IL-10 expressing cells for enhancing cancer immunotherapy

Abstract

The present invention relates generally to the field of anti-cancer therapy, and in particular to the use of adoptive T cell transfer therapy to treat cancer, especially solid tumors. More specifically, the present invention relates to immune cells comprising one or more recombinant constructs, at least one of which encodes interleukin-10, a fragment or variant thereof.

Description

[Technical field]

[0001] The present invention relates generally to the field of anti-cancer therapy, and in particular to the use of adoptive T cell transfer therapy to treat cancer, especially solid tumors. More specifically, the present invention relates to immune cells comprising one or more recombinant constructs, at least one of which encodes interleukin-10, a fragment or variant thereof. [Background technology]

[0002] Chimeric antigen receptor (CAR) T cells and T cell receptor (TCR) transgenic (genetically transduced) T cells (both referred to as "adoptive T cell transfer" therapy) are adoptive transfer immunotherapies based on genetically engineered T cells. For example, CAR T cells have shown promising results in the clinic, especially in hematological malignancies, but progress in solid tumors has been limited (Lim, WA and June, CH The Principles of Engineering Immune Cells to Treat Cancer. Cell 168, 724-740 (2017)).

[0003] CAR T cells in the tumor microenvironment (TME) have been reported to exhibit loss of effector function and proliferative capacity, defined as T cell "exhaustion," which can result from persistent antigen stimulation and other metabolic stresses in solid tumors (Schietinger, A. et al., Tumor-Specific T Cell Dysfunction Is a Dynamic Antigen-Driven Differentiation Program Initiated Early during Tumorigenesis. Immunity 45, 389-401 (2016); Vodnala, SK et al., T cell stemness and dysfunction in tumors are triggered by a common mechanism. Science 363, (2019)).

[0004] It has been reported that exhausted T cells exhibit suppressed mitochondrial respiration, and such poor metabolic fitness may enhance T cell exhaustion and impair their antitumor immune response. Metabolic intervention during the expansion phase of adoptively transferred CAR T cells has been shown to regulate in vivo differentiation and improve antitumor responses (Alizadeh, D. et al., IL15 Enhances CAR T Cell Antitumor Activity by Reducing mTORC1 Activity and Preserving Their Stem Cell Memory Phenotype. Cancer Immunol.Res. 7, 759-772(2019)).

[0005] However, this type of intervention resulted in suboptimal antitumor effects, which could be caused either by a lack of constitutive supply of the applied cytokines or by a limited metabolic reprogramming capacity to fully rescue T cells from exhaustion. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Lim, WA and June, CH The Principles of Engineering Immune Cells to Treat Cancer. Cell 168, 724-740 (2017) [Non-Patent Document 2] Schietinger, A. et al., Tumor-Specific T Cell Dysfunction Is a Dynamic Antigen-Driven Differentiation Program Initiated Early during Tumorigenesis. Immunity 45, 389-401 (2016) [Non-Patent Document 3] Vodnala, SK et al., T cell stemness and dysfunction in tumors are triggered by a common mechanism. Science 363, (2019) [Non-Patent Document 4] Alizadeh, D. et al., IL15 Enhances CAR T Cell Antitumor Activity by Reducing mTORC1 Activity and Preserving Their Stem Cell Memory Phenotype. Cancer Immunol.Res. 7, 759-772 (2019) Summary of the Invention [Problem to be solved by the invention]

[0007] Thus, there remains an urgent need for engineered processes that support the metabolic compatibility, proliferation, and survival of immune cells within the TME, as well as the development of effective immune cells with enhanced anti-tumor activity. [Means for solving the problem]

[0008] The present invention provides an immune cell expressing interleukin-10, a fragment or a variant thereof, said immune cell comprising one or more recombinant constructs, at least one of which encodes interleukin-10, a fragment or a variant thereof.

[0009] Further provided are nucleic acid sequences encoding one or more recombinant constructs of the present invention.

[0010] Further provided is a plasmid or vector comprising a nucleic acid sequence of the invention.

[0011] Further provided is a pharmaceutical composition comprising i) an immune cell of the invention, ii) a nucleic acid of the invention, and / or iii) a plasmid or vector of the invention and at least one pharma- ceutically acceptable carrier or diluent.

[0012] Also provided is a method for the treatment and / or prevention of cancer, comprising the step of administering a pharmaceutical composition of the present invention to a subject in need thereof.

[0013] Also provided is a method of treating and / or preventing cancer in a subject, comprising the steps of: (i) removing and isolating immune cells, preferably natural T cells, from said subject or providing immune cells, preferably natural T cells; (ii) genetically engineering said T cells with at least one recombinant construct encoding interleukin-10, a fragment or variant thereof, and a second recombinant construct encoding a chimeric antigen receptor (CAR), a T cell receptor (TCR) or any other synthetic tumor targeting motif or antigen; (iii) expanding ex vivo into a larger population of engineered T cells; and (iv) reintroducing into said patient or subject.

[0014] Also provided is a method of enhancing anti-tumor activity in a subject comprising the steps of: (i) removing and isolating immune cells, preferably natural T cells, from said subject or providing immune cells, preferably natural T cells; (ii) genetically engineering said T cells with at least one recombinant construct encoding interleukin-10, a fragment or variant thereof, and a second recombinant construct encoding a chimeric antigen receptor (CAR), a T cell receptor (TCR) or any other synthetic tumor targeting motif or antigen; (iii) expanding ex vivo into a larger population of engineered T cells; and (iv) reintroducing into said patient or subject. [Brief description of the drawings]

[0015] [Figure 1 (1)]HER2-specific CAR T cells co-expressing IL-10 (IL-10 HER2 CAR T) enhance OXPHOS of CAR T cells upon antigen stimulation and promote CAR T cell proliferation. (a) Schematic diagram of HER2-directed second-generation CAR (HER2 CAR) and HER2-directed second-generation CAR modified to express mouse IL-10 after the 2A element (HER2 CAR-IL-10). [Figure 1 (2)] HER2-specific CAR T cells co-expressing IL-10 (IL-10 HER2 CAR T) enhance OXPHOS of CAR T cells upon antigen stimulation and promote CAR T cell proliferation. (b) Transduction with HER2 CAR or HER2 CAR-IL-10 construct was performed by retroviral vector. CAR expression levels were analyzed by flow cytometry. Numbers in the histograms represent the percentage of HER2 CAR positive staining cells. Similar results were obtained from 10 independent experiments. [Figure 1 (3)] HER2-specific CAR T cells co-expressing IL-10 (IL-10 HER2 CAR T) enhance OXPHOS of CAR T cells upon antigen stimulation and promote CAR T cell proliferation. (c) CAR T cells were co-cultured with mitomycin C-treated MC38-HER2 (HER2-expressing MC38 colon cancer cells) for 3 days. Culture supernatants were examined for the concentration of IL-10 by enzyme-linked immunosorbent assay (ELISA). (d, e) CAR T cells were labeled with the cell tracker CFSE and co-cultured with mitomycin C-treated MC38-HER2 cells at an effector:target (E:T) ratio of 1:1 for the indicated time periods. (d) Absolute number of viable HER2 CAR T or IL-10 HER2 CAR T on different days. (e) Percentage of HER2 CAR T or IL-10 HER2CAR T cell division in the presence of phosphate-buffered saline (PBS), isotype control antibody, or anti-IL-10 mAb. Data represent the mean ± SEM. Similar results were obtained from at least three independent experiments. [Figure 1 (4)]HER2-specific CAR T cells co-expressing IL-10 (IL-10 HER2 CAR T) enhance OXPHOS of CAR T cells upon antigen stimulation and promote CAR T cell proliferation. (f) Representative oxygen consumption rate (OCR) trace of CAR T cells stimulated for 24 h with MC38-HER2 at an E:T ratio of 5:1. (g) Representative extracellular acidification rate (ECAR) trace of cells treated as in (f). Similar results were obtained from at least three independent experiments. [Figure 1 (5)] HER2-specific CAR T cells co-expressing IL-10 (IL-10 HER2 CAR T) enhance OXPHOS of CAR T cells upon antigen stimulation and promote CAR T cell proliferation. (h) Statistical analysis of basal OCR from (f). (i) Statistical analysis of basal ECAR from (g). Data represent mean ± SEM. Similar results were obtained from at least three independent experiments. [Figure 1 (6)] HER2-specific CAR T cells co-expressing IL-10 (IL-10 HER2 CAR T) enhance OXPHOS of CAR T cells upon antigen stimulation and promote CAR T cell proliferation. (j) Ratio of OCR / ECAR from (f) and (g). Data represent mean ± SEM. Similar results were obtained from at least three independent experiments. [Diagram 2] IL-10 HER2 CAR T cells enhance the antitumor activity of CAR T cells in a pyruvate-dependent manner. (a) Cytotoxicity assay using MC38-HER2 cells as targets. Target cells were mixed with HER2 CAR T cells or IL-10 HER2 CAR T cells at the indicated E / T ratios. (b, c, d) 48-h co-cultures of non-transduced control T cells (Ctrl T), HER2 CAR T in the presence of 0 and 145 ng / mL murine recombinant IL-10 (mIL-10), or IL-10 HER2 CAR T cells with MC38-HER2 cells at an E:T ratio of 0.5:1. (b) MC38-HER2 tumor cell killing percentage, (c) number of viable CAR T cells were analyzed by flow cytometry. (d) The proportion of polyfunctional CAR T cells in the co-culture or resting phase was assessed by intracellular cytokine staining. [Diagram 3] IL-10 HER2 CAR T therapy eradicates established murine MC38-HER2 colon adenocarcinoma. C57BL / 6 mice were inoculated subcutaneously with MC38-HER2 cells (3×105) and received intravenous (iv) adoptive cell transfer of HER2 CAR T cells (3×106), IL-10 HER2 CAR T cells (3×106), or HER2 CAR T cells (3×106) followed by iv administration of mIL-10 (1 μg), respectively, on day 6. The mean tumor growth curves (a) and survival curves (b) of each treatment group are shown. The number of long-term survivors out of the total number of mice in the group is shown. (c) Surviving mice from the IL-10 HER2 CAR T monotherapy treatment group were subcutaneously rechallenged with MC38-HER2 (1×106) cells 90 days after the primary inoculation. Naive WT mice (n=5) were inoculated with the same number of tumor cells as controls. Survival curves to rechallenge and the number of long-term surviving mice are shown. Data represent the mean±SEM. [Figure 4(1)] TRP-1 IL-10 CAR T therapy extends survival in a murine B16F10 melanoma model. (a) Schematic diagram of TRP-1-directed second generation CAR (TRP-1 CAR) and TRP-1-directed second generation CAR modified to express murine IL-10 after the 2A element (IL-10 TRP-1 CAR). (b) Transduction with TRP-1 CAR or IL-10 TRP-1 CAR constructs was performed by retroviral vector. CAR expression levels were analyzed by flow cytometry. Numbers in the histograms represent the percentage of c-Myc tag positive staining cells. Similar results were obtained from 10 independent experiments. [Figure 4 (2)]TRP-1 IL-10 CAR T therapy extends survival in a murine B16F10 melanoma model. (c, d) 48-hour co-culture of TRP-1 CAR T or IL-10 TRP-1 CAR T cells with B16F10 cells at an E:T ratio of 0.5:1 in the presence of 0 and 145 ng / mL murine recombinant IL-10 (mIL-10). (c) Percentage of B16F10 tumor cell killing was analyzed by flow cytometry. (d) Number of surviving CAR T cells was analyzed by flow cytometry. Data represent mean ± SEM. [Figure 4 (3)] TRP-1 IL-10 CAR T therapy prolongs survival in a murine B16F10 melanoma model. (e, f) C57BL / 6 mice were inoculated subcutaneously with B16F10 melanoma cells (3×105) and received adoptive cell transfer iv on day 6 of TRP-1 CAR T cells (3×106), IL-10 TRP-1 CAR T cells (3×106), respectively. The mean tumor growth curves for each treatment group (e) and survival curves for each treatment group (f) are shown. Data represent the mean ± SEM. [Figure 5(1)] Complete regression of a pre-established murine 4T1-Luc-EGFRvIII metastatic breast cancer model by treatment with IL-10 EGFRvIII CAR T cells. (a) Schematic diagram of EGFRvIII-directed second generation CAR (EGFRvIII CAR) and EGFRvIII-directed second generation CAR modified to express murine IL-10 after the 2A element (IL-10 EGFRvIII CAR). (b) CAR expression levels were analyzed by flow cytometry. Numbers in the histograms represent the percentage of c-Myc tag positive staining cells. Similar results were obtained from 10 independent experiments. [Figure 5 (2)]Complete regression of a pre-established murine 4T1-Luc-EGFRvIII metastatic breast cancer model by treatment with IL-10 EGFRvIII CAR T cells. (c) CAR T cells were co-cultured with mitomycin C-treated 4T1-Luc-EGFRvIII for 3 days. Culture supernatants were examined for IL-10 concentration by ELISA. (d, e) 48-hour co-cultures of EGFRvIII CAR T in the presence of Ctrl T, 0 and 145 ng / mL mIL-10, or IL-10 EGFRvIII CAR T cells with 4T1-Luc-EGFRvIII cells at an E:T ratio of 0.5:1. (d) 4T1-Luc-EGFRvIII tumor cell killing percentage was analyzed by flow cytometry. (e) Number of viable CAR T cells was analyzed by flow cytometry. Data represent mean ± SEM. [Figure 5 (3)] Complete regression of a pre-established murine 4T1-Luc-EGFRvIII metastatic breast cancer model by treatment with IL-10 EGFRvIII CAR T cells. (f-h) BALB / c mice were injected iv with 4T1-Luc-EGFRvIII cells (5x104) and received adoptive cell transfer of EGFRvIII CAR T cells (3x106), or IL-10 EGFRvIII CAR T cells (3x106), or EGFRvIII CAR T cells (3x106) followed by iv administration of mIL-10 (1μg) on ​​day 6, respectively. (f) Individual mean radiance (p / s / cm2 / sr) of mouse groups at different time points. (g) Survival curves of each treatment group and the number of long-term survivors out of the total number of mice in the group are shown. (h) Number of CAR T cells in blood on day 15 quantified by flow cytometry. Data represent mean ± SEM. [Figure 6]IL-10 Pmel TCR T therapy prolongs survival in a murine B16F10 melanoma model. (a) Transduction with IL-10 constructs was performed by retroviral vector. IL-10 expression levels were analyzed by ELISA. (b-f) C57BL / 6 mice were inoculated subcutaneously with B16F10 melanoma cells (3×105) and received adoptive cell transfer of Pmel T cells (10×106) or IL-10 Pmel T cells (10×106) iv on day 6, respectively. (b-d) Individual tumor growth curves of PBS control group (b), Pmel T cell therapy (c) and IL-10 Pmel T cell therapy (d). (e,f) Mean tumor growth curves (e) and survival curves (f) for each treatment group are shown. Data represent mean ± SEM. [Figure 7(1)] In vitro characterization of IL-10 CD19 human CAR T. (a) Schematic diagram of CD19-directed second generation CAR (CD19 CAR) and CD19-directed second generation CAR modified to express human IL-10 (IL-10 CD19 CAR). (b) CAR expression levels were analyzed by flow cytometry. Similar results were obtained from 10 independent experiments. [Figure 7(2)] In vitro characterization of IL-10 CD19 human CAR T. (c) Secreted IL-10 concentration was detected by ELISA. (d) Percentage of tumor cell killing in co-cultures of Ctrl T, CD19 CAR T, or IL-10 CD19 CAR T cells with target tumor cells at an E:T ratio of 1:32 was analyzed by LDH assay. (e) Mean tumor growth curves. NSG mice were inoculated (sc) with PANC1-CD19 human epidermoid carcinoma cells (2x106) and received adoptive cell transfer of CD19 hCAR T cells (1x106) or IL-10 CD19 hCAR T cells (1x106) iv on day 8 (n=9 / group). Data represent mean ± SEM. [Figure 8]IL-10 expression maintains mitochondrial fitness of CAR-T cells. C57BL / 6 mice were inoculated with MC38-HER2 tumor cells (1×106, sc), sublethally lymphodepleted by irradiation on day 5, and iv transferred with IL-10 HER2 CAR-T cells (3×106) or HER2 CAR-T cells (3×106) with or without iv IL-10 (1 μg) on ​​day 6 (n=5 / group). On day 14, mice were sacrificed and the indicated tissues were processed. CAR-T cells were subjected to mitochondrial phenotype analysis by flow cytometry or sorted for electron microscopy analysis. Mitochondrial mass and membrane potential of CAR-T cells were examined by staining with MitoTracker Green (MG) and MitoTracker Deep Red (MDR), respectively. a. Frequency of HER2 CAR-T cells with dysfunctional mitochondria. b. Ratio of MDR / MG in tumor-infiltrating HER2 CAR-T cells from the indicated treatment groups. c. Representative electron microscopy images of selected intratumoral HER2 CAR-T cells from the indicated treatment groups. d-f. Quantification of mitochondria per cell (d), cristae per mitochondrion (e), and total cristae length per mitochondrial area (f) in the selected intratumoral HER2 CAR-T cells shown in (c). All data represent the mean ± sem and are analyzed by unpaired Student's t-test (d, e, f) or one-way ANOVA with Tukey's multiple comparison test (a, b). [Figure 9]Surviving mice after treatment with IL-10 HER2 CAR-T cells or IL-10 TRP-1 CAR-T cells as described in Figure 5 were rechallenged sc with MC38-HER2 (1x106) and B16F10 (1x105) cells, respectively, 90 days after primary tumor inoculation. Naive WT mice (n=5 / group) were inoculated with the same number of tumor cells as controls. a. Experimental timeline. b, c. Survival curves and numbers of long-term surviving mice rejecting a second tumor challenge in the MC38-HER2 tumor model (b) and the B16F10 tumor model (c) are shown. [Figure 10] Mice treated with IL-10 CAR-T cells induced stem cell-like memory. C57BL / 6 mice were inoculated with MC38-HER2 cells (1x106, sc), sublethally lymphodepleted by irradiation on day 5, and iv transferred with IL-10 HER2 CAR-T cells (3x106) or HER2 CAR-T cells (3x106) with or without iv IL-10 (1μg) on ​​day 6 (n=5 / group). On day 18, mice were sacrificed for phenotypic analysis of CAR-T cells in spleen and peripheral blood by flow cytometry. a, b. Mean frequency of CD62L+CD44- among total CAR-T cells in spleen (a) and mean frequency of Sca-1+CD122+ among CD62L+CD44- CAR-T cells (b). c. Representative flow cytometry plots and mean MFI of Sca-1 expression in CAR-T cells in spleen. d. Representative flow cytometry plots showing the phenotype of CAR-T cells in spleen and blood (e). Frequency of IL-7Rα+KLRG-1- among total CAR-T cells in spleen (d) and blood (e). All data represent mean ± sem and are analyzed by one-way ANOVA with Tukey's multiple comparison test (a-e). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The publications and applications discussed herein are provided solely for their disclosure prior to the filing date of this application. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publications by virtue of prior invention. Additionally, the materials, methods, and examples are illustrative only and are not intended to be limiting.

[0017] In case of conflict, the present specification, including definitions, will control. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter of this invention belongs. As used herein, the following definitions are provided to facilitate understanding of the present invention.

[0018] The term "comprise / comprising" is generally used in the sense of include / including, i.e. allowing for the presence of one or more features or components. The terms "comprise" and "comprising" also encompass the more restrictive terms "consist" and "consisting," respectively, as well as "consist / consisting essentially of."

[0019] As used in this specification and claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0020] As used herein, "at least one" means "one or more," "two or more," "three or more," etc. For example, at least one means one or more constructs, and refers to one construct, two constructs, three constructs, etc.

[0021] As used herein, the terms "subject" / "subject in need" or "patient" / "patient in need" are well-recognized in the art and are used interchangeably herein to refer to a mammal, including a dog, cat, rat, mouse, monkey, cow, horse, goat, sheep, pig, camel, and most preferably a human. In some cases, the subject is a subject in need of treatment or a subject suffering from a disease or disorder. However, in other aspects, the subject can be a healthy subject. The term does not denote a particular age or sex. Thus, it is intended to encompass adult and newborn subjects, whether male or female. Preferably, the subject is a human, most preferably a human at risk of suffering from cancer.

[0022] According to the present invention, the cancer is solid or liquid cancer.In one embodiment, the cancer is solid cancer.Preferably, the solid cancer is selected from the non-limiting group including lung cancer, breast cancer, ovarian cancer, cervical cancer, uterine cancer, head and neck cancer, glioblastoma, hepatocellular carcinoma, colon cancer, rectal cancer, colorectal cancer, kidney cancer, prostate cancer, gastric cancer, bronchial cancer, pancreatic cancer, bladder cancer, liver cancer, brain cancer and skin cancer, particularly melanoma, or one or more combinations thereof.

[0023] The terms "nucleic acid", "polynucleotide" and "oligonucleotide" are used interchangeably and refer to any type of polymer of deoxyribonucleotides (e.g. DNA, cDNA, ...) or ribonucleotides (e.g. RNA, mRNA, ...), or a combination of deoxyribonucleotides and ribonucleotides (e.g. DNA / RNA) in a linear or circular conformation, in single- or double-stranded form. These terms are not intended to be limiting with respect to the length of the polymer, and can encompass known analogues of natural nucleotides, as well as nucleotides modified in the base, sugar and / or phosphate moieties (e.g. phosphorothioate backbones). In general, analogues of a particular nucleotide have the same base-pairing specificity; i.e., an analogue of A will base-pair with T.

[0024] The term "vector," as used herein, refers to a nucleic acid (DNA or RNA) molecule, such as a viral vector, or a plasmid or other vehicle, which contains one or more heterologous nucleic acid sequences of the invention, and which is preferably designed for the purposes of transfer (transduction) and / or amplification between different host cells.

[0025] The terms "expression vector," "gene transfer vector," and "gene therapy vector" refer to any vector effective to introduce and express one or more nucleic acids of the invention in a cell, preferably under the control of a promoter. A cloning or expression vector may contain additional elements in addition to a promoter, such as, for example, regulatory elements and post-transcriptional regulatory elements.

[0026] As used herein, interleukin-10 (IL-10) refers to a member of the IL-10 family of cytokines. IL-10 is generally considered to be immunosuppressive because it reduces tissue damage caused by uncontrolled inflammatory responses. "IL-10, fragments or variants thereof" preferably includes sequences including native human IL-10, as well as sequences of fragments and variants of IL-10 as described in Mumm et al., 2011, Cancer Cell, 20, 781-796; Guo et al., 2012, Protein Expr. Purif., 83, 152-156 (2012); Zheng et al., 1997, J. Immunol., 158, 4507-13; Qiao et al., 2019, Cancer Cell 35, 901-915; Guo et al., 2021, Nat Immunol 22, 746-756, the contents of which are incorporated herein by reference in their entirety. In one embodiment, the IL-10 sequence is the human IL-10 amino acid sequence set forth in SEQ ID NO:1.

[0027] The term "variant" in the context of IL-10 refers to one or more biologically active derivatives of IL-10, preferably of the human IL-10 sequence of the present invention. In general, the term "variant" refers to a molecule having a native sequence and having one or more additions, substitutions (generally conservative in nature) and / or deletions compared to the native molecule, but to the extent that the modifications do not destroy its biological activity and are "substantially homologous" to the reference molecule (Gorby et al., Sci. Signal. 13, eabc0653, 2020; Saxton et al., Science 371, eabc8433, 2021). In general, the sequence of such a variant has a high degree of sequence homology or identity to the reference sequence, e.g., greater than 25%, generally greater than 50% to 70%, even more specifically 80% or 85% or more, e.g. at least 90% or 95% or more sequence homology or sequence identity when the two sequences are aligned. Spencer, Juliet V et al. reported that spliced ​​forms of IL-10 retain biological activity or properties despite having only 27% sequence identity to hIL-10 (Spencer, Juliet V et al., "Stimulation of B lymphocytes by cmvIL-10 but not LAcmvIL-10," Virology Vol. 374, 1 (2008): 164-9. doi:10.1016 / j.virol.2007.11.031, the contents of which are incorporated herein by reference in their entirety).

[0028] As used herein, a "fragment" of IL-10 of the present invention, preferably human IL-10, refers to a sequence that contains a shorter nucleotide length than the respective polypeptide or nucleic acid sequence. Preferably, the sequence or fragment contains 90% shorter, preferably 60% shorter, in particular 30% shorter, nucleotide length than the respective polypeptide or nucleic acid sequence.

[0029] While focusing on developing novel and efficient approaches to treat tumors with CAR engineered T cells, the inventors surprisingly showed that metabolically engineered IL-10 expressing CAR T redirected the fate of CAR T cells away from exhaustion and towards a memory-like state, leading to eradication of established solid tumors and durable cures in the majority of treated mice. These promising results demonstrated the great potential of IL-10 expressing CAR T or any other engineered immune cells to enhance the efficacy of adoptive cell therapy in the clinic.

[0030] The present invention in one aspect provides an immune cell or population of immune cells expressing interleukin-10, a fragment or a variant thereof, hi one aspect, the immune cell is an isolated immune cell.

[0031] As used herein, the term "immune cells" includes any type of immune cell classified as lymphocytes, neutrophils, and monocytes / macrophages, whether recombinant (engineered) or not. In a preferred embodiment, the immune cells are selected from the non-limiting group including T cells, chimeric antigen receptor (CAR)-T cells, T cell receptor (TCR)-transgenic T cells, tumor infiltrating lymphocytes (TIL), NK cells, NK-T cells, CAR-NK cells, CAR-NKT cells, TCR-transgenic NK cells, TCR-transgenic NK-T cells, dendritic cells, macrophages, CAR-macrophages, or any synthetic tumor-specific immune cells. The immune cell population of immune cells includes: Autologous, i.e. using the patient's own immune cells, or allogeneic, i.e. derived from donor blood, umbilical cord blood or pluripotent stem cells (such as iPSCs, which may be genetically engineered); or ·Heterogeneity It can be.

[0032] In the latter two cases, strategies to reduce allorejection should also be considered, which will be obvious to those skilled in the art.

[0033] Preferably, said immune cells comprise one or more recombinant constructs, at least one of which encodes interleukin-10, a fragment or a variant thereof.

[0034] In one embodiment, the construct encoding interleukin-10, a fragment or variant thereof comprises or encodes the amino acid sequence of SEQ ID NO:1, or a fragment or variant thereof.

[0035] In one embodiment, a recombinant construct encoding interleukin-10, a fragment or variant thereof is linked to a second recombinant construct encoding a CAR, TCR, or any other synthetic tumor targeting motif.

[0036] Also preferably, the second recombinant construct encodes a chimeric antigen receptor (CAR), a T cell receptor (TCR) or any other synthetic tumor targeting motif. Non-limiting examples of synthetic tumor targeting motifs include, for example, the tumor targeting peptides listed in Tables 2 and 3 of Liu R, Li X, Xiao W, Lam KS (tumor targeting peptides from combinatorial libraries [amendment published in Adv Drug Deliv Rev. March 9, 2018], which tables are incorporated herein by reference in their entirety.

[0037] Those skilled in the art know that tumor targeting motifs, such as tumor targeting peptides, can be detected, for example, from phage display libraries, through screening approaches selected from in vitro, in vivo or ex vivo selection approaches (see, for example, Figure 1 in Liu R, Li X, Xiao W, Lam KS). Cancer-associated proteins, specific cancer cell lines, patient tissues, and tumor xenograft mouse models can be used as screening probes.

[0038] Preferably, the linkage is via a sequence encoding a self-cleaving peptide (e.g., peptide 2A (see, e.g., Takahashi, K. and Yamanaka, S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663-676, which is incorporated herein by reference in its entirety), or an internal ribosome entry site (IRES) sequence). For example, upon cleavage of the self-cleaving peptide, IL-10, a fragment or variant thereof is secreted or membrane-bound by or on immune cells, preferably in the tumor microenvironment.

[0039] In one embodiment, the recombinant construct comprises a nucleic acid encoding a CAR that encodes a polypeptide of an extracellular antigen recognition domain, a transmembrane region, an intracellular T cell activation domain and / or an intracellular region of a single-chain fragment variant (scFv).

[0040] The extracellular antigen recognition domain of the single chain fragment variant (scFv) is preferably derived from an antibody or a ligand or receptor. In some instances, the extracellular domain comprises a hinge portion. Various hinges, such as the CD8 hinge, can be used in accordance with the present invention.

[0041] Typically, the extracellular antigen recognition domain of the antibody-derived single chain fragment variant (scFv) recognizes an antigen selected from the non-limiting group including c-MET, TRP-1, CD19, CD20, BCMA, CD133, CD171, CD70, CEA, EGFR, EGFR-vIII, EpCAM, EphA2, FAP, GD2, GPC3, HER2, HER3, IL-13Ra2, mesothelin, MUC1, claudin 18.2, PSCA, PSMA, ROR1, and VEGFR2, or a combination of one or more thereof. In a preferred embodiment, the extracellular antigen recognition domain in the CAR of the invention is a CD8 or CD28 transmembrane domain scFv that recognizes HER2 (SEQ ID NO: 2), a CD8 or CD28 transmembrane domain scFv that recognizes TRP-1 (SEQ ID NO: 3), a CD8 or CD28 transmembrane domain scFv that recognizes EGFR-vIII (SEQ ID NO: 4), or a CD8 or CD28 transmembrane domain scFv that recognizes CD19 (SEQ ID NO: 5), e.g., linked to a hinge.

[0042] The transmembrane region and hinge are usually fused to the extracellular domain of the CAR. The transmembrane region and hinge can also be fused to the intracellular domain of the CAR. In some cases, the transmembrane domain can be selected or modified by amino acid substitution to avoid such domains binding to the transmembrane domain of the same or different surface membrane protein to minimize interaction with other members of the receptor complex. The transmembrane domain can be derived from either natural or synthetic sources. If the source is natural, the domain can be derived from any membrane-associated or transmembrane protein. Particularly useful transmembrane regions in the present invention include CD28, CD28T, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), inducible T cell costimulator (ICOS), lymphocyte function-associated antigen-1 (LFA-1, CD1-la / CD18), CD3γ, CD3δ, CD3ε, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Igα (CD79a), DAP-10, Fcγ receptors, MHC class 1 molecules, TNF receptor proteins, immunoglobulin proteins, cytokine receptors, integrins, Signaling Lymphocytic Activation Molecules (SLRs), and the like. Molecules)(SLAM protein), activated NK cell receptor, BTLA, Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM(LIGHTR), KIRDS2, SLAMF7, NKp80( KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL-2Rβ, IL-2Rγ, IL-7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl ld, ITGAE, CD103, ITGAL, CDl la, LFA-1, ITGAM, CDl lb, ITGAX, CDlThe ligand may be derived from (or may include or correspond to) lc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, a ligand that specifically binds to CD83, or any combination thereof. CD19a.

[0043] Optionally, a short linker may form the connection between any or some of the extracellular, transmembrane, and intracellular domains of the CAR. In a preferred embodiment, the transmembrane domain and hinge in the CAR of the invention are the CD8 transmembrane domain and hinge. In one embodiment, the CD8 transmembrane domain and hinge comprises the transmembrane portion and hinge of the amino acid sequence of SEQ ID NO: 6, a fragment or variant thereof.

[0044] The intracellular T cell activation domain is capable of activating a T cell upon binding of the antigen binding molecule to its target. It will be understood that the intracellular domain typically further comprises one or more costimulatory molecules as described herein.

[0045] In a further aspect, the T cell activation domain comprises a CD3, preferably CD3 zeta, more preferably CD3 zeta (CD3ζ), of the amino acid sequence of SEQ ID NO: 7, a fragment or a variant thereof.

[0046] As used herein, a "costimulatory molecule" refers to a molecule that provides a signal that mediates a T cell response, including, but not limited to, proliferation, activation, differentiation, etc. A costimulatory molecule can provide a signal in addition to the primary signal provided by an activation molecule described herein.

[0047] The intracellular (cytoplasmic) region of the engineered T cells of the invention can provide for activation of at least one of the normal effector functions of an immune cell. Effector function of a T cell may refer to, for example, cytolytic activity or helper activity.

[0048] Suitable intracellular domains include, but are not limited to, CD28, CD28T, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), inducible T cell costimulatory factor (ICOS), lymphocyte function associated antigen-1 (LFA-1, CD1-la / CD18), CD3γ, CD3δ, CD3ε, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Igα (CD79a), DAP-10, Fcγ receptor, MHC class 1 molecule, TNF receptor protein, immunoglobulin protein, cytokine receptor body, integrin, signaling lymphocyte activation molecule (SLAM protein), activating NK cell receptor, BTLA, Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL-2Rβ, IL-2Rγ, IL-7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 ld, ITGAE, CD103, ITGAL, CDl la, LFA-1, ITGAM, CDl lb, ITGAX, CDl lc, ITGBl, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT It will be understood that the present invention includes (i.e., includes) signaling domains derived from (or corresponding to) a ligand that specifically binds to AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, CD83, or any combination thereof.

[0049] An example of a combination includes 4-1BB and the CD28 intracellular domain.

[0050] In a preferred embodiment, the intracellular domain of the CAR comprises the 4-1BB intracellular region.

[0051] Exemplary CAR constructs according to the present invention are as shown in Figures 1a, 4a, and 7a.

[0052] Where the second recombinant construct encodes a transgenic TCR, this TCR preferably recognises an antigen selected from the non-limiting group including gp100, NY-ESO-1, MAGE-A3 and TRP-1, or a combination of one or more of these.

[0053] In one embodiment, the construct encoding interleukin-10, a fragment or variant thereof is contained within a sequence encoding Fc, human serum albumin (HSA), or an antibody fusion protein.

[0054] In one aspect of the invention, the immune cells or populations of immune cells described herein are for use in the prevention and / or treatment of cancer. The cancer can be either a solid or liquid cancer.

[0055] Preferably, the cancer is a solid cancer selected from the non-limiting group including lung cancer, breast cancer, ovarian cancer, cervical cancer, uterine cancer, head and neck cancer, glioblastoma, hepatocellular carcinoma, colon cancer, rectal cancer, colorectal cancer, kidney cancer, prostate cancer, gastric cancer, bronchial cancer, pancreatic cancer, bladder cancer, liver cancer, brain cancer, lymphoma and skin cancer, in particular melanoma, or a combination of one or more thereof. More preferably, the solid cancer is selected from the group including lymphoma, breast cancer, gastric cancer and melanoma.

[0056] The present invention further provides nucleic acid sequences encoding one or more of the recombinant constructs described herein, including the SEQ ID NOs disclosed herein.

[0057] The present invention further provides plasmids or vectors comprising a nucleic acid sequence encoding one or more of the recombinant constructs described herein, including the SEQ ID NOs disclosed herein.

[0058] Any vector known in the art may be suitable for the present invention. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a retroviral vector (such as pMSGV), a DNA vector, a murine leukemia virus vector, a SFG vector, an RNA vector, an adenoviral vector, a baculoviral vector, an Epstein-Barr virus vector, a papovavirus vector, a vaccinia virus vector, a herpes simplex virus vector, an adenovirus-associated vector (AAV), a lentiviral vector (such as pGAR), or any combination thereof.

[0059] The present invention also contemplates compositions and pharmaceutical compositions. In one aspect of the invention, a pharmaceutical composition of the invention comprises a therapeutically effective amount of i) an immune cell or population of immune cells as described herein, ii) a nucleic acid as described herein, and / or iii) a plasmid or vector as described herein, and at least one pharma- ceutically acceptable carrier and / or diluent.

[0060] The term "therapeutically effective amount" as used herein means, within the scope of sound medical judgment, an amount of immune cells, nucleic acid, plasmid or vector that is high enough to significantly positively alter the symptoms and / or condition to be treated, yet low enough to avoid serious side effects (a reasonable risk / benefit ratio).

[0061] A therapeutically effective amount of the immune cells, nucleic acids, plasmids or vectors described herein is selected according to a variety of factors, including the type, species, age, weight, sex and medical condition of the patient or subject; the severity of the condition or disease (e.g., cancer) to be treated; the route of administration; and the renal and hepatic function of the patient or subject. One of skill in the art can readily determine and prescribe the effective amount of the immune cells, nucleic acids, plasmids or vectors required to prevent, combat or arrest the progression of a disease, such as cancer.

[0062] "Pharmaceutically acceptable carrier or diluent" means a carrier or diluent that is generally safe, non-toxic and useful for preparing a desired pharmaceutical composition, and includes a carrier or diluent that is acceptable for human pharmaceutical use.

[0063] The immune cells or populations of immune cells of the present invention may be administered alone or as a pharmaceutical composition. The pharmaceutical composition of the present invention may comprise the immune cells or populations of cells, such as T cells, described herein in combination with one or more pharma- ceutical or physiologically acceptable carriers or diluents. Such compositions may include buffers, such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrates, such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids, such as glycine; antioxidants; chelating agents, such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The compositions of the present invention are preferably formulated for intravenous administration.

[0064] The pharmaceutical compositions (solutions, suspensions, etc.) may contain one or more of the following: sterile diluents, such as water for injection, saline, preferably saline, Ringer's solution, isotonic sodium chloride, fixed oils, such as synthetic mono- or diglycerides, polyethylene glycol, glycerin, propylene glycol or other solvents that can function as solvents or suspending media; antibacterial agents, such as benzyl alcohol or methylparabens; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetates, citrates or phosphates, and agents for adjusting tonicity, such as sodium chloride or dextrose. Parenteral preparations can be enclosed in glass or plastic ampoules, disposable syringes or multiple dose vials. Pharmaceutical compositions for injection are preferably sterile.

[0065] The pharmaceutical compositions of the present invention can further comprise at least one additional therapeutic agent or therapy. A variety of other additional therapeutic agents may be used in conjunction with the compositions described herein. In one embodiment, the at least one additional therapeutic agent or therapy is an anti-cancer agent or therapy useful for treating cancer, preferably a solid cancer. Preferably, the one or more anti-cancer therapies will be selected from the group comprising radiation therapy, chemotherapy, immune checkpoint inhibitors, immunotherapy and hormonal therapy, or a combination of one or more thereof.

[0066] Preferably, the immune checkpoint inhibitor is selected from the group comprising a PD-1 inhibitor, a PD-L1 inhibitor, and a CTLA-4 inhibitor, or a combination of one or more thereof.

[0067] For example, potentially useful additional therapeutic agents include PD-1 inhibitors such as nivolumab (Opdivo®), pembrolizumab (Keytruda®), pembrolizumab, pidilizumab, and atezolizumab.

[0068] For example, potentially useful additional therapeutic agents include PD-L1 inhibitors such as atezolizumab, avelumab, AMP-224, MEDI-0680, RG-7446, GX-P2, durvalumab, KY-1003, KD-033, MSB-0010718C, TSR-042, ALN-PDL, STI-A1014, CX-072, and BMS-936559.

[0069] Non-limiting examples of CTLA-4 inhibitors include ipilimumab (Yervoy) (also known as BMS-734016, MDX-010, MDX-101) and tremelimumab (formerly ticilimumab, CP-675,206).

[0070] Chemotherapy according to the present invention can involve agents that damage DNA and / or prevent cells from proliferating, such as genotoxins.

[0071] The genotoxin may be selected from the group including alkylating agents, antimetabolites, DNA cutters, DNA binders, topoisomerase poisons and spindle poisons. Examples of alkylating agents are lomustine, carmustine, streptozocin, mechlorethamine, melphalan, uracil nitrogen mustard, chlorambucil, cyclosporine, ifosfamide, cisplatin, carboplatin, mitomycin, thiotepa, dacarbazine, procarbazine, hexamethylmelamine, triethylenemelamine, busulfan, pipobroman, mitotane and other platin derivatives.

[0072] An example of a DNA cutter is bleomycin.

[0073] The topoisomerase poison may be selected from the group including topotecan, irinotecan, camptothecin sodium salt, daunorubicin, doxorubicin, idarubicin, mitoxantrone, teniposide, adriamycin and etoposide.

[0074] Examples of DNA binders are dactinomycin and mithramycin, while the spindle poison may be selected from the group including vinblastine, vincristine, navelbine, paclitaxel and docetaxel.

[0075] The chemotherapy of the present invention may involve an antimetabolite selected from the following compounds: methotrexate, trimetrexate, pentostatin, cytarabine, ara-CMP, fludarabine phosphate, hydroxyurea, fluorouracil, floxuridine, chlorodeoxyadenosine, gemcitabine, thioguanine, and 6-mercaptopurine.

[0076] Radiation therapy refers to the use of high-energy radiation to shrink tumors and kill cancer cells. Examples of radiation therapy include, but are not limited to, external radiation therapy and internal radiation therapy (also called brachytherapy).

[0077] External radiation therapy is the most common and typically involves directing a beam of direct or indirect ionizing radiation to the tumor or cancer site. The radiation beam, photons, cobalt or microparticle therapy is focused on the tumor or cancer site, but it is almost impossible to avoid exposing normal healthy tissue. The energy source for external radiation therapy is selected from the group including direct or indirect ionizing radiation (e.g., x-rays, gamma rays and particle beams or combinations thereof).

[0078] Internal radiation therapy involves implanting a radiation emitting source, such as beads, wires, pellets, capsules, etc., within the body, at or near the tumor site. The energy source for internal radiation therapy is selected from the group of radioisotopes including iodine (Iodine-125 or Iodine-131), strontium-89, phosphorus, palladium, cesium, indium, phosphate, or cobalt radioisotopes, and combinations thereof. Such implants can be removed after treatment or left inactive in the body. Types of internal radiation therapy include, but are not limited to, interstitial brachytherapy and intracavitary brachytherapy (high dose rate, low dose rate, pulsed dose rate).

[0079] Currently less common forms of internal radiation therapy involve biological carriers of radioisotopes, such as by radioimmunotherapy, in which tumor-specific antibodies bound to radioactive substances are administered to the patient or subject. The antibodies bind to tumor antigens, thereby effectively delivering a dose of radiation to the relevant tissue.

[0080] Methods for administering radiation therapy are well known to those of skill in the art.

[0081] A variety of other additional therapeutic agents may be used in conjunction with the compositions described herein.

[0082] Additional therapeutic agents suitable for use in combination with the present invention include ibrutinib (Imbruvica®), ofatumumab (Arzerra®), rituximab (Rituxan®), bevacizumab (Avastin®), trastuzumab (Herceptin®), trastuzumab emtansine (KADCYLA®), and rituximab (Rituxan®). Kadcyla®), imatinib (Gleevec®), cetuximab (Erbitux®), panitumumab (Vectibix®), catumaxomab, ibritumomab, ofatumumab, tositumomab, brentuximab, alemtuzumab, gemtuzumab, erlotinib, gefitinib, vandetanib, afatinib, lapatinib, neratinib, axitinib, Tinib, masitinib, pazopanib, sunitinib, sorafenib, toceranib, lestaurtinib, axitinib, cediranib, lenvatinib, nintedanib, pazopanib, regorafenib, semaxanib, sorafenib, sunitinib, tivozanib, toceranib, vandetanib, entrectinib, cabozantinib, imatinib, dasatinib, nilotinib, ponatinib, radotinib, bosutinib, lestaurtinib These include, but are not limited to, tinib, ruxolitinib, pacritinib, cobimetinib, selumetinib, trametinib, binimetinib, alectinib, ceritinib, crizotinib, aflibercept, adipotide, denileukin diftitox, mTOR inhibitors such as everolimus and temsirolimus, hedgehog inhibitors such as sonidegib and vismodegib, CDK inhibitors such as CDK inhibitors (palbociclib).

[0083] In a further embodiment, the additional therapeutic agent may be an anti-inflammatory agent.Anti-inflammatory agents or anti-inflammatory drugs include, but are not limited to, steroids and glucocorticoids (including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone), non-steroidal anti-inflammatory drugs (NSAIDS), such as aspirin, ibuprofen, naproxen, methotrexate, sulfasalazine, leflunomide, anti-TNF drugs, cyclophosphamide and mycophenolic acid.Exemplary NSAIDs include ibuprofen, naproxen, naproxen sodium, Cox-2 inhibitors, and sialylate.Exemplary analgesics include acetaminophen, oxycodone, tramadol or propoxyphene hydrochloride. Exemplary glucocorticoids include cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, or prednisone. Exemplary biological response modifiers include molecules directed against cell surface markers (e.g., CD4, CD5, etc.), cytokine inhibitors, such as TNF antagonists (e.g., etanercept (ENBREL®), adalimumab (HUMIRA®) and infliximab (REMICADE®), chemokine inhibitors, and adhesion molecule inhibitors. Biological response modifiers include monoclonal antibodies and recombinant forms of molecules. Exemplary DMARDs include azathioprine, cyclophosphamide, cyclosporine, methotrexate, penicillamine, leflunomide, sulfasalazine, hydroxychloroquine, gold preparations (oral (auranofin) and intramuscular), and minocycline.

[0084] The present invention further contemplates a method for the treatment and / or prevention of cancer.

[0085] The term "treatment" or "treating" means (i) inhibiting the disease, i.e., halting the progression of clinical symptoms, and / or (ii) relieving the disease, i.e., causing regression of clinical symptoms; This means administering to a subject a composition, pharmaceutical composition, therapeutic agent, compound, etc. of the present disclosure for the purpose of:

[0086] As used herein, the terms "prevention" or "preventing" refer to the administration of a composition, pharmaceutical composition, therapeutic agent, compound, etc. of the present disclosure to a subject for the purpose of preventing a disease, i.e., not developing clinical symptoms of the disease.

[0087] In the context of the present invention, the disease is a cancer, preferably a solid tumor as disclosed herein.

[0088] In one embodiment, a method for treating and / or preventing cancer in a patient or subject includes the steps of (i) removing and isolating immune cells, preferably native T cells, from said patient or subject, (ii) genetically engineering said T cells with one recombinant construct encoding interleukin-10, a fragment or variant thereof, and a second recombinant construct encoding a chimeric antigen receptor (CAR), a T cell receptor (TCR), or any other synthetic tumor targeting motif or antigen, (iii) expanding ex vivo into a larger population of engineered T cells, and (iv) reintroducing said engineered T cells into said patient or subject. After the engineered T cells are reintroduced into the patient or subject, they mediate an immune response against cells expressing the tumor targeting motif or antigen described herein. This immune response includes secretion of IL-10, a fragment or variant thereof, and other cytokines by T cells, clonal expansion of T cells that recognize the tumor targeting motif or antigen, and T cell-mediated specific killing of target positive cells.

[0089] In one aspect, the method of treating and / or preventing cancer comprises the steps of: (i) removing and isolating or providing immune cells, preferably natural T cells, from a patient or subject; (ii) genetically engineering said T cells with at least one recombinant construct encoding interleukin-10, a fragment or variant thereof, and a second recombinant construct encoding a chimeric antigen receptor (CAR), a T cell receptor (TCR) or any other synthetic tumor targeting motif or antigen; (iii) expanding ex vivo into a larger population of engineered T cells; and (iv) reintroducing them into said patient or subject.

[0090] In one embodiment, the method for treating and / or preventing cancer in a subject comprises administering to a subject in need thereof a pharmaceutical composition of the present invention.

[0091] In one embodiment, the above-mentioned methods of treatment and / or prevention can further comprise administering at least one additional therapeutic agent or therapy, preferably an anti-cancer agent or therapy, more preferably a therapeutically effective amount or dose of an anti-cancer agent or therapy, wherein the one or more anti-cancer agents or therapies are selected from the non-limiting group including radiation therapy, chemotherapy, immune checkpoint inhibitors, immunotherapy and hormonal therapy, or one or more combinations thereof, as described above.

[0092] Various known techniques can be used to prepare the polynucleotides, polypeptides, vectors, antigen-binding molecules, immune cells, compositions, and the like according to the present invention.

[0093] Prior to the in vitro manipulation or genetic modification of immune cells as described herein, cells may be obtained and isolated from a subject. In some aspects, the immune cells include T cells. T cells may be obtained from several sources, including peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain aspects, T cells may be obtained from a unit of blood collected from a subject using any number of techniques known to those skilled in the art, such as FICOLL™ separation. Cells may be obtained from the circulating blood of an individual, preferably by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In certain embodiments, cells collected by apheresis may be washed to remove the plasma fraction and placed in an appropriate buffer or medium for subsequent processing. The cells may be washed with PBS. As will be appreciated, washing steps may be used. After washing, the cells may be resuspended in a variety of biocompatible buffers or other saline solutions with or without buffers. In certain aspects, undesirable components of the apheresis sample may be removed.

[0094] In certain embodiments, T cells are isolated from PBMCs by lysing red blood cells and depleting monocytes, for example, using centrifugation through a PERCOLL™ gradient. + , CD4 + , CD8 + , CD45RA + , and CD45RO +Specific subpopulations of T cells, such as T cells, can be further isolated by positive or negative selection techniques known in the art. For example, enrichment of a T cell population by negative selection can be achieved using a combination of antibodies against surface markers unique to the cells to be negatively selected. One method for use in the present invention is negative magnetic immunoadherence, or cell sorting and / or selection by flow cytometry using a cocktail of monoclonal antibodies against cell surface markers present on the cells to be negatively selected. For example, negative selection can be used to enrich for CD4 + To enrich for cells, the monoclonal antibody cocktail typically includes antibodies against CD14, CD20, CD11B, CD16, HLA-DR, and CD8. Flow cytometry and cell sorting may also be used to isolate cell populations of interest for use in the present invention.

[0095] PBMCs may be used directly for genetic modification with immune cells (such as CAR or TCR) using the methods described herein. In certain aspects, after isolating PBMCs, T lymphocytes can be further isolated and both cytotoxic and helper T lymphocytes can be sorted into subpopulations of naive, memory, and effector T cells, either before or after genetic modification and / or expansion.

[0096] In some embodiments, CD8 + Cells differentiate between these types of CD8 cells by identifying cell surface antigens associated with naive, central memory, and effector cells, respectively. + The cells are further sorted.

[0097] The immune cells described herein can be genetically modified after isolation using known methods, or the immune cells can be activated and expanded (e.g., TIL cells) or, in the case of progenitor cells, differentiated in vitro before being genetically modified. In another embodiment, immune cells such as T cells are genetically modified with a chimeric antigen receptor described herein (e.g., transduced with a viral vector comprising one or more nucleotide sequences encoding a CAR) and then activated and / or expanded in vitro. Methods for activating and expanding T cells are known in the art and are described, for example, in U.S. Pat. No. 6,905,874; U.S. Pat. No. 6,867,041; U.S. Pat. No. 6,797,514; and PCT Publication WO 2012 / 079000, the contents of which are incorporated herein by reference in their entireties. Generally, such methods include contacting PBMCs or isolated T cells with stimulatory and costimulatory molecules, such as anti-CD3 and anti-CD28 antibodies, typically attached to beads or other surfaces, in culture medium with appropriate cytokines, such as IL-2. In other embodiments, T cells may be activated and stimulated for proliferation using feeder cells and appropriate antibodies and cytokines, using methods such as those described in U.S. Pat. No. 6,040,177; U.S. Pat. No. 5,827,642; and WO2012129514, the contents of which are incorporated herein by reference in their entireties.

[0098] Certain methods for making the constructs and engineered immune cells of the present invention are described, for example, in PCT Application No. PCT / US2015 / 14520, the contents of which are incorporated herein by reference in their entirety.

[0099] It will be understood that PBMCs can further comprise other cytotoxic lymphocytes such as NK cells or NKT cells. An expression vector carrying a recombinant construct of the invention disclosed herein can be introduced into a population of human donor T cells, NK cells or NKT cells. Successfully transduced T cells carrying this expression vector can be sorted using flow cytometry to isolate CD3 positive T cells, which can then be further expanded to increase the number of these CAR-expressing T cells in addition to cell activation using anti-CD3 antibodies and IL-2 or other methods known in the art as described elsewhere herein. Standard procedures are used for cryopreservation of CAR-expressing T cells for storage and / or preparation for use in human subjects. In one embodiment, the in vitro transduction, culture and / or expansion of T cells is performed in the absence of non-human animal derived products such as fetal bovine serum and fetal bovine serum.

[0100] For cloning the polynucleotide of the present invention, the vector may be introduced into a host cell (autologous, allogeneic or heterologous) to allow the vector to replicate itself, thereby amplifying copies of the polynucleotide contained therein. The cloning vector of the present invention may generally contain sequence components including, but not limited to, an origin of replication, a promoter sequence, a transcription initiation sequence, an enhancer sequence, and a selection marker. These elements may be appropriately selected by those skilled in the art. For example, an origin of replication may be selected to promote the autonomous replication of the vector in the host cell.

[0101] The term "autologous" refers to any material derived from the same individual into which it is subsequently reintroduced. The term "allogeneic" refers to any material derived from one individual and then introduced into another individual of the same species, including, for example, allogeneic T cell transplantation.

[0102] In certain aspects, the disclosure provides an isolated host cell containing a vector provided herein. A host cell comprising the vector may be useful for expression or cloning of a polynucleotide contained in the vector. Suitable host cells can include, but are not limited to, oncolytic viruses, prokaryotic cells, fungal cells, yeast cells, or higher eukaryotic cells such as mammalian cells. Prokaryotic cells suitable for this purpose include eubacteria, such as gram-negative or gram-positive organisms, such as Enterobacteriaceae, such as Escherichia, such as E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, such as Salmonella typhimurium, Serratia, such as Serratia marcescens, Serratia marcescans, and Shigella, as well as Bacillus (bacilli), such as B. subtilis and B. licheniformis, Pseudomonas, such as P. aeruginosa, and Streptomyces.

[0103] Vectors can be introduced into host cells using any suitable method known in the art, including, but not limited to, DEAE-dextran mediated delivery, calcium phosphate precipitation, cationic lipid mediated delivery, liposome mediated transfection, electroporation, microprojectile bombardment, receptor mediated gene delivery, polylysine, histones, chitosan, and peptide mediated delivery. Standard methods for transfection and transformation of cells for expression of a vector of interest are well known in the art.

[0104] Also contemplated is a method of enhancing anti-tumor activity in a subject comprising the steps of: (i) removing and isolating immune cells, preferably natural T cells, from said subject or providing immune cells, preferably natural T cells; (ii) genetically engineering said T cells with at least one recombinant construct encoding interleukin-10, a fragment or variant thereof, and a second recombinant construct encoding a chimeric antigen receptor (CAR), a T cell receptor (TCR) or any other synthetic tumor targeting motif or antigen; (iii) expanding ex vivo into a larger population of engineered T cells; and (iv) reintroducing into said patient or subject.

[0105] Kits for carrying out one or more of the methods of the present invention are also contemplated.

[0106] Kits are further contemplated that contain the compositions or pharmaceutical compositions of the present invention in one or more containers. The compositions may be in liquid form or may be frozen. Suitable containers for the compositions include, for example, bottles, vials, syringes, and test tubes. The containers can be made of a variety of materials, including glass or plastic. The kits may further include instructions that may include information or instructions about prescription, drug amounts, composition, etc.

[0107] One of skill in the art will understand that the invention described herein is not limited to the use of IL-10 expressing immune cell (such as CAR T or TCR T) transplantation therapy to treat cancer. IL-10 expressing CAR T cells can be considered as one exemplary strategy for tumor targeted delivery of IL-10 to enhance anti-tumor immunity, while other strategies, such as stem cells (Liu, L. et al., Mechanoresponsive stem cells to target cancer metastases through biophysical cues. Sci.Transl.Med. 9, eaan2966(2017)), blood platelets (Wang, C. et al., In situ activation of platelets with checkpoint inhibitors for post-surgical cancer immunotherapy. Nat.Biomed.Eng. 1, (2017)), oncolytic viruses (Rivadeneira, D. B. et al., Oncolytic Viruses Engineered to Enforce Leptin Expression Reprogram Tumor-Infiltrating T Cell Metabolism and Promote Tumor Clearance. Immunity 51, 548-560.e4(2019)), mRNA (cancer vaccines, e.g. Sahin, U. et al., Personalized RNA It can be expected that tumor-targeted delivery of IL-10 by mutanome vaccines mobilize poly-specific therapeutic immunity against cancer. Nature 547, 222-226(2017)) or nanotechnology (Tang, L. et al., Enhancing T cell therapy through TCR-signaling-responsive nanoparticle drug delivery. Nat. Biotechnol. 36, 707-716(2018)) is also encompassed for enhancing anti-tumor immunity as described herein.

[0108] The invention described herein may be subject to variations and modifications other than those specifically described. It should be understood that the invention includes all such variations and modifications to the extent that they do not depart from the spirit or essential characteristics thereof. The invention includes all of the steps, features, compositions and compounds referred to or shown in this specification, individually or collectively, as well as any and all combinations of the steps or features described above, or any two or more of them. Therefore, the present disclosure should be considered in all respects as illustrative and not limiting, the scope of the invention being indicated by the appended claims, and all changes that come within the meaning and range of equivalents are intended to be embraced therein. Various references are cited throughout this specification, each of which is incorporated herein by reference in its entirety. The above description will be more fully understood with reference to the following examples. EXAMPLES

[0109] HER2-targeted IL-10 CAR T cells We first investigated whether CAR T with ectopic IL-10 expression could modulate metabolic fitness and improve their antitumor activity. IL-10 HER2 CAR constructs were generated by fusing HER2 CAR and IL-10 gene fragments with 2A self-cleaving peptide into retroviral vector pMSGV (Fig. 1a). The cell surface expression of HER2 CAR in IL-10 HER2 CAR T was nearly equivalent to that in conventional HER2 CAR T cells (Fig. 1b). The IL-10 levels produced by IL-10 HER2 CAR were measured by ELISA (Fig. 1c). Since IL-10 is known to enhance the proliferation of CD8 T cells upon antigen stimulation (Guo, Y. et al., Metabolic reprogramming of terminally exhausted CD8+ T cells by IL-10 enhances anti-tumor immunity. Nat.Immunol. 22, 746-756 (2021)), we investigated the absolute number and cell division number of CAR T cells upon antigen stimulation. When co-cultured with mitomycin-treated MC38-HER2 mouse colon cancer cells, the number and cell division of IL-10 HER2 CAR T cells were significantly higher compared to the number and cell division of HER2 CAR T cells (Figure 1d, e). The enhanced division of IL-10 HER2 CAR T cells was completely attenuated by anti-IL-10 antibody (Figure 1e), indicating that IL-10 is essential for improving the proliferation of IL-10 HER2 CAR T cells. In the MC38-HER2 cell co-culture system, the basal oxygen consumption rate (OCR) of IL-10 HER2 CAR T cells was elevated, whereas the extracellular acidification rate (ECAR) remained unchanged compared to HER2 CAR T cells (Fig. 1f-i). On the other hand, IL-10 HER2 CAR T cells showed a basal OCR comparable to that of HER2 CAR T in the presence of mIL-10. The ratio of OCR to EACR of both HER2 CAR T and IL-10 HER2 CAR T in the presence of mIL-10 was significantly increased (Fig. 1j), suggesting that IL-10 signaling actively promoted oxidative phosphorylation (OXPHOS) in CAR T cells.As a result of metabolic reprogramming, the tumor lytic ability of IL-10 HER2 CAR T was significantly enhanced (Figure 2a), while the antigen-specific proliferation ability, killing efficiency and multifunctionality of CAR T cells were significantly enhanced in the IL-10 HER2 CAR T group in a co-culture environment (E:T ratio 0.5:1 for 48 h) mimicking the in vivo scenario (Figures 2b-d). These data confirm that the IL-10 CAR T of the present invention can reprogram CAR T cell metabolism to promote cell proliferation by increasing OXPHOS, indicating that the IL-10 CAR T of the present invention could also promote the proliferation of tumor-infiltrating CAR T cells in the TME through metabolic intervention.

[0110] CD8 with various reagents + Enhancement of OXPHOS or inhibition of glycolytic metabolism in T cells may mediate the upregulation of CD8 + It promoted T cell proliferation, memory development, and antitumor function (Sukumar, M. et al., Inhibiting glycolytic metabolism enhances CD8+ T cell memory and antitumor function. J. Clin. Invest. 123, 4479-4488 (2013)). Based on the observed metabolic regulatory function of IL-10 HER2 CAR T cells, we next investigated whether in vivo metabolic intervention of CAR T cells could be achieved to enhance efficacy against solid tumors.

[0111] In a treatment setting where MC38-HER2 tumors were pre-established, IL-10 HER2 CAR T cells (3 × 10 6Adoptive transfer of HER2 CAR T monotherapy consistently induced complete tumor regression and durable cure in 80% of treated mice (Fig. 3a-b). In contrast, HER2 CAR T monotherapy had minimal effects on tumor growth inhibition. Adoptive transfer of HER2 CAR T with intravenous administration of mIL-10 only transiently controlled tumor growth and failed to induce tumor regression. Moreover, all long-term surviving mice treated with IL-10 HER2 CAR T cells rejected rechallenge with MC38-HER2 cells 2 months after cessation of treatment, indicating induction of antitumor immune memory (Fig. 3c).

[0112] IL-10 CAR T cells targeting TRP-1 To test the robustness of IL-10 CAR T cell therapy, we next evaluated whether it could also control the less immunogenic and more aggressive murine B16F10 melanoma model. We then generated IL-10 CAR T cells targeting TRP-1 and confirmed CAR expression by flow cytometry (Fig. 4a, b). The in vitro cell proliferation and antitumor activity of IL-10 TRP-1 CAR T cells were superior to those of conventional TRP-1 CAR T cells (Fig. 4c, d). B16F10 murine melanoma-bearing mice were lymphodepleted by irradiation (4 Gy) prior to CAR T cell implantation. IL-10 TRP-1 CAR T led to significant tumor regression and eventual disappearance in the majority of tumor-bearing mice, whereas TRP-1 CAR T showed only transient tumor growth inhibition without sustained therapeutic effects (Fig. 4e). In addition, 60% of mice treated with IL-10 TRP-1 CAR T therapy exhibited long-term survival (Fig. 4f).

[0113] IL-10 CAR T cells targeting EGFRvIII Next, we further extended the intervention IL-10 CAR T to the highly aggressive and metastatic 4T1-Luc-EGFRvIII (stably transfected with EGFRvIII and luciferase (Luc)) mouse breast cancer model. We prepared IL-10 CAR T cells targeting EGFRvIII. CAR expression and IL-10 production were confirmed by flow cytometry and ELISA, respectively (Fig. 5a-c). The in vitro cell proliferation and antitumor activity of IL-10 EGFRvIII CAR T cells were significantly improved compared with those of EGFRvIII CAR T cells (Fig. 5d, e). BALB / c mice were transfected with 4T1-Luc-EGFRvIII tumor cells (5 × 10 4 ) were injected i.v. to allow lung metastases to develop. Mice were treated with EGFRvIII CAR T cells (3 × 10 6 ) resulted in transient tumor inhibition (Fig. 5f). Remarkably, IL-10 EGFRvIII CAR T cells completely eradicated tumors and led to durable cures in 100% of treated mice (Fig. 5g). Consistent with their robust antitumor efficacy, IL-10 EGFRvIII CAR T cells induced a significantly higher density of CAR T cells in the circulation (Fig. 5h).

[0114] These results demonstrated that IL-10 HER2 CAR T exhibited improved in vivo proliferation, enhanced functionality, and ultimately contributed to the superior efficacy of IL-10-expressing CAR T cells.

[0115] IL-10 TCR T(Pmel) cells To further extend the IL-10-expressing T cell strategy to tumor-specific T cell receptor (TCR) transgenic T cells (TCR T) for the highly aggressive B16F10 mouse melanoma tumor model, we prepared IL-10-expressing Pmel T cells (IL-10 Pmel T) as previously described and confirmed the expression of IL-10 production by ELISA (Fig. 6a). Mice bearing B16F10 mouse melanoma received adoptive transfer of PBS control, Pmel T or IL-10 Pmel T cells. IL-10Pmel T cells led to significant tumor regression in most tumor-bearing mice, whereas Pmel T cells showed only minor tumor growth inhibition (Fig. 6b-e). In addition, mice treated with IL-10-expressing Pmel T cells showed improved survival compared to mice treated with Pmel T cells (Fig. 6f). Similarly, this IL-10-expressing TCR T cell strategy could also be used to enhance the efficacy of adoptive transfer therapy of TILs against solid tumors.

[0116] Human IL-10 CAR T cells targeting CD19 Finally, this IL-10-expressing CAR T strategy was extended to human CAR T. An IL-10 CD19 CAR construct was generated by fusing the CD19 CAR and human IL-10 gene fragment with the 2A self-cleaving peptide into a lentiviral vector (Fig. 7a). The cell surface expression of CD19 CAR in IL-10 CD19 CAR T was slightly higher than that in conventional CD19 CAR T cells (Fig. 7b). The IL-10 levels produced by IL-10 CD19 CAR were measured by ELISA (Fig. 7c). Accordingly, the tumor lysis ability of IL-10 CD19 CAR T was also enhanced (Fig. 7d), consistent with the results observed in mouse CAR T. Moreover, in immune-deficient NSG mice bearing established PANC1-CD19 human pancreatic tumors, all mice treated with IL-10-expressing CD19 hCAR T cells showed complete remission without relapse (Figure 7e), suggesting that IL-10-expressing human CAR T cells have superior antitumor capacity against solid tumors in xenograft models.

[0117] IL-10-expressing CAR-T cells maintain mitochondrial fitness Impairment of mitochondrial fitness has been shown to enhance T cell exhaustion. Interestingly, we found that IL-10 expression maintained mitochondrial fitness in tumor-infiltrating CAR-T cells, substantially reducing the frequency of dysfunctional mitochondria (4.8%) in IL-10 HER2 CAR-T cells compared to HER2 CAR-T cells alone (27.4%) or HER2 CAR-T cells combined with exogenous IL-10 (21.7%) (Figure 8a). IL-10 expression also increased the ratio of MDR to MG in IL-10 HER2 CAR-T cells (Figure 8b). EM imaging analysis of mitochondrial ultrastructure provided further evidence of condensed mitochondria with tubular shape, well-structured cristae, increased cristae number, and expanded length of cristae per mitochondrion in tumor-infiltrating IL-10 HER2 CAR-T cells compared to conventional HER2 CAR-T cells (Figure 8c-f).

[0118] IL-10 expression in CAR-T cells induces sustained antitumor immunity and promotes stemness To investigate whether IL-10-expressing CAR-T cells develop antitumor immune memory, we rechallenged surviving mice 3 months after adoptive CAR-T cell transfer. Impressively, 100% of long-term surviving mice treated with IL-10 HER2 CAR-T cells or IL-10 TRP-1 CAR-T cells rejected a second challenge with the original tumor cells (Fig. 9a-c). Motivated by this robust immune memory response, we investigated the memory phenotype of IL-10-expressing CAR-T cells in lymphoid tissues and circulation. Flow cytometry analysis of CAR-T cells 12 days after treatment (initially transferred CAR-T cells were all CD44hi) revealed that IL-10 HER2 CAR T cells dominated the spleen and peripheral blood as a population with a Tscm phenotype (CD62LhiCD44lo and stem cell antigen-1 (Sca-1)). + CD122 +The results showed that IL-10 HER2 CAR-T cells in the spleen were enriched for CD62LhiCD44lo T cells (defined by Sca-1+CD122+Tscm) compared to HER2 CAR-T cells alone (Figure 10a and b). IL-10 HER2 CAR-T cells in the spleen showed approximately 3.2-fold higher frequency of CD62LhiCD44lo T cells compared to HER2 CAR-T cells alone, and the majority (approximately 71.2%) of CD62LhiCD44lo T cells in IL-10 HER2 CAR-T cells were Sca-1+CD122+Tscm (Figure 10a and b). In addition, IL-10 HER2 CAR-T cells showed substantially increased expression of Sca-1 compared to HER2 CAR-T cells alone or HER2 CAR-T cells plus exogenous IL-10 (Figure 10c). This finding was further confirmed by the observation that IL-10 HER2 CAR T cells were composed of approximately 3.7-fold and 2.6-fold higher percentages of IL-7Rα+KLRG1 long-lived memory precursor T cells than HER2 CAR-T cells in the spleen and blood, respectively (Figure 10d and e). In addition, we observed that treatment with IL-10 CD19 hCAR-T cells appeared to be enriched for Tscm compared to CD19 hCAR-T cells. Altogether, these results suggest that IL-10 signaling can induce the formation of Tscm CAR-T cells in mice and humans that contribute to long-term antitumor immunity.

[0119] array [ka] [ka]

Claims

1. 1. An immune cell that expresses interleukin-10, a fragment or a variant thereof, said immune cell comprising one or more recombinant constructs, at least one of which encodes interleukin-10, a fragment or a variant thereof.

2. 2. The immune cell of claim 1, wherein the second recombinant construct encodes a chimeric antigen receptor (CAR), a T cell receptor (TCR), or any other synthetic tumor-targeting motif.

3. The immune cell according to claim 1, wherein the immune cell is a T cell, a chimeric antigen receptor (CAR)-T cell, a T cell receptor (TCR)-transgenic T cell, a tumor-infiltrating lymphocyte (TIL), an NK cell, an NK-T cell, a CAR-NK cell, a CAR-NKT cell, a TCR-transgenic NK cell, a TCR-transgenic NK-T cell, a dendritic cell, a macrophage, a CAR-macrophage, or any synthetic tumor-specific immune cell.

4. 2. The immune cell of claim 1, wherein the construct encoding interleukin-10, a fragment or a variant thereof comprises or encodes the sequence of SEQ ID NO: 1, or a fragment or variant thereof.

5. 3. The immune cell of claim 2, wherein the recombinant construct encoding interleukin-10, a fragment or variant thereof, is linked to the second recombinant construct encoding a CAR, a TCR, or any other synthetic tumor targeting motif.

6. 6. The immune cell of claim 5, wherein the recombinant construct encoding interleukin-10, a fragment or variant thereof, is linked to the second recombinant construct encoding a CAR, TCR or any other synthetic tumor targeting motif via a sequence encoding a self-cleaving peptide (e.g., peptide 2A).

7. The immune cell of claim 5, wherein the second recombinant construct encoding the CAR comprises an extracellular antigen recognition domain of a single chain fragment variant (scFv) derived from an antibody.

8. The immune cell of claim 5 , wherein the second recombinant construct encoding the CAR comprises a nucleic acid encoding a polypeptide of a transmembrane region.

9. The immune cell of claim 5, wherein the second recombinant construct encoding the CAR comprises a nucleic acid encoding a polypeptide of the intracellular T cell activation domain of CD3ζ.

10. The immune cell of claim 5, wherein the second recombinant construct encoding the CAR comprises 4-1BB or CD28, or a combination of 4-1BB and CD28 intracellular regions.

11. i) the extracellular antigen recognition domain of a single chain fragment variant (scFv) derived from said antibody recognizes an antigen selected from the group comprising c-MET, CD7, CD19, CD20, CD22, CD38, CD123, CD133, CD171, CD70, BCMA, CEA, EGFR-VIII, EpCAM, EphA2, FAP, GD2, GPC3, HER2, IL-13Ra2, mesothelin, MUC1, PSCA, PSMA, ROR1, VEGFR2, claudin 18.2; or ii) the TCR recognizes an antigen selected from the group consisting of gp100, NY-ESO-1, MAGE-A3 and TRP-1, or a combination of one or more thereof; The immune cell of claim 7.

12. 2. The immune cell of claim 1, wherein said interleukin-10, a fragment or a variant thereof is secreted or membrane-bound by said immune cell, preferably in the tumor microenvironment.

13. 2. The immune cell of claim 1, wherein the construct encoding interleukin-10, a fragment or a variant thereof is contained within or outside a sequence encoding an Fc, HSA, or antibody fusion protein.

14. The immune cell of claim 1 for use in the prevention and / or treatment of cancer, wherein the cancer is a solid cancer or a liquid cancer.

15. 15. The immune cell of claim 14, wherein the solid cancer is selected from the group comprising lung cancer, breast cancer, ovarian cancer, cervical cancer, uterine cancer, head and neck cancer, glioblastoma, hepatocellular carcinoma, colon cancer, rectal cancer, colorectal cancer, kidney cancer, prostate cancer, gastric cancer, bronchial cancer, pancreatic cancer, bladder cancer, liver cancer, brain cancer and skin cancer, in particular melanoma, or a combination of one or more thereof.

16. 10. The immune cell of claim 1, wherein the immune cell is autologous, allogeneic, or xenogeneic.

17. The immune cell of claim 7, wherein the extracellular antigen-recognition domain of the single-chain fragment variant (scFv) derived from the antibody recognizes an antigen selected from the group including HER2, TRP-1, EGFRvIII, and CD19.

18. The immune cell of claim 17, wherein the amino acid sequence of the scFv that recognizes HER2 is shown in SEQ ID NO: 2, a fragment or a variant thereof.

19. The immune cell according to claim 17, wherein the amino acid sequence of the scFv that recognizes TRP-1 is represented by SEQ ID NO: 3, a fragment or a variant thereof.

20. The immune cell of claim 17, wherein the amino acid sequence of the scFv that recognizes EGFRvIII is set forth in SEQ ID NO: 4, a fragment or a variant thereof.

21. The immune cell of claim 17, wherein the amino acid sequence of the scFv that recognizes CD19 is set forth in SEQ ID NO: 5, a fragment or a variant thereof.

22. The immune cell of claim 8, wherein the amino acid sequence of the CD8 transmembrane domain and hinge polypeptide is set forth in SEQ ID NO: 6, a fragment or variant thereof.

23. The immune cell of claim 9, wherein the amino acid sequence of the polypeptide of the intracellular T cell activation domain of CD3ζ is set forth in SEQ ID NO: 7, a fragment or a variant thereof.

24. a therapeutically effective amount of i) an immune cell according to any one of claims 1 to 23, ii) a nucleic acid encoding one or more recombinant constructs according to any one of claims 1 to 23, and / or iii) 24. A pharmaceutical composition comprising a plasmid or vector containing a nucleic acid sequence encoding one or more recombinant constructs according to any one of claims 1 to 23, and at least one pharmaceutically acceptable carrier and / or diluent.

25. 25. The pharmaceutical composition of claim 24, further comprising at least one additional therapeutic agent or therapy.

26. 26. The pharmaceutical composition of claim 25, wherein the at least one additional therapeutic agent or therapy is an anti-cancer agent or therapy useful in treating cancer, preferably solid cancers, and / or an anti-inflammatory agent.

27. 27. The pharmaceutical composition of claim 26, wherein the anti-cancer therapy is selected from the group comprising radiation therapy, chemotherapy, immune checkpoint inhibitors, immunotherapy and hormonal therapy, or a combination of one or more thereof.

28. 28. The pharmaceutical composition of claim 27, wherein the immune checkpoint inhibitor is selected from the group comprising a PD-1 inhibitor, a PD-L1 inhibitor, and a CTLA-4 inhibitor, or a combination of one or more thereof.

29. 25. A method for treating and / or preventing cancer in a subject, comprising administering a pharmaceutical composition according to claim 24.

30. 1. A method of treating and / or preventing cancer in a subject, comprising the steps of: (i) removing and isolating or providing immune cells, preferably naive T cells, from said subject; (ii) genetically engineering said T cells with at least one recombinant construct encoding interleukin-10, a fragment or variant thereof, and a second recombinant construct encoding a chimeric antigen receptor (CAR), a T cell receptor (TCR), or any other synthetic tumor targeting motif or antigen; (iii) expanding ex vivo into a larger population of engineered T cells; and (iv) reintroducing them into the patient or subject.

31. 1. A method of enhancing anti-tumor activity in a subject, comprising the steps of: (i) removing and isolating or providing immune cells, preferably naive T cells, from said subject; (ii) genetically engineering said T cells with at least one recombinant construct encoding interleukin-10, a fragment or variant thereof, and a second recombinant construct encoding a chimeric antigen receptor (CAR), a T cell receptor (TCR), or any other synthetic tumor-targeting motif or antigen; (iii) expanding ex vivo into a larger population of engineered T cells; and (iv) reintroducing them into the patient or subject.

Images