (EMR2) containing an EGF-like module, lymphocyte antigen 75 (LY75), phosphatidylinositol proteoglycan-3 (GPC3), Fc receptor-like 5 (FCRL5), and immunoglobulin lambda-like polypeptide 1 (IGLL1).

In some embodiments, the tumor antigen bound by the encoded CAR molecule is selected from one or more of the following: TSHR, CD171, CS-1, CLL-1, GD3, Tn Ag, FLT3, CD38, CD44v6, B7H3, KIT, IL-13Rα2, IL-11Ra, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-β, SSEA-4, MUC1, EGFR, NCAM, CAIX, LMP2, EphA2, fucosyl GM1, SLE, GM3, TGS5, HMWMAA, o-acetyl GD2, folate receptor β, TEM1/CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53 mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMPR SS2ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B1, MYCN, RhoC, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, and IGLL1.

In certain embodiments, the tumor antigen bound by the encoded CAR molecule is selected from one or more of the following: TSHR, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, and OR51E2.

In some specific embodiments, the target antigen of the CAR-T cell is selected from CD19, IL-13Rα2, EphA2 and EGFRvIII.

In a specific embodiment, the target antigen of the CAR-T cell is CD19. Examples of such CAR-T cells are described in Ying Zhang et al., Co-expression IL-15 receptor alpha with IL-15 reduces toxicity via limiting IL-15 systemic exposure during CAR-T immunotherapy. J Transl Med. 2022 Sep27; 20(1):432.

In a specific embodiment, the target antigen of the CAR-T cell is IL-13Rα2. Examples of such CAR-T cells are described in Chinese Patent Application No. 202210019437.4.

In a specific embodiment, the target antigen of the CAR-T cell is EphA2. Examples of such CAR-T cells are described in Chinese patent applications No. 202110919075.X and An Z, etc., Antitumor activity of the third generation EphA2 CAR-T cells against glioblastoma is associated with interferon gamma induced PD-L1.Oncoimmunology.2021 Aug 16；10(1):1960728.

In a specific embodiment, the target antigen of the CAR-T cell is EGFRvIII. Examples of such CAR-T cells are described in Chinese Patent Application No. 202211140506.3.

In some specific embodiments, the CAR molecule comprises a transmembrane domain selected from the group consisting of: α, β, or ζ transmembrane domains of a T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, IL2Rβ, IL2Rγ, IL7Rα, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD , CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG/Cbp, NKp44, NKp30, NKp46, NKG2D, and/or NKG2C transmembrane domain. Preferably, the transmembrane domain is a CD28 transmembrane domain or a CD8 transmembrane domain or a CD4 transmembrane domain.

In some embodiments, the extracellular target antigen binding domain of the CAR molecule is connected to the transmembrane domain by a hinge region. In one embodiment, the hinge region comprises the amino acid sequence of a CD8 hinge. In another embodiment, the hinge region comprises the amino acid sequence of an IgG4 hinge.

In some specific embodiments, the CAR molecule comprises an intracellular segment. In some specific embodiments, the intracellular segment comprises a costimulatory signaling domain, and the costimulatory signaling domain comprises a functional signaling domain of a protein selected from one or more of the following: CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand specifically binding to CD83, CDS, ICA M-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LF A-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 ( Preferably, the co-stimulatory signaling domain is a CD28 co-stimulatory domain and/or a 4-1BB co-stimulatory domain.

In some specific embodiments, the intracellular segment comprises a stimulatory signaling domain selected from the signaling domains of CD3ζ, CD3γ, CD3δ, CD3ε, common FcRγ (FCER1G), FcRβ (FcεR1b), CD79a, CD79b, FcγRIIa, DAP10 and DAP12, preferably, the stimulatory signaling domain is a CD3ζ signaling domain. In some specific embodiments, the intracellular segment further comprises an IL15 sequence.

In some embodiments, the CAR-T cell optionally contains at least one other modification in addition to the exogenously introduced CAR molecule, such as other exogenous genes introduced through the same/different vector as the CAR molecule, and/or modification of the original genomic sequence, such as through gene editing technology, etc.

In some embodiments, the placental mesenchymal stem cells of the present invention enhance the anti-tumor function of CD19-IL15 CAR-T cells and IL13 CAR-T cells, but inhibit the anti-tumor function of uPAR CAR-T cells. In some embodiments, mesenchymal stem cells promote the proliferation and activation of CD19-IL15 CAR-T cells and promote the release of cytokines IL2 and IL4. IL-2 is involved in shaping the transcriptional and metabolic processes that determine the fate of T cells. It is an important cytokine for T cell activation and proliferation and is considered a means of treating cancer. While IL-4 is generally considered a typical Th2 cytokine, it has been reported that it can promote the conversion of CD4 ⁺ T cells in human thymus and umbilical cord blood into CD8 ⁺ T cells, and promote the frequency and function of memory CD8+T cells, thereby promoting rather than weakening Th1 cell immune responses. In some embodiments, mesenchymal stem cells promote an increase in the proportion of TCM phenotype cells in CD3 ⁺ T cells, including CD4 ⁺ and CD8 ⁺ T cells. In one embodiment, mesenchymal stem cells significantly upregulate the expression of TCF-7, a transcription factor associated with T cell differentiation and involved in the differentiation and development of TCM phenotype cells. It is well known in the art that CAR-T cells with less differentiated phenotypes, such as TN and TCM phenotypes, are associated with increased characteristics of self-renewal, proliferation, and survival. Therefore, the increase in such cells/CAR-T cells tend to differentiate into such cells, which will enhance the in vivo activity of CAR-T cells or cell populations.

The terms "tumor" and "cancer" are not mutually exclusive and are used interchangeably herein, covering solid tumors and blood tumors, referring to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues. In certain embodiments, cancers suitable for treatment by the methods of the present invention include blood cancers, for example, selected from chronic lymphocytic leukemia (CLL), acute leukemia, acute lymphocytic leukemia (ALL), B-cell acute lymphocytic leukemia (B-ALL), T-cell acute lymphocytic leukemia (T-ALL), chronic myeloid leukemia (CML), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B-cell Lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell-follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin lymphoma such as Burkitt lymphoma, Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom's macroglobulinemia, or one or more of the following: Cancer can also include solid tumors, for example selected from the group consisting of colon cancer, rectal cancer, renal cell carcinoma, liver cancer, non-small cell carcinoma of the lung, small intestine cancer, esophageal cancer, melanoma, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, Hodgkin lymphoma, non-Hodgkin lymphoma, cancer of the endocrine system, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, solid tumors in children, bladder cancer, cancer of the kidney or ureter, renal pelvis cancer, central nervous system tumors (CNS), primary CNS lymphoma, tumor angiogenesis, spinal tumors, brain stem gliomas, pituitary adenomas, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers, combinations of said cancers and metastatic forms of said cancers.

The term "pharmaceutical composition" refers to a mixture of mesenchymal stem cells as an active ingredient and a pharmaceutically acceptable carrier (as appropriate). The pharmaceutical composition facilitates administration of the active ingredient to a patient.

As used herein, the term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, stabilizer, dispersant, suspending agent, diluent, excipient, thickener, solvent or encapsulating material, which is involved in carrying or transporting the useful compounds of the present invention in or to the patient's body so that it can perform its intended function. Typically, such a construct can be carried to or transported from one organ or part of the body to another organ or part of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation (including the musk extract described in the present invention) and not harmful to the patient. Some examples of materials that can be used as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerol, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffers, such as magnesium hydroxide and aluminum hydroxide; surfactants; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; phosphate buffer; and other nontoxic compatible substances used in pharmaceutical formulations. As used herein, "pharmaceutically acceptable carriers" also include any and all coatings, antibacterial and antifungal agents, and absorption delaying agents that are compatible with the activity of the compounds of the invention and are physiologically acceptable to patients. Supplementary active compounds may also be incorporated into the composition. "Pharmaceutically acceptable carriers" may further include pharmaceutically acceptable salts of the compounds useful in the present invention. Other ingredients that may be included in the pharmaceutical compositions of the present invention are known in the art and are described, for example, in Remington"s Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, PA), which is incorporated herein by reference.

In some embodiments, the pharmaceutical composition is prepared for intravenous injection. In some embodiments, the pharmaceutical composition includes mesenchymal stem cells prepared for intravenous injection. In some embodiments, the pharmaceutical composition includes mesenchymal stem cells and CAR-T cells prepared for intravenous injection. Pharmaceutical compositions suitable for injection include sterile aqueous solutions (water-soluble) or dispersions and sterile powders for immediate preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, antibacterial water, or fluids that are easy to inject. Injectable compositions must be stable under manufacturing and storage conditions and must prevent contamination by microorganisms such as bacteria and fungi. The carrier can be a solvent or a dispersion medium, containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol and liquid polyethylene glycol, etc.), and suitable mixtures thereof. For example, appropriate fluidity can be maintained by using a coating such as lecithin, by maintaining the desired particle size in the case of a dispersion, and by using a surfactant. The action of microorganisms can be prevented by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

In some embodiments, the pharmaceutical composition is prepared for co-culturing with the mesenchymal stem cells of the present invention (e.g., preferably, placental stem cells) before the therapeutic cells therein are used for intravenous injection, so that the therapeutic effect is enhanced in vitro, and this state is continued after administration in vivo. Therefore, the pharmaceutical composition contains mesenchymal stem cells, therapeutic cells (e.g., CAR-T cells), and a culture medium suitable for co-culturing the two, and optionally, a culture container suitable for co-culturing the two.

In some embodiments, the pharmaceutical composition is suitable for modifying cells from the subject itself (e.g., transgenic modification), and after modification, necessary expansion culture and co-culture with the mesenchymal stem cells of the present invention (e.g., preferably, placental stem cells) are performed, so that the therapeutic effect is enhanced in vitro, and this state is continued after administration in vivo. Therefore, the pharmaceutical composition contains mesenchymal stem cells, necessary reagents for modifying cells from the subject itself (e.g., transfection reagents, transgenic constructs, preferably, viral vector constructs), and suitable culture media, and optionally, a culture container suitable for co-culture of the two.

As used herein, "treat" refers to slowing down, interrupting, blocking, alleviating, stopping, reducing, or reversing the progression or severity of existing symptoms, disorders, conditions, or diseases. The therapeutic effects on cancer/tumors generally include, but are not limited to, for example, reduction in tumor volume, reduction in the number of cancer cells, reduction in the number of metastases, increase in life expectancy, reduction in cancer cell proliferation, reduction in cancer cell survival, or improvement in various physiological symptoms associated with cancerous disorders.

As used herein, "prevention" includes inhibition of the occurrence or development of a disease or disorder or symptoms of a particular disease or disorder. In some embodiments, subjects with a family history of cancer are candidates for preventive regimens. Generally, in the context of cancer, the term "prevention" refers to the administration of a drug before the signs or symptoms of cancer occur, particularly in a subject at risk for cancer.

As used herein, "enhancement" or "promotion" of therapy means being able to enhance the therapeutic effect of existing immunotherapy (e.g., cell therapy, preferably, CAR-T cell therapy), wherein existing immunotherapy can refer to any therapy already used or known in the art, preferably a therapy for cancer, more preferably CAR-T cell therapy. Enhanced efficacy means that compared to patients treated with existing therapies that have not been enhanced by the mesenchymal stem cells of the present invention, patients who have received existing therapies enhanced by the mesenchymal stem cells of the present invention have slowed down, interrupted, blocked, relieved, stopped, reduced, or reversed the progression or severity of existing symptoms, illnesses, conditions, or diseases, or have fewer side effects, better treatment experience, and higher quality of life, or in short, have achieved or substantially achieved a better treatment process or outcome than patients who have received existing therapies that have not been enhanced by the mesenchymal stem cells of the present invention.

In some specific embodiments, mesenchymal stem cells promote the secretion of cytokines by the therapeutic cells (e.g., preferably, CAR-T cells) to kill tumor cells. In other specific embodiments, mesenchymal stem cells enhance the direct killing effect of therapeutic cells (e.g., preferably, CAR-T cells) on target cells. In some specific embodiments, mesenchymal stem cells promote the proliferation and expansion of therapeutic cells (e.g., preferably, CAR-T cells), that is, the number increases. In some specific embodiments, mesenchymal stem cells promote the persistence of therapeutic cells (e.g., preferably, CAR-T cells), that is, the time of survival/existence in the body is prolonged. In some specific embodiments, mesenchymal stem cells enhance the ability of therapeutic cells (e.g., preferably, CAR-T cells) to reach the tumor area. In some specific embodiments, the efficacy enhancer promotes therapeutic cells (e.g., preferably, CAR-T cells) to penetrate the body barrier (e.g., blood-brain barrier) so that more therapeutic cells (e.g., preferably, CAR-T cells) reach the tumor (e.g., glioma) area, that is, increase the number of therapeutic cells (e.g., preferably, CAR-T cells) that penetrate the body barrier (e.g., blood-brain barrier). In some preferred embodiments, the enhancement is achieved by increasing the persistence of immune effector cells and/or maintaining them in a poorly differentiated state and/or more differentiation into memory effector cell subtypes.

(1) Enhance the proliferation ability of immune effector cells in vivo;

(2) increase the in vivo persistence of single immune effector cells;

(4) Enhanced killing of cells carrying target antigens;

(6) Increase the formation of memory T cells;

(7) Increased target antigen neutralization/inhibition;

(8) Delaying lesion progression/reducing lesion size;

(1) Enhance the proliferation ability of immune effector cells in vivo;

(2) increase the in vivo persistence of single immune effector cells;

(3) Enhanced killing of cells carrying target antigens;

(5) Increase the formation of memory T cells;

(6) Delaying lesion progression/reducing lesion size;

(3) increase the formation of memory T cells; and

In some embodiments, the ratio of the number of therapeutic cells to mesenchymal stem cells used in cell therapy is 100:1, or 90:1, or 80:1, or 70:1, or 60:1, or 50:1, or 40:1, or 30:1, or 20:1, or 10:1, or 5:1, or 4:1, or 2:1, or 1:1, or 1:2, or 1:4, or 1:5, or 1:10, or a ratio therebetween.

Treatment methods/combination therapies

In one aspect, the present invention relates to the use of mesenchymal stem cells for enhancing the efficacy of immunotherapy. In some embodiments, immunotherapy is cell therapy, that is, a therapy that administers therapeutic cells. In some more specific embodiments, therapeutic cells are nonspecific immune effector cells. In some more specific embodiments, therapeutic cells are specific immune effector cells, preferably, therapeutic cells are T cells, more preferably, therapeutic cells are modified T cells, and most preferably, therapeutic cells are CAR-T cells. In some embodiments, human T cells are CD8+T cells. In other specific embodiments, therapeutic cells are CAR-NK, TCR-T, TIL, CAR-DC, etc. cells.

In another aspect, the present invention provides a method comprising administering immune effector cells to a subject in need, wherein the immune effector cells are optionally modified, preferably comprising a CAR molecule and/or a vector molecule comprising a coding sequence of the CAR molecule, and the cells have been co-cultured with mesenchymal stem cells in vitro/ex vivo, and/or co-administered with mesenchymal stem cells to the body. Preferably, the immune effector cells are CAR-T cells. In one embodiment, the subject has a condition described herein, for example, the subject suffers from cancer, for example, the subject has a cancer expressing a target antigen described herein. In one embodiment, the subject is a human.

In another aspect, the present invention relates to a method for treating a subject with a disease associated with the expression of a cancer-associated antigen as described herein, comprising administering an immune effector cell to a subject in need thereof, the immune effector cell being optionally modified, preferably comprising a CAR molecule and/or a vector molecule comprising a coding sequence of the CAR molecule, and the cell has been co-cultured with mesenchymal stem cells in vitro/ex vivo, and/or co-administered with mesenchymal stem cells in vivo. Preferably, the immune effector cell is a CAR-T cell.

In another aspect, the present invention provides a method for treating a subject suffering from a disease associated with the expression of a tumor antigen, comprising administering to a subject in need an immune effector cell, the immune effector cell being optionally modified, preferably comprising a CAR molecule and/or a vector molecule comprising a coding sequence for the CAR molecule, and the cell having been co-cultured with mesenchymal stem cells in vitro/ex vivo, and/or co-administered with mesenchymal stem cells in vivo. Preferably, the immune effector cell is a CAR-T cell.

In another aspect, the present invention provides a method for enhancing the in vivo proliferation ability of immune effector cells after administration and/or increasing the in vivo persistence of a single immune effector cell after administration, the method comprising co-culturing the immune effector cells with mesenchymal stem cells in vitro/ex vivo, and/or co-administering them with mesenchymal stem cells to the body. Therefore, in a specific embodiment, the present invention provides a method for enhancing the in vivo proliferation ability of immune effector cells after administration in vitro/ex vivo and/or increasing the in vivo persistence of a single immune effector cell after administration, the method comprising co-culturing the immune effector cells with mesenchymal stem cells in vitro/ex vivo. In another specific embodiment, the present invention provides a method for enhancing the in vivo proliferation ability of immune effector cells after administration in vivo and/or increasing the in vivo persistence of a single immune effector cell after administration, the method comprising co-administering the immune effector cells with mesenchymal stem cells to the body. Preferably, the immune effector cells are modified. Preferably, the immune effector cells are T cells. More preferably, the immune effector cells are CAR-T cells. Preferably, the above method increases the persistence of immune effector cells after administration in a subject.

The present invention also provides a method for regulating the differentiation of immune effector cells in vivo after administration, the method comprising co-culturing the immune effector cells with mesenchymal stem cells in vitro/ex vivo, and/or co-administering them with mesenchymal stem cells in vivo. Therefore, in a specific embodiment, the present invention provides a method for regulating the differentiation of immune effector cells in vivo after administration in vitro/ex vivo, the method comprising co-culturing the immune effector cells with mesenchymal stem cells in vitro/ex vivo. In another specific embodiment, the present invention provides a method for regulating the differentiation of immune effector cells in vivo after administration in vivo, the method comprising co-administering the immune effector cells with mesenchymal stem cells in vivo. Preferably, the immune effector cells are modified. Preferably, the immune effector cells are T cells. More preferably, the immune effector cells are CAR-T cells. Preferably, the above method keeps the immune effector cells in a low differentiated state after being administered to the subject.

The present invention also provides a method for promoting the differentiation of immune effector cells to memory cell subtypes in vivo after administration, the method comprising co-culturing the immune effector cells with mesenchymal stem cells in vitro/ex vivo, and/or co-administering them with mesenchymal stem cells in vivo. Therefore, in a specific embodiment, the present invention provides a method for promoting the differentiation of immune effector cells to memory cell subtypes in vivo after administration in vitro/ex vivo, the method comprising co-culturing the immune effector cells with mesenchymal stem cells in vitro/ex vivo. In another specific embodiment, the present invention provides a method for promoting the differentiation of immune effector cells to memory cell subtypes in vivo after administration in vivo, the method comprising co-administering the immune effector cells with mesenchymal stem cells in vivo. Preferably, the immune effector cells are modified. Preferably, the immune effector cells are T cells. More preferably, the immune effector cells are CAR-T cells. Preferably, the above method allows immune effector cells to differentiate into more memory effector cell subtypes after being administered to a subject.

In another aspect, the present invention provides a composition comprising immune effector cells and mesenchymal stem cells, for treating a subject with a disease associated with the expression of a tumor antigen such as a disease as described herein, wherein the immune effector cell comprises a CAR molecule and/or a vector molecule comprising a coding sequence of the CAR molecule, and the cell has been co-cultured with mesenchymal stem cells in vitro/ex vivo, and/or co-administered with mesenchymal stem cells to the body. Preferably, the immune effector cell is a CAR-T cell.

In certain embodiments of any of the above methods or uses, the mesenchymal stem cells can be delivered in combination with the immune effector cells, administered before the immune effector cells are administered, administered simultaneously with the immune effector cells, and administered after the immune effector cells are administered. Alternatively, the immune effector cells are co-cultured in vitro/ex vivo with the mesenchymal stem cells before being administered to the subject, and after co-culture, the mesenchymal stem cells are delivered in combination with/not with the immune effector cells. Alternatively, mesenchymal stem cells can be administered after an extended period of time after the immune effector cells are administered. In one embodiment, mesenchymal stem cells and immune effector cells or cell groups are administered to the subject at the same time (e.g., administered on the same day) or immune effector cells or cell groups are administered (e.g., 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks or more) after administration.

In any of the above-mentioned methods or embodiments of the use, other therapeutic agents/treatment methods known in the art may be further combined, such as, but not limited to, immune checkpoint inhibitors, targeted drugs, chemotherapy drugs, surgical treatment, radiotherapy, other CAR-T enhancers, and the like.

In any of the above methods or embodiments of the use, other therapeutic agents/treatment methods known in the art may be further combined, such as, but not limited to, immune checkpoint inhibitors, targeted drugs, chemotherapy drugs, surgical treatment, radiotherapy, other CAR-T enhancers, and the like.

In any embodiment of the above method or use, during the co-culture/combined delivery, the therapeutic cells and mesenchymal stem cells are present in a ratio approximately selected from the following: mesenchymal stem cells: therapeutic cells = 5: 1, 4: 1, 3: 1, 2: 1, 1: 1, 1: 2, 1: 3, 1: 4, 1: 5, 1: 6, 1: 7, 1: 8, 1: 9, 1: 10, 1: 11, 1: 12, 1: 13, 1: 14, 1: 15, 1: 16, 1: 17, 1: 18, 1: 19, 1: 20, 1: 30, 1: 40, 1: 50, 1: 60, 1: 70, 1: 80, 1: 90, 1: 100, or any ratio therebetween.

In any of the above methods or embodiments of purposes, through co-culture/combined delivery, the killing efficacy of the immune effector cells to the target tumor is enhanced relative to the same immune effector cells that are not co-cultured with mesenchymal stem cells and not delivered in combination. In a preferred embodiment, the killing efficacy is enhanced by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 120%, 140%, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 100 times, 500 times, 1000 times or higher, as evaluated by any index known in the art for assessing disease improvement and/or tumor regression.

In certain embodiments of any of the above methods or uses, the disease associated with a tumor antigen, e.g., a tumor antigen as described herein, is selected from a proliferative disease, such as a cancer or a malignant tumor or a precancerous lesion, such as myelodysplasia, myelodysplastic syndrome or preleukemia, or a non-cancer-related indication associated with the expression of a tumor antigen as described herein. In one embodiment, the disease is a cancer as described herein above. In some embodiments, the tumor antigen is a tumor antigen as described herein above.

In another aspect, the present invention provides a method comprising administering a modified immune effector cell to a subject in need, the cell comprising a CAR molecule and/or a vector molecule comprising a coding sequence of the CAR molecule, and the cell has been co-cultured with mesenchymal stem cells in vitro/ex vivo, and/or co-administered with mesenchymal stem cells to the body. Preferably, the immune effector cell is a CAR-T cell. In one embodiment, the subject has a condition described herein, for example, the subject suffers from cancer, for example, the subject has a cancer expressing a target antigen described herein. In one embodiment, the subject is a human.

In some preferred embodiments, co-culture/co-administration with the mesenchymal stem cells increases the therapeutic efficacy of the immune effector cells.

Pharmaceutical use/pharmaceutical composition

In another aspect, the present invention provides a pharmaceutical composition for enhancing the efficacy of cell therapy, wherein the pharmaceutical composition comprises mesenchymal stem cells. The cell therapy administers therapeutic cells to subjects in need, and the therapeutic cells are nonspecific immune effector cells. Preferably, the immune effector cells comprise a CAR molecule and/or a vector molecule comprising a coding sequence of the CAR molecule, and more preferably the immune effector cells are CAR-T cells.

In another aspect, the present invention provides a pharmaceutical composition for treating a subject suffering from a disease such as a disease described herein associated with the expression of a tumor antigen, wherein the pharmaceutical composition comprises immune effector cells and mesenchymal stem cells, and the immune effector cells have been co-cultured with mesenchymal stem cells in vitro/ex vivo, and/or co-administered with mesenchymal stem cells in vivo. Preferably, the immune effector cells comprise a CAR molecule and/or a vector molecule comprising a coding sequence of the CAR molecule, and more preferably the immune effector cells are CAR-T cells.

In any of the above embodiments, the pharmaceutical composition may further comprise one or more pharmaceutically acceptable or physiologically acceptable carriers, diluents or excipients, which make the pharmaceutical composition suitable for administration via the intended route, such as, but not limited to, intravenous injection or local tumor injection, etc.

Therefore, in another aspect, the present invention provides use of mesenchymal stem cells in preparing an agent for enhancing the therapeutic effect of cell therapy.

All patents, patent applications, publications, technical documents and/or scholarly articles and other references cited or referred to herein are incorporated by reference in their entirety to the extent permitted by law.

Example

The present invention will now be described with reference to the following examples. These examples are provided for illustrative purposes only, and the present invention is not limited to these examples, but encompasses all variations that are obvious based on the teaching provided herein.

Example 1. Materials and methods

This study (including each of its parts, such as those cited or referenced therein) was approved by the Institutional Review Board of Beijing Shijitan Hospital and informed consent was obtained from all participants. Placentas were obtained from healthy donors of Beijing Shijitan Hospital affiliated to Capital Medical University. All protocols for processing human placentas, isolation and culture of mesenchymal stem cells, in vitro cell experiments, and in vivo xenograft mouse experiments were approved by the Ethics Committee of Beijing Shijitan Hospital.

Isolation and culture of placental mesenchymal stem cells (pMSC)

Placental tissue was processed according to the method of Papait A et al. (Mesenchymal Stromal Cells from Fetal and Maternal Placenta Possess Key Similarities and Differences: Potential Implications for Their Applications in Regenerative Medicine. Cells. 2020 Jan 6; 9(1): 127) with slight modifications. First, the placental tissue was soaked in a preheated phosphate-buffered saline (PBS) solution containing 10% penicillin and streptomycin for 10 minutes, then washed twice with PBS and cut into 1 mm ³ with scissors. Mononuclear cells were obtained through a 100 μm sterile cell filter, and lymphocytes were separated by density gradient centrifugation. Cells were cultured at a density of 1×10 ⁶ cells/cm ² in DMEM medium containing 20% FBS, 2mM L-glutamine and 100U/ml penicillin/streptomycin at 37°C in a humidified atmosphere containing 5% (v/v) CO _2. After about 6 to 8 days, many colonies had formed. Unattached cells were removed and fresh medium was added. After the confluence reached 85%, the adherent cells were digested with 0.25% (w/v) trypsin/EDTA and re-plated at a cell density of 5×10 ³ cells/cm ^2. Placental cells were cultured continuously under the same conditions and expanded for 2 to 6 passages (Passage, P). All experiments were performed in a sterile environment with cells harvested between passages 3 and 6 (i.e., P3 and P6) and performed in a biosafety cabinet whenever possible.

Expansion and Characterization of Placental Mesenchymal Stem Cells (pMSC)

According to the method of Lechanteur et al. (Clinical-scale expansion of mesenchymal stromal cells: a large banking experience. J Transl Med. 2016 May 20; 14(1): 145.), the expansion of pMSCs was evaluated by the cumulative population doublings per generation. The first generation of mesenchymal stem cells was plated in a six-well plate at a cell density of 5×10 ³ cells/cm ^2. Then the cells were harvested every 5 days, counted by trypan blue staining, and re-plated at the same cell density of 5×10 ³ cells/cm ^2. The process was repeated until the 6th generation.

The characterization of pMSCs was performed according to the guidelines proposed by the International Society for Cellular Therapy (ISCT) (see: Viswanathan S et al., Mesenchymal stem versus stromal cells: International Society for Cell & Gene Therapy

Mesenchymal Stromal Cell committee position statement on nomenclature. Cytotherapy. 2019 Oct; 21(10): 1019-1024.). The immunophenotype of cultured pMSCs was evaluated by flow cytometry. A total of 1×10 ⁶ cells at passage 3 were incubated at room temperature in the dark for 15 minutes. Data acquisition and analysis were performed using a FACSCanto-II flow cytometer and flowjo v.10 software (BD Biosciences). The differentiation induction ability of pMSCs was evaluated by culturing in a specific differentiation induction medium for 3-4 weeks. The induction and detection of osteogenic differentiation were evaluated according to the following references: Jaiswal N et al., Osteogenic differentiation of purified, culture-expanded human mesenchymal stem cells in vitro. J Cell Biochem 1997; 64: 295-312. and Xu X et al., Dysregulated systemic lymphocytes affect the balance of osteogenic/adipogenic differentiation of bone mesenchymal stem cells after local irradiation. Stem Cell Res Ther 2017; 8: 71. Mesenchymal stem cells were stained with Alizarin Red to detect calcium deposition, and the mRNA level expression of specific transcription factors related to the induction of osteoblast formation was further quantitatively evaluated. Total mRNA was extracted from differentiated mesenchymal stem cell cultures and controls using Trizol, and quantitative RT-PCR (qRT-PCR) analysis was performed on a Roche Light Cycler 480II using SYBR Green PCR Master Mix (Fisher Scientific SL) and primers. The reactions were performed in triplicate, and the gene expression values were normalized to the expression values of GAPDH.

Cell lines

Human B-type acute lymphoblastic leukemia cell line NALM-6 and human Burkitt lymphoma cell Raji as well as retroviral packaging cell lines PG13 and Phoenix ECO were purchased from ATCC. NALM-6 and Raji cell lines engineered to express GFP and firefly luciferase (Luc) by retroviral transduction were named NALM-6-GL and Raji-GL, respectively. These cell lines were well cultured in 1640 medium (Lonza) containing 10% fetal bovine serum (FBS, Biosera), 100 U/mL penicillin, and 100 μg/mL streptomycin (EallBio Life Sciences). Retroviral producer cell lines were cultured in 1640 medium without penicillin and streptomycin containing 10% FBS.

Generation of chimeric antigen receptor-T cells

Three specific targeted CAR-T cells are used in embodiments of the present invention, including CAR-T cells targeting the blood tumor antigen CD19, and CAR-T cells targeting two solid tumor antigens uPAR and IL13Rα2, respectively. CAR is transferred into T cells via a retroviral vector. Examples of CAR-T cells targeting the blood tumor antigen CD19 are described in Ying Zhang et al., Co-expression of IL-15 receptor alpha with IL-15 reduces toxicity via limiting IL-15 systemic exposure during CAR-T immunotherapy. J Transl Med. 2022 Sep 27; 20(1):432. Examples of CAR-T cells targeting two solid tumor antigens uPAR and IL13Rα2 are described in Amor, C. et al., Senolytic CAR T cells reverse senescence-associated pathologies. Nature 583, 127–132 (2020). and Chinese patent application No. 202210019437.4. The universal preparation method of CAR-T cells can refer to, for example, the previous paper (An Z et al. Antitumor activity of the third generation EphA2 CAR-T cells against glioblastoma is associated with interferon gamma induced PD-L1. Oncoimmunology. 2021 Aug 16; 10(1): 1960728).

Specifically, the detailed structure of CAR and the preparation process of CAR-T cells are shown in Figures 6 and 7. The structure of the CAR consists of three parts, namely, the extracellular domain containing the anti-CD19 scFV sequence, the transmembrane domain of CD28, and the intracellular domain of CD28, 4-1BB, CD3ζ and IL15 sequences connected by P2A. Our previous studies have found that the intracellular domain of primary human CD8+T cells containing CD28, 4-1BB and/or CD3ζ is better than that containing only one or two domains in promoting cytokine release (see: Zhong XS et al., Chimeric antigen receptors combining 4-1BB and CD28 signaling domains augment PI3kinase/AKT/Bcl-XL activation and CD8+T cell-mediated tumor eradication. Mol Ther. 2010 Feb; 18(2): 413-20.).

Human peripheral blood mononuclear cells (PBMCs) from healthy donors were isolated by gradient centrifugation using lymphocyte separation medium (MP Biomedicals). T cells in PBMCs were stimulated with anti-CD3/CD28 T cell activation Dynabeads (Invitrogen). After 48 h of bead activation, T cells were transduced with retroviral supernatant by centrifugation on Retronectin (Takara)-coated plates. T cells were immunoblotted for CAR expression on day 7. T cells were cultured in X-VIVO-15 medium containing 5% human AB serum (SIGMA), 100 U/ml IL-2, 100 U/mL penicillin, and 100 μg/mL streptomycin (EallBio Life Sciences).

Flow cytometric analysis

Flow cytometry was performed using a BD FacsCanto II Plus instrument (BD Biosciences) and analyzed using FlowJo v.10 software (Treestar, Inc. Ashland, OR). T cells were detected using APC-conjugated anti-human CD3 antibodies (BD Biosciences), V450-conjugated anti-human CD4 (BD Biosciences), PE-Cy7-conjugated anti-human CD8 (BD Biosciences), and PE-Cy7-conjugated anti-human CCR7 (BD Biosciences). PE-Cy5-conjugated anti-human CD95 (BD Biosciences), Alexa Fluor 700-conjugated anti-human CD27 (BD Biosciences), and goat anti-mouse IgG (Fab specific) F(ab')2 fragment antibodies (Sigma). CAR was detected by staining with FITC-labeled goat anti-mouse IgG (H+L) antibodies (Sigma). MSC cells were stained with PE-Cy7-conjugated anti-human CD105 (ThermoFisher Scientific) and V450-conjugated anti-human CD45 (BD Biosciences). T cell degranulation was detected by BV421-conjugated anti-human CD107a-APC (ThermoFisher Scientific). After incubation with antibodies, cells were washed with PBS, resuspended in PBS containing 1% FBS, and analyzed on an analyzer.

Detection of CD107a degranulation of CAR-T cells

CAR-T cells were co-cultured with MSC cells at a ratio of 10:1 for 24 hours, and then CAR-T cells were collected and co-cultured with NALM-6 cells at a ratio of 1:1 in a 24-well plate, where each well contained 1 μl of anti-human CD107a antibody (BD Bioscience) and Golgi Stop ^TM (BD Bioscience). After 6 hours, the cells were harvested and incubated with anti-human CD3 antibody (BD Bioscience), and the CD107a degranulation of CAR-T cells was detected by flow cytometry analysis. CD19-IL15 CAR-T cells were used as an example. The experiment was divided into four groups: (1) CAR-T cells that were not co-cultured with MSCs in advance as a control; (2) CAR-T cells that were co-cultured with MSCs in advance; (3) CAR-T cells that were co-cultured with NALM-6 cells; (4) CAR-T cells that were first co-cultured with MSCs and then collected and co-cultured with NALM-6 cells.

Carboxyfluorescein succinimidyl ester dilution test

CAR-T cells were prepared using cells from three healthy donors, co-cultured with the NALM-6 cell line, and T cell proliferation was analyzed using flow cytometry based on carboxyfluorescein succinimidyl ester (CFSE) dye. CFSE-labeled CAR-T cells were first co-cultured with MSC cells in a round-bottom 24-well plate at a density of 1×10 ⁶ :1×10 ⁵ cells/ml (i.e., a ratio of 10:1) for 24 hours in triplicate. CAR-T cells were then collected and co-cultured with NALM-6 at a ratio of 10:1 for 3 days. T cell proliferation was determined by CFSE dye dilution of CD3-positive cells using a BD FacsCanto II Plus instrument (BD Biosciences). Flow cytometric data were analyzed using Flow Jo v.10 software (Tree star, Inc. Ashland, OR). The groups in this experiment were the same as those in the CD107a degranulation experiment.

Cytotoxicity assay

Two methods were used to detect the cytotoxic function of CAR-T cells. One was to detect the GFP signal of target cells by flow cytometry, and the other was to detect the chemiluminescence of target cells using a live imaging analysis system. CAR-T cells were first co-cultured with MSC cells at a ratio of 10:1 for 24 hours, then re-collected and co-cultured with NALM-6-GL and Raji-GL cell lines at various E:T ratios (1:1 and 0.5:1) in 24-well plates. After 24 hours, cells were collected and target cells were detected using a BD FacsCanto II Plus instrument (BD Biosciences), and data were analyzed using Flow Jo v.10 software (Treestar, Inc. Ashland, OR). For the chemiluminescence method, cells (96-well plates) were added with substrates, and the signals were collected using a PerkinElmer photochemical imaging system. The intensity of the fluorescence signal was analyzed using a live imaging analysis system to evaluate the cytotoxicity of CAR-T cells. The experiment included three groups: (1) target cells only; (2) CAR-T cells co-cultured with target cells without prior co-culture with MSCs; and (3) CAR-T cells co-cultured with MSCs beforehand and then collected for co-culture with target cells.

Cytokine production assay

CAR-T cells were first co-cultured with MSC cells at a ratio of 10:1 for 24 hours, and then re-collected and co-cultured with NALM-6-GL and Raji-GL cell lines at a ratio of E:T = 1:1 on a 24-well plate. After 24 hours of co-culture, the supernatant was collected for cytokine detection. The expression of human interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), IL-4, IL-6, IL-10, and IL-17A in the supernatant of co-cultured cells was evaluated using a commercial cytometric bead array (CBA) kit (BD Biosciences) according to the manufacturer's procedures. The level of human IFN-γ was also measured by using

The ELISA kit (R&D, Minnesota, USA) was used for evaluation according to the manufacturer's instructions.

To detect the cytokine IL2, CAR-T cells were cultured in medium without IL2 for 3 days and then co-cultured with mesenchymal stem cells and target cells in the same medium without IL2. The supernatant was collected and used

The release of IL2 was detected by ELISA kit (R&D, Minnesota, USA).

Cell Subpopulation Analysis

CAR-T cells were co-cultured with MSC cells at a ratio of 10:1 for 5 days, then re-collected and co-cultured with NALM-6-GL and Raji-GL cell lines at an E:T ratio of 10:1 in 24-well plates for 24 hours. The co-cultured cells were then collected for flow cytometry analysis of T cell subsets.

Xenograft mouse model with intracerebral injection of NALM-GL cells

Female NOD-SCID mice aged six to eight weeks were purchased from Charles River Laboratories and carefully raised under pathogen-free conditions. After one week of adaptive feeding, NOD-SCID mice were injected with 1×10 ⁶ NALM-6-GL cells via the tail vein to construct a tumor xenograft mouse model. A total of 12 mice were randomly divided into three groups. The control group was a model group injected with NALM-6-GL only (N=4). The experimental group was a CAR-T cell treatment group, including CAR-T cells co-cultured with mesenchymal stem cells (N=4) and CAR-T cells not co-cultured with mesenchymal stem cells (N=4). The experimental group was also injected with 1×10 ⁷ /day CAR-T cells via the tail vein 24 hours after model construction for three consecutive days. The progression of the tumor was monitored weekly by bioluminescence imaging using an in vivo imaging analysis system (IVIS, Xenogen, Alameda, CA, USA). According to the experimental animal management regulations of Beijing Shijitan Hospital affiliated to Capital Medical University, mice were euthanized after reaching the euthanasia criteria.

Statistical Analysis

All data included at least three biological replicates and were analyzed using GraphPad Prism 7 software (GraphPad Software, San Diego, CA). Data are presented as mean ± SEM, and differences were assessed using unpaired t-tests. Overall survival of xenografted mice was measured using the Kaplan-Meier method, and group comparisons were performed using Cox proportional hazards regression analysis. Differences were considered statistically significant if P < 0.05.

Example 2. Expansion and Characterization of Placental Mesenchymal Stem Cells

We isolated and cultured mesenchymal stem cells from the placentas of eight normal donors and determined whether the cells were mesenchymal stem cells by identifying their morphology, immunophenotype, and differentiation potential, referring to the standardized criteria listed by ISCT. Adherent cells were successfully cultured from all samples using standard culture conditions and methods for mesenchymal stem cells. These cells adhered to the plastic and showed a uniform spindle-forming fibroblast morphology (Figure 1). We calculated the growth dynamics from P1 to P6 by trypan blue staining and found that the proliferation of mesenchymal stem cells from different donor placentas was basically the same (Figure 2). We detected the immunophenotype of mesenchymal stem cells from P3 by flow cytometry. The results showed that CD105 (one of the markers of MSC) was positive (>95%), while CD45 (a hematopoietic cell marker) was negative (Figure 3). Next, we evaluated the induction differentiation potential of MSC in vitro. Mesenchymal stem cells from P3 were exposed to osteogenic induction medium and cultured for 3-4 weeks. Calcium deposition was then detected by alizarin red staining. The results showed that significant calcium deposition was detected in osteoblast-induced cells ( FIG. 4 ).

Before exploring the effect of MSC on CAR-T cell function, we first detected whether common tumor target antigens were expressed on the surface of MSC. The results of flow cytometry showed that uPAR was expressed on the surface of mesenchymal stem cells, but CD19 and IL13Rα2 were not expressed (Figure 5).

Example 3. Generation of CAR-T cells

The three CAR molecules described above were constructed according to the method described in Example 1 and transduced into T cells to obtain CAR-T cells, which were verified by flow cytometry.

Example 4. pMSCs enhance the function of CD19-IL15 targeted CAR-T cells in vitro

We evaluated the effects of MSCs on CD19-IL15 CAR-T function from four aspects, including CAR-T proliferation, CD107a activation, cytokine release, and cytotoxicity.

In the experiment to explore the effect of MSC on the proliferation of CD19-IL15 CAR-T cells, CAR-T cells were first co-cultured with MSC at an E:T = 10:1 ratio for 24 hours, and then the CAR-T cells were collected, labeled with CFSE, and co-cultured with NALM-6 cells at an E:T ratio of 1:1. Three days later, the cells were harvested and incubated with an appropriate amount of anti-human CD3 antibody for flow cytometry analysis. Using CFSE labeling as a control, CAR-T cells that were not co-cultured with MSC were co-cultured with/without NALM-6 cells under the same conditions. The results showed that MSC promoted the proliferation of CAR-T cells (Figure 8).

Next, we explored the effect of MSC on CD107a activation of CAR-T cells. CAR-T cells were also co-cultured with MSCs at a ratio of 10:1 for 24 hours in advance, and then co-cultured with/without NALM-6 at a ratio of 1:1 for 6 hours. CAR-T cells that were not co-cultured with MSCs and co-cultured with/without NALM-6 served as controls. Compared with the control group, when CAR-T cells were co-cultured with MSCs in advance and then co-cultured with NALM-6, the proportion of CD107a-positive cells showed a significant increase (Figure 9, 22.7±1.36 vs 30.47±1.26, P=0.0138).

We then collected the supernatants of the co-cultured cells and assayed the release of cytokines by cytometry bead array kit and ELISA. CAR-T cells were co-cultured with MSCs for 24 h and then re-collected and co-cultured with/without NALM-6 or Raji cells overnight. As shown in Figure 10A (results involving NALM-6) and B (results involving Raji), the release of cytokines IL10, IL4 and IL6, IFN-γ, TNF-α, and IL17 by CD19-IL15 CAR-T cells during the anti-tumor process did not change significantly, regardless of whether they were co-cultured with MSCs. When CD19-IL15 CAR-T cells attacked NALM-6 cells, it was found that the secretion of IL4 and IL10 in CAR-T cells co-cultured with MSCs increased significantly, while the secretion of IFN-γ, IL17A, and TNF-α did not change significantly. When CD19-IL15 CAR-T cells attacked Raji cells, it was found that the secretion of IL4 and IL17A in CAR-T cells co-cultured with mesenchymal stem cells was significantly increased, while the secretion of IFN-γ, IL17A and IL10 did not change significantly.

Finally, we evaluated the effect of MSC on the cytotoxicity of CD19-IL15 CAR-T cells. CAR-T cells were co-cultured with MSCs at a ratio of 10:1 for 24 hours, and then CAR-T cells were collected and co-cultured with NALM-6-GL and Raji-GL cells at various E:T ratios. The survival of tumor cells was analyzed by flow cytometry to detect GFP fluorescence (see Figures 11 and 12, respectively) and luciferase activity by IVIS imaging system (see Figure 13, A and B, respectively) to evaluate cytotoxic function. As shown in the above figures, the killing ability of CD19-IL15 CAR-T cells was significantly enhanced after co-culture with mesenchymal stem cells.

Example 5. Mesenchymal stem cells enhance the function of CAR-T cells targeting IL13Rα2 in vitro

We also took IL13Rα2-targeted CAR-T cells as an example to explore the effect of MSC on the function of CAR-T cells targeting glioma. We evaluated the effect of mesenchymal stem cells on the function of CAR-T cells targeting IL13Rα2 glioma from three aspects: CD107a activity, cytokine release, and killing ability. The detailed experimental method is consistent with the previous embodiment, that is, the research method for evaluating the effect of MSC on the function of CD19-IL15 CAR-T cells. As shown in Figures 14-16, at an E:T ratio of 1:1, MSC did not cause an increase in CD107a activation of IL13Rα2 CAR-T cells, nor did it affect the release of IFN-γ and IL6 (Figures 14, 15), but it increased the killing ability of CAR-T cells (Figure 16).

Example 6. Mesenchymal stem cells inhibited the function of uPAR-targeted CAR-T cells in vitro

We found that MSCs have different functions on different CAR-T cells. MSCs caused a decrease in the killing ability of uPAR-targeted CAR-T cells. We explored the effect of MSCs on the function of uPAR-targeted CAR-T cells and found that MSCs enhanced the activation of CD107a (Figure 17) and promoted the release of cytokine IFN-γ (Figure 18), but severely hindered its cytotoxic function (Figures 19-20). When the E:T ratio was 2:1, MSCs reduced the killing ability of CAR-T cells by 22%. When the E:T ratio was 2:1, mesenchymal stem cells reduced the killing ability of CAR-T cells by 22% and severely damaged the killing ability; at an E:T ratio of 1:1, the killing ability was severely impaired, resulting in a decrease in killing ability of up to 92%. Further studies showed that when the E:T ratio was 1:1, uPAR CAR-T cells killed up to 77% of mesenchymal stem cells (Figure 21).

Example 7. MSC regulates T cell differentiation

We explored how mesenchymal stem cells regulate the function of CAR-T cells, and used CD19-IL15 CAR-T cells as the research object to find the possible mechanism by which mesenchymal stem cells promote CAR-T function. We found that mesenchymal stem cells affected the differentiation of CAR-T cells. CAR-T cells were co-cultured with MSC for one day and collected with or without co-culture with NALM-6 cells. CAR-T cells were then collected to detect T cell subsets. As shown in Figures 22-23, compared with CD19-IL15 CAR-T cells, the proportion of TCM phenotype in CD3+ cells was significantly increased in CAR-T cells co-cultured with MSC. Further analysis found that its proportion in CD4+ cells and CD8+ cells was also significantly increased. The results of qRT-PCR showed that the expression of TCF-7, a transcription factor related to T cell differentiation, was significantly upregulated (Figure 24). At the same time, we detected the secretion of IL2, a necessary cytokine for T cell proliferation and differentiation. CAR-T cells were co-cultured with MSCs for 24 hours in advance and then co-cultured with NALM-6 and Raji cells in X-VIVO-15 medium without IL2 overnight. The supernatant was collected and the IL2 content was detected by ELISA. As shown in Figure 25, compared with the CD19-IL15 CAR-T cell group, the IL2 released by MSC-treated CAR-T cells did not change significantly, but when co-cultured with tumor cells such as NALM-6 and Raji, the IL2 secreted by MSC-treated CAR-T cells increased significantly.

Example 8. Mesenchymal stem cells enhance the anti-tumor ability of CD19-IL15 CAR-T in vivo

A xenograft mouse model was constructed by tail vein injection of NALM-6-eGFP-Luc (NALM-6-GL) cells. As shown in FIG26 , 1×10 ⁶ NALM-6-GL cells were injected one day in advance, and 1×10 ⁷ CD19-IL15 CAR-T cells were injected through the tail vein on day 0. The CAR-T cells injected into the experimental group mice were co-cultured with mesenchymal stem cells in vitro for 1 day in advance, while the CAR-T cells injected into the control group mice were not co-cultured with MSCs. In addition, a non-treatment control group without CAR-T injection was set up. Fluorescence imaging of mice was performed regularly. As shown in FIG27 and FIG28 , the tumor burden in the CAR-T treatment group was significantly lower than that in the control group, but there was no significant difference between the CAR-T group treated with mesenchymal stem cells and the CAR-T group alone. The survival of the mice was observed and counted. The results showed that after co-culture of CAR-T cells with mesenchymal stem cells, the survival time of mice was significantly prolonged ( FIG29 ).

discuss

In the above examples, placental mesenchymal stem cells enhanced the anti-tumor function of CD19-IL15 CAR-T cells and IL13 CAR-T cells, but inhibited the anti-tumor function of uPAR CAR-T cells. Specifically, the results showed that mesenchymal stem cells promoted the proliferation and activation of CD19-IL15 CAR-T cells and promoted the release of cytokines IL2 and IL4. IL-2 is involved in shaping the transcriptional and metabolic processes that determine the fate of T cells. It is an important cytokine for T cell activation and proliferation and is considered a means of treating cancer. IL-4 is generally considered to be a typical Th2 cytokine, but it has been reported that it can promote the conversion of CD4+T cells in human thymus and umbilical cord blood into CD8+T cells and promote the number and function of memory CD8+T cells, thereby promoting rather than weakening Th1 cell immune responses. We also found that mesenchymal stem cells promoted an increase in the proportion of TCM phenotype cells in CD3+T cells, including CD4+ and CD8+T cells. TCF-7, a transcriptional activator involved in the differentiation and development of TCM phenotype cells, was significantly increased in expression. It is well known that CAR-T cells with less differentiated phenotypes, such as TN and TCM phenotypes, are associated with increased properties of self-renewal, proliferation, and survival.

The efficacy of CAR-T cell therapy is closely related to the activity and persistence of CAR-T cells. Therefore, all the above results well explain why mesenchymal stem cells can enhance the anti-tumor ability of CD19-IL15 CAR-T cells.

This paper also evaluated whether mesenchymal stem cells can promote the anti-tumor efficacy of CD19-IL15 CAR-T cells in a xenograft mouse model. The results showed that CAR-T cells co-cultured with mesenchymal stem cells in vitro significantly prolonged the survival time of NALM-6-GL tumor-bearing mice. In previous reports, BM-MSCs were co-cultured with CD19CAR-T and tumor cells, and it was found that MSCs could not protect tumor cells or affect the anti-tumor ability of CAR-T cells (Zanetti SR et al., Bone marrow MSCs from pediatric patients with B-ALL highly immunosuppress T-cell responses but do not compromise CD19-CAR T-cell activity. J Immunother Cancer 2020; 8: e001419.). However, in this paper, CD19-IL15 CAR-T cells were first stimulated by MSCs and then collected and co-cultured with tumor cells, and the results showed that their anti-tumor ability was significantly enhanced. CAR-T cells undergo a series of changes under MSC stimulation, such as phenotype, cytokine release, activation and proliferation ability, leading to the enhancement of their anti-tumor function.

In this experiment, we also found that mesenchymal stem cells inhibited the function of uPAR CAR-T cells. Previous flow cytometry results showed that MSCs expressed uPAR but did not express CD19 and IL13Rα2. Therefore, it is speculated that CD19-IL15 CAR-T cells and IL13Rα2 CAR-T cells did not kill mesenchymal stem cells when co-cultured with mesenchymal stem cells (data not shown), but uPAR CAR-T cells can attack mesenchymal stem cells through their target antigen specificity. As a result, uPAR CAR-T cells may be exhausted due to killing MSCs, resulting in a decrease in their anti-tumor ability. This phenomenon shows that whether mesenchymal stem cells have an enhancing effect on the function of CAR-T cells is a key factor in whether mesenchymal stem cells express the corresponding target antigen.

Claims

Use of mesenchymal stem cells for enhancing immunotherapy against a disease, wherein the mesenchymal stem cells do not express or substantially do not express the antigen targeted by the immunotherapy on their surface.
The use of claim 1, wherein the immunotherapy is cell therapy.
The use of claim 2, wherein the therapeutic cells used in the cell therapy are immune effector cells, optionally modified immune effector cells.
The use of claim 3, wherein the immune effector cells are T cells.
The use of claim 3, wherein the immune effector cells are CAR-T cells.
The use of claim 3, wherein the immune effector cells are selected from one or more of TIL, CAR-NK, TCR-T, CAR-DC, DC loaded with antigen, and B cells.
The use according to any one of claims 1 to 6, wherein the disease is a tumor or an autoimmune disease.
The use according to claim 7, wherein the disease is a tumor.
The use of claim 8, wherein the immunotherapy is directed against one or more tumor-associated antigens expressed by the tumor.
The use of claim 9, wherein the one or more tumor-associated antigens are selected from CD19, CD123, CD22, CD30, CD171, CS-1 (also known as CD2 subset 1, CRACC, SLAMF7, CD319, and 19A24), C-type lectin-like molecule-1 (CLL-1 or CLECL1), CD33, epidermal growth factor receptor variant III (EGFRvIII), ganglioside G2 (GD2), ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer), TNF receptor family member B cell maturation (BCMA), Tn antigen ((Tn Ag) or (GalNAcα-Ser/Thr)), prostate specific membrane antigen (PSMA), receptor tyrosine kinase ROS-like orphan receptor 1 (ROR1), Fms-like tyrosine kinase 3 (FLT3), tumor-associated glycoprotein 72 (TAG72), CD38, CD44v6, carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EPCAM), B7H3 (CD276), KIT (CD117), interleukin 13 receptor subunit alpha-2 (IL-13Rα2 or CD213A2), mesothelin, interleukin 11 receptor alpha (IL-11Ra), prostate stem cell antigen (PSCA), testisin serine 21 (testisin or PRSS21), vascular endothelial growth factor receptor 2 (VEGFR2), Lewis (Y) antigen, CD24, platelet-derived growth factor receptor beta (PDGFR-β), stage-specific embryonic antigen-4 (SSEA-4), CD20, folate receptor alpha, receptor tyrosine protein Kinase ERBB2 (Her2/neu), cell surface associated mucin 1 (MUC1), epidermal growth factor receptor (EGFR), neural cell adhesion molecule (NCAM), prostase, prostatic acid phosphatase (PAP), mutated elongation factor 2 (ELF2M), ephrin B2, fibroblast activation protein alpha (FAP), insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX), proteasome (Prosome, Macropain) subunit, beta type, 9 (LMP2), glycoprotein 100 (gp100), oncogene fusion protein composed of breakpoint cluster region (BCR) and Alelson murine leukemia virus oncogene homolog 1 (AB1) (bcr-abl), tyrosinase, ephrin type A receptor 2 (EphA2), fucosyl GM1 , sialyl Lewis adhesion molecule (sLe), ganglioside GM3 (aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer), transglutaminase 5 (TGS5), high molecular weight melanoma associated antigen (HMWMAA), o-acetyl GD2 ganglioside (OAcGD2), folate receptor β, tumor endothelial marker 1 (TEM1/CD248), tumor endothelial marker 7 related (TEM7R), Claudin 6 (CLDN6), thyroid stimulating hormone receptor (TSHR), G protein-coupled receptor class 5 group C, member D (GPRC5D), X chromosome open reading frame 61 (CXORF61), CD97, CD179a, anaplastic lymphoma kinase (ALK), polysialic acid, placenta-specific 1 (PLAC1), globoH hexose moiety of glycoceramide (GloboH), breast differentiation antigen (NY-BR-1), uroplakin 2 (UPK2), hepatitis A virus cell receptor 1 (HAVCR1), adrenergic receptor β3 (ADRB3), pannexin 3 (PANX3), G protein-coupled receptor 20 (GPR20), lymphocyte antigen 6 complex, locus K9 (LY6K), olfactory receptor 51E2 (OR51E2), TCRγ alternate reading frame protein (TARP), Wilms tumor protein (WT1), cancer/testis antigen 1 (NY-ESO-1), cancer/testis antigen 2 (LAGE-1A), melanoma-associated antigen 1 (MAGE-A1), ETS translocation variant gene 6, located on chromosome 12p (ETV6-AML), sperm protein 17 (SPA17), X antigen family, member 1A (XA GE1), angiopoietin binding cell surface receptor 2 (Tie2), melanoma cancer testis antigen-1 (MAD-CT-1), melanoma cancer testis antigen-2 (MAD-CT-2), FOS-related antigen 1, tumor protein p53 (p53), p53 mutant, prostein, survivin, telomerase, prostate cancer tumor antigen-1 (PCTA-1 or galectin 8), melanoma antigen recognized by T cells 1 (MelanA or MART1), rat sarcoma (Ras) mutant, human telomerase reverse transcriptase (hTERT), sarcoma translocation breakpoints, melanoma inhibitor of apoptosis (ML-IAP), ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene), N-acetylglucosaminyltransferase V (NA17), paired box protein Pax-3 (PAX3), androgen receptor, cyclin B1, V-myc avian Myelocytic neoplastic disease viral oncogene neuroblastoma-derived homolog (MYCN), Ras homolog family member C (RhoC), tyrosinase-related protein 2 (TRP-2), cytochrome P450 1B1 (CYP1B1), CCCTC binding factor (zinc finger protein)-like (brother of BORIS or regulator of imprinting sites), squamous cell carcinoma antigen recognized by T cells 3 (SART3), paired box protein Pax-5 (PAX5), proacrosin binding protein sp32 (OY-TES1), lymphocyte-specific protein tyrosine kinase (LCK), A kinase anchoring protein 4 (AKAP-4), synovial sarcoma, X breakpoint 2 (SSX2), receptor for advanced glycation end products (RAGE-1), renal uibiguitous 1 (RU1), renal uibiguitous 2 (RU2), legumain, human papilloma HPV E6 (HPV E6), human papillomavirus E7 (HPV E7), intestinal carboxylesterase, mutant heat shock protein 70-2 (mut hosp 70-2), CD79a, CD79b, CD72, leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), Fc fragment of IgA receptor (FCAR or CD89), leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2), CD300 molecule-like family member f (CD300LF), C-type lectin domain family 12 member A (CLEC12A), bone marrow stromal cell antigen 2 (BST2), EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2), lymphocyte antigen 75 (LY75), phosphatidylinositol proteoglycan-3 (GPC3), Fc receptor-like 5 (FCRL5), and immunoglobulin lambda-like polypeptide 1 (IGLL1) One or more.
The use of claim 9, wherein the one or more tumor-associated antigens are selected from TSHR, CD171, CS-1, CLL-1, GD3, Tn Ag, FLT3, CD38, CD44v6, B7H3, KIT, IL-13Rα2, IL-11Ra, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-β, SSEA-4, MUC1, EGFR, NCAM, CAIX, LMP2, EphA2, fucosyl GM1, SLE, GM3, TGS5, HMWMAA, o-acetyl GD2, folate receptor β, TEM1/CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, A One or more of DRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53 mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, ERG (TMPRSS2ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B1, MYCN, RhoC, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, and IGLL1.
The use of claim 9, wherein the one or more tumor-associated antigens are selected from one or more of CD19, IL-13Rα2, EphA2 and EGFRvIII.
Use of mesenchymal stem cells for enhancing the in vivo proliferation capacity of immune effector cells after administration and/or increasing the in vivo persistence of single immune effector cells after administration, wherein the mesenchymal stem cells do not express or substantially do not express antigens targeted by the immune effector cells on their surface.
The use of claim 13, wherein the immune effector cells are modified.
The use of claim 13, wherein the immune effector cells are T cells, optionally CAR-T cells.
Use of mesenchymal stem cells for regulating the in vivo differentiation of immune effector cells after administration and/or promoting the in vivo differentiation of immune effector cells into memory cell subtypes after administration, wherein the surface of the mesenchymal stem cells does not express or substantially does not express the antigens targeted by the immune effector cells.
The use of claim 16, wherein the immune effector cells are modified.
The use of claim 16, wherein the immune effector cells are T cells, optionally CAR-T cells.
Use of mesenchymal stem cells in the preparation of anti-tumor drugs, wherein the drugs also contain immune effector cells, and the surface of the mesenchymal stem cells does not express or substantially does not express antigens targeted by the immune effector cells.
The use of claim 19, wherein the immune effector cells are modified.
The use of claim 19, wherein the immune effector cells are T cells, optionally CAR-T cells.
Use of mesenchymal stem cells for preparing one or more of the following:

(1) Agents that enhance the in vivo proliferation capacity of immune effector cells and/or the in vivo persistence of single immune effector cells;

(2) immune effector cell differentiation inhibitors that keep immune effector cells in a low-differentiation state;

(3) Agents that increase the formation of memory immune effector cell subtypes;

(4) An agent for enhancing the efficacy of cell (preferably immune effector cell) therapy;

The mesenchymal stem cells do not express or substantially do not express the antigen targeted by the cells on their surface.
The use of claim 22, wherein the immune effector cells are modified.
The use of claim 22, wherein the immune effector cells are T cells, optionally CAR-T cells.
The invention relates to the use of mesenchymal stem cells in combination with immune effector cells for preparing a drug for treating cancer, wherein the surface of the mesenchymal stem cells does not express or substantially does not express the antigen targeted by the immune effector cells.
The use of claim 25, wherein the immune effector cells are modified.
The use of claim 25, wherein the immune effector cells are T cells, optionally CAR-T cells.
A pharmaceutical composition comprising mesenchymal stem cells and immune effector cells, and optionally further comprising a suitable pharmaceutically acceptable carrier, wherein the surface of the mesenchymal stem cells does not express or substantially does not express antigens targeted by the immune effector cells, and the drug is used to treat tumors or autoimmune diseases.
The pharmaceutical composition of claim 28, wherein the immune effector cells are modified.
The pharmaceutical composition of claim 28, wherein the immune effector cells are T cells, optionally CAR-T cells.
A method for treating a tumor, the method comprising administering immune effector cells to a subject having the tumor, wherein the cells have been co-cultured with mesenchymal stem cells in vitro/ex vivo, and/or co-administered with mesenchymal stem cells in vivo, wherein the immune effector cells are directed against a tumor-associated antigen of the tumor, and the mesenchymal stem cells do not express or substantially do not express the antigen directed against by the immune effector cells on their surface.
The method of claim 31, wherein the immune effector cells are modified.
The method of claim 31, wherein the immune effector cell is a T cell, optionally a CAR-T cell.
The method of claim 31, wherein the tumor-associated antigen is selected from CD19, CD123, CD22, CD30, CD171, CS-1 (also known as CD2 subset 1, CRACC, SLAMF7, CD319, and 19A24), C-type lectin-like molecule-1 (CLL-1 or CLECL1), CD33, epidermal growth factor receptor variant III (EGFRvIII), ganglioside G2 (GD2), ganglioside GD3 (aNeu5Ac (2-8) aNeu5Ac (2-3) bDGalp (1-4) bDGlcp (1-1) Cer), TNF receptor family member B cells Maturation (BCMA), Tn antigen ((Tn Ag) or (GalNAcα-Ser/Thr)), prostate-specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), Fms-like tyrosine kinase 3 (FLT3), tumor-associated glycoprotein 72 (TAG72), CD38, CD44v6, carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EPCAM), B7H3 (CD276), KIT (CD117), interleukin 13 receptor subunit α-2 (IL-13Rα2 or CD213A2), mesothelin, interleukin 11 receptor α (IL-11Ra), prostate stem cells Antigen (PSCA), testisin or PRSS21, vascular endothelial growth factor receptor 2 (VEGFR2), Lewis (Y) antigen, CD24, platelet-derived growth factor receptor beta (PDGFR-β), stage-specific embryonic antigen-4 (SSEA-4), CD20, folate receptor alpha, receptor tyrosine protein kinase ERBB2 (Her2/neu), cell surface-associated mucin 1 (MUC1), epidermal growth factor receptor (EGFR), neural cell adhesion molecule (NCAM), prostase, prostatic acid phosphatase (PAP), mutated elongation factor 2 (ELF2 M), ephrin B2, fibroblast activation protein alpha (FAP), insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX), proteasome (Prosome, Macropain) subunit, beta type, 9 (LMP2), glycoprotein 100 (gp100), oncogene fusion protein composed of breakpoint cluster region (BCR) and Alelson murine leukemia virus oncogene homolog 1 (AB1) (bcr-abl), tyrosinase, ephrin type A receptor 2 (EphA2), fucosyl GM1, sialyl Lewis adhesion molecule (sLe), ganglioside GM3 (aNeu5Ac(2 -3)bDGalp(1-4)bDGlcp(1-1)Cer), transglutaminase 5 (TGS5), high molecular weight melanoma associated antigen (HMWMAA), o-acetyl GD2 ganglioside (OAcGD2), folate receptor β, tumor endothelial marker 1 (TEM1/CD248), tumor endothelial marker 7 related (TEM7R), Claudin 6 (CLDN6), thyroid stimulating hormone receptor (TSHR), G protein coupled receptor class C 5 group, member D (GPRC5D), X chromosome open reading frame 61 (CXORF61), CD97, CD179a, anaplastic lymphoma kinase ( ALK), polysialic acid, placenta-specific 1 (PLAC1), hexose moiety of globoH glycoceramide (GloboH), breast differentiation antigen (NY-BR-1), uroplakin 2 (UPK2), hepatitis A virus cell receptor 1 (HAVCR1), adrenergic receptor β3 (ADRB3), pannexin 3 (PANX3), G protein-coupled receptor 20 (GPR20), lymphocyte antigen 6 complex, locus K9 (LY6K), olfactory receptor 51E2 (OR51E2), TCRγ alternate reading frame protein (TARP), nephroblastoma protein (WT1 ), cancer/testis antigen 1 (NY-ESO-1), cancer/testis antigen 2 (LAGE-1A), melanoma-associated antigen 1 (MAGE-A1), ETS translocation variant gene 6, located on chromosome 12p (ETV6-AML), sperm protein 17 (SPA17), X antigen family, member 1A (XAGE1), angiopoietin binding cell surface receptor 2 (Tie2), melanoma cancer testis antigen-1 (MAD-CT-1), melanoma cancer testis antigen-2 (MAD-CT-2), FOS-associated antigen 1, tumor protein p53 (p53), p53 mutant, prostein, survivin, telomerase, prostate adenocarcinoma tumor antigen-1 (PCTA-1 or galectin 8), melanoma antigen recognized by T cells 1 (MelanA or MART1), rat sarcoma (Ras) mutant, human telomerase reverse transcriptase (hTERT), sarcoma translocation breakpoints, melanoma inhibitor of apoptosis (ML-IAP), ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene), N-acetylglucosaminyltransferase V (NA17), paired box protein Pax-3 (PAX3), androgen receptor, cyclin B1, V-myc avian myelocytic virus oncogene neuroblastoma-derived homolog (MYCN), Ras homolog family family member C (RhoC), tyrosinase-related protein 2 (TRP-2), cytochrome P450 1B1 (CYP1B1), CCCTC binding factor (zinc finger protein)-like (brother of regulator of BORIS or imprinting sites), squamous cell carcinoma antigen recognized by T cells 3 (SART3), paired box protein Pax-5 (PAX5), proacrosin binding protein sp32 (OY-TES1), lymphocyte-specific protein tyrosine kinase (LCK), A kinase anchoring protein 4 (AKAP-4), synovial sarcoma, X breakpoint 2 (SSX2), receptor for advanced glycation end products (RAGE-1), renal uibiguitous 1 (RU1), renal uibiguitous 2 (RU2), legumain, human papillomavirus E6 (HPV E6), human papilloma HPV E7 (HPV E7), intestinal carboxylesterase, mutated heat shock protein 70-2 (mut hosp 70-2), CD79a, CD79b, CD72, leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), Fc fragment of IgA receptor (FCAR or CD89), leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2), CD300 molecule-like family member f (CD300LF), C-type lectin domain family 12 member A (CLEC12A), bone marrow stromal cell antigen 2 (BST2), EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2), lymphocyte antigen 75 (LY75), phosphatidylinositol proteoglycan-3 (GPC3), Fc receptor-like 5 (FCRL5), and immunoglobulin lambda-like polypeptide 1 (IGLL1) One or more.
The method of claim 31, wherein the tumor-associated antigen is selected from TSHR, CD171, CS-1, CLL-1, GD3, Tn Ag, FLT3, CD38, CD44v6, B7H3, KIT, IL-13Rα2, IL-11Ra, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-β, SSEA-4, MUC1, EGFR, NCAM, CAIX, LMP2, EphA2, fucosyl GM1, SLE, GM3, TGS5, HMWMAA, o-acetyl GD2, folate receptor β, TEM1/CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADR One or more of B3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53 mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, ERG (TMPRSS2ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B1, MYCN, RhoC, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, and IGLL1.
The method of claim 31, wherein the tumor-associated antigen is selected from one or more of CD19, IL-13Rα2, EphA2 and EGFRvIII.
A method for enhancing the in vivo proliferation capacity of immune effector cells after administration and/or increasing the in vivo persistence of a single immune effector cell after administration, the method comprising co-culturing the immune effector cells with mesenchymal stem cells in vitro/ex vivo, and/or co-administering the immune effector cells with mesenchymal stem cells into the body, wherein the mesenchymal stem cells do not express or substantially do not express the antigen targeted by the immunotherapy on their surface, and optionally the method is in vivo/in vitro/ex vivo.
The method of claim 37, wherein the immune effector cells are modified.
The method of claim 37, wherein the immune effector cell is a T cell, optionally a CAR-T cell.
A method for regulating the in vivo differentiation of immune effector cells after administration and/or promoting the differentiation of immune effector cells into memory cell subtypes in vivo after administration, the method comprising co-culturing the immune effector cells with mesenchymal stem cells in vitro/ex vivo, and/or co-administering the immune effector cells with mesenchymal stem cells into the body, wherein the surface of the mesenchymal stem cells does not express or substantially does not express the antigen targeted by the immunotherapy, and optionally the method is in vivo/in vitro/ex vivo.
The method of claim 40, wherein the immune effector cells are modified.
The method of claim 40, wherein the immune effector cell is a T cell, optionally a CAR-T cell.
The use or method or pharmaceutical composition of any preceding claim, wherein the mesenchymal stem cells are of placental origin.
The use or method or pharmaceutical composition of any preceding claim, wherein the mesenchymal stem cells express CD105 and/or do not express CD45.
The use or method or pharmaceutical composition of any preceding claim, wherein the mesenchymal stem cells are co-cultured in vitro with therapeutic cells used in cell therapy.
The use or method or pharmaceutical composition of any of the preceding claims, wherein the mesenchymal stem cells are co-administered into a subject with therapeutic cells used in cell therapy, wherein the mesenchymal stem cells are administered before, simultaneously with, or after the therapeutic cells.
The use or method or pharmaceutical composition of any preceding claim, wherein the ratio of the number of mesenchymal stem cells to the therapeutic cells used in cell therapy is 10:1, or 5:1, or 4:1, or 2:1, or 1:1, or 1:2, or 1:4, or 1:5, or 1:10, or 1:20.