Methods and compositions for cellular immunotherapy

Abstract

The present invention provides methods and compositions for conferring and / or enhancing cellular immunotherapy-mediated immune responses, such as by adoptively transferring genetically modified tumor-specific CD8+ T cells in the presence of genetically modified tumor-specific, subset-specific CD4+ T cells, wherein the CD4+ T cells confer and / or enhance the ability of the CD8+ T cells to maintain antitumor reactivity and increase and / or maximize tumor-specific proliferation of tumor-specific CD8+ T cells of interest. Also disclosed are pharmaceutical preparations made by the methods and methods of using the same.

Description

[0001] This application was filed as a PCT international patent application on March 23, 2012, with Fred Hutchinson Cancer Research Center, a U.S. national corporation, as the applicant in all designated countries except the United States. The applicants designated in the United States are Stanley R. Riddell, a Canadian citizen, and Michael Hudecek, a German citizen. This application claims priority to U.S. Patent Application No. 61 / 466,552, filed on March 23, 2011, the disclosure of which is incorporated herein by reference in its entirety. Invention Field

[0002] This invention relates to the field of biomedicine, and in particular to methods for cancer treatment. Specifically, embodiments of the invention relate to methods and compositions for performing cellular immunotherapy.

[0003] Statement on Federally Funded Research

[0004] This invention was completed with government support, specifically a grant (R01CA18029) from the U.S. Department of Health and Human Services, the National Institutes of Health, and the Leukemia and Lymphoma Society (SCORE). The U.S. government holds certain rights to this invention. Background of the Invention

[0005] Studies in rodents have shown that adoptive immunotherapy using antigen-specific T cells is effective for cancer and infection, and there is evidence that this therapy may be effective in humans. 1-8 For clinical applications, it is necessary to isolate T cells with the desired antigen specificity, or to modify T cells to express receptors that target infected or transformed cells, and then expand these cells in culture. 9-14 The transfer of T-cell clones is promising because it allows for control over specificity and function, and facilitates assessment of in vivo persistence, toxicity, and efficacy. Furthermore, in allogeneic stem cell transplantation settings, administering donor-derived T-cell clones targeting pathogens or malignant cells to the recipient avoids graft-versus-host disease that can occur with the injection of unselected donor T-cells. 3,4,15 However, clinical studies have clearly shown that cultured T cells, especially cloned CD8 cells, are less effective. + The efficacy of T cells is frequently limited because they cannot persist after adoptive transfer. 16,17 .

[0006] The lymphocyte pool from which T cells are derived for adoptive immunotherapy contains both naive and long-lived antigen-stimulated memory T cells (T cells). M). T M It can be further divided into phenotype, homing properties, and central memory with different functions (T). CM ) and effect memory (T) EM Cell subsets 18 CD8 + T CM The cells express CD62L and CCR7 on their surface, which promote migration to lymph nodes and rapidly proliferate upon re-exposure to the antigen. CD8 + T EM Lacking cell surface CD62L and preferentially migrating to surrounding tissues, it exhibits direct effector functions. 19 In response to antigen stimulation, CD8 + T CM and T EM All differentiated into cytolytic effector T cells (T cells) expressing high levels of granzyme and perforin. E However, these are short-lived cells. 20 Therefore, the poor T cell viability in clinical immunotherapy trials may simply stem from their differentiation into T cells destined to die during in vitro culture. E 17,21,22 It is necessary to identify cell populations and methods to improve the in vivo viability of adoptive T cells. Invention Overview

[0007] In one aspect, the present invention relates to methods and compositions for conferring and / or enhancing cellular immunotherapy-mediated immune responses, such as by adoptive transfer of tumor-specific, subset-specific genetically modified CD4+ T cells, wherein the CD4+ T cells confer and / or enhance the ability of CD8+ T cells to maintain antitumor reactivity and increase and / or maximize tumor-specific proliferation.

[0008] In one embodiment, the present invention provides a method for cellular immunotherapy in an individual suffering from a disease or condition by administering a genetically modified cytotoxic T lymphocyte preparation and a genetically modified helper T lymphocyte preparation that provide a cellular immune response to the individual, wherein the cytotoxic T lymphocyte preparation comprises CD8+ T cells having a chimeric antigen receptor having an extracellular antibody variable domain specific to an antigen associated with the disease or condition and an intracellular signaling domain, such as a co-stimulatory domain, of a T cell or other receptor; and the genetically modified helper T lymphocyte preparation exhibits a pronounced Th1 phenotype and produces other cytokines, inducing direct tumor recognition and enhancing the ability of the genetically modified cytotoxic T lymphocyte preparation to mediate a cellular immune response, wherein the helper T lymphocyte preparation comprises CD4+ T cells having a chimeric antigen receptor having an extracellular antibody variable domain specific to an antigen associated with the disease or condition and an intracellular signaling domain of a T cell receptor. Various modifications may be made to the above method. For example, the chimeric antigen receptors modified on the CD4+ T cells and CD8+ T cells can be the same or different. In an alternative embodiment, the T cells can be modified with recombinant T cell receptors (TCRs). TCRs can be specific to any antigen, pathogen, or tumor. TCRs targeting many tumor antigens exist in melanoma (e.g., MART1, gp100), leukemia (e.g., WT1, minor histocompatibility antigens), and breast cancer (e.g., HER2, NY-BR1).

[0009] In another embodiment, the present invention provides an adoptive cellular immunotherapy composition comprising a genetically modified CD8+ cytotoxic T lymphocyte preparation that elicits a cellular immune response, and a genetically modified helper T lymphocyte preparation, wherein the cytotoxic T lymphocyte preparation comprises CD8+ T cells having a chimeric antigen receptor having an extracellular variable domain antibody specific to a disease or symptom-associated antigen and an intracellular signaling domain of a T cell or other receptor, such as a co-stimulatory domain; the genetically modified helper T lymphocyte preparation induces a pronounced Th1 phenotype and produces other cytokines, triggers direct tumor recognition, and enhances the ability of the genetically modified cytotoxic T lymphocyte preparation to mediate a cellular immune response, wherein the helper T lymphocyte preparation comprises CD4+ T cells having a chimeric antigen receptor having an extracellular antibody variable domain specific to an antigen associated with the disease or symptom and an intracellular signaling domain of a T cell receptor.

[0010] In yet another embodiment, the present invention provides an adoptive cellular immunotherapy composition having a chimeric antigen receptor-modified tumor-specific CD8+ cytotoxic T lymphocyte preparation that elicits a cellular immune response, wherein the cytotoxic T lymphocyte preparation comprises CD8+ T cells having a chimeric antigen receptor comprising an extracellular single-chain antibody specific to an antigen associated with the disease or condition and an intracellular signaling domain of a T cell receptor; and antigen-reactive chimeric antigen receptor-modified naïve CD4+ T helper cells derived from CD45RO-negative, CD62L-positive CD4-positive T cells; and a pharmaceutically acceptable carrier.

[0011] In another embodiment, the present invention provides an adoptive cellular immunotherapy composition comprising an antigen-specific CD8+ cytotoxic T lymphocyte preparation comprising CD8+ T cells derived from a patient that elicit a cellular immune response and antigen-reactive chimeric antigen receptor-modified CD4+ T helper cells, the CD4+ T helper cells eliciting a Th1 cytokine response and enhancing a CD8+ immune response against a pathogen, wherein the helper T lymphocyte preparation comprises CD4+ T cells having a chimeric antigen receptor having an extracellular antibody variable domain specific to the disease or symptom-associated antigen and an intracellular signaling domain of a T cell receptor.

[0012] In another embodiment, the present invention provides an adoptive cellular immunotherapy composition comprising antigen-reactive chimeric antigen receptor-modified CD4+ T helper cells that elicit direct tumor recognition and enhance CD8+ immune responses to pathogens, wherein the helper T lymphocyte formulation comprises CD4+ T cells having a chimeric antigen receptor comprising a disease- or symptom-associated antigen-specific extracellular antibody variable domain and an intracellular signaling domain of a T cell receptor.

[0013] In another aspect, the present invention provides a method for preparing an adoptive immunotherapy composition by obtaining a chimeric antigen receptor-modified tumor-specific CD8+ cytotoxic T lymphocyte preparation and an antigen-responsive chimeric antigen receptor that elicit a cellular immune response, and obtaining modified initial CD4+ T helper cells that elicit a Th1 cytokine response, wherein the modified cytotoxic T lymphocyte preparation comprises CD8+ T cells having a chimeric antigen receptor having an extracellular antibody variable domain specific to the disease or symptom-related antigen and an intracellular signaling module of a T cell receptor; wherein the modified helper T lymphocyte preparation comprises CD4+ cells having a chimeric antigen receptor having an extracellular antibody variable domain specific to the disease or symptom-related antigen and an intracellular signaling module of a T cell receptor.

[0014] In another embodiment, the present invention provides a method for preparing an adoptive cell immunotherapy composition, comprising obtaining modified naïve CD4+ helper T cells that elicit a Th1 cytokine response, wherein the modified helper T lymphocyte preparation comprises CD4+ T cells having a chimeric antigen receptor, the chimeric antigen receptor comprising an extracellular antibody variable domain specific to the disease or condition-associated antigen and an intracellular signaling domain of a T cell receptor; and combining the modified naïve CD4+ T helper cells with an antigen-specific central memory CD8+ cytotoxic T lymphocyte preparation having a chimeric antigen receptor having an extracellular antibody variable domain specific to the disease or condition-associated antigen and an intracellular signaling domain of a T cell or other receptor.

[0015] In one embodiment, the present invention provides a method for cellular immunotherapy in an individual suffering from a disease or condition, comprising administering a genetically modified helper T lymphocyte preparation to the individual, wherein the modified helper T lymphocyte preparation comprises CD4+ T cells having a chimeric antigen receptor, the chimeric antigen receptor comprising an extracellular antibody variable domain specific to an antigen associated with the disease or condition and an intracellular signaling module of a T cell receptor.

[0016] These and other embodiments of the invention will be further described in the accompanying specification, drawings and claims. Attached Figure Description

[0017] Figure 1 The phenotype and analysis of chimeric antigen receptor (CAR) expression in a CAR transduced with a lentiviral ROR1-CAR are shown, with untransduced CD8+ T cell lines serving as controls. The ROR1-CAR cassette contains a truncated EGFR as a transduction marker and can be detected by staining with an anti-EGFR monoclonal antibody. The truncated Fc-ROR1 fusion protein binds directly to the antigen-binding domain of the ROR1-CAR and selectively stains the transduced ROR1-CAR but not the untransduced control T cell lines. ROR1-CAR expression on the cell surface of CD8+ T cells is measured directly by binding to the ROR1-Fc fusion protein and indirectly by the expression of the truncated EGFR encoded downstream of the 2A sequence in the vector.

[0018] Figure 2 Shown in 51In the Cr release assay, CD8+ T cells expressing the ROR1-specific chimeric antigen receptor exhibited cytolytic activity against a group of human ROR1-positive tumor cell lines (K562), primary tumor cells (B-CLL), and autologous normal B-cells. Consistent with the uniform expression of ROR1 in malignant but not mature normal B cells, genetically modified CD8+ROR1-CAR T cells lysed only ROR1+ tumor cells and not mature normal B cells. CD8+ROR1-CAR T cells demonstrated specific cytolytic activity against ROR1-positive tumor cells, including primary CLL, but not against normal B cells.

[0019] Figure 3 The phenotype and CAR expression of ROR1-CAR-transduced CD4+ T cell lines are shown, with untransfected CD4+ T cell lines serving as controls. ROR1-CAR expression on the cell surface of CD4+ T cells was measured by specific binding to the ROR1-Fc fusion protein. The truncated FcROR1 fusion protein, rather than the individual Fc protein, directly binds to ROR1-CAR, and selective staining of ROR1-CAR-transduced, rather than untransduced, control CD4+ T cell lines confirmed ROR1-CAR expression on the cell surface and its binding to the ROR1-protein. ROR1-CAR expression on the cell surface of CD4+ T cells was measured by specific binding to the ROR1-Fc fusion protein, but not to the control Fc fusion protein.

[0020] Figure 4 (i.e.) Figures 4A-4B Together) showed in 51 In the Cr release assay, CD4+ROR1-CAR T cells exhibited weak but specific cytolytic activity against a subset of ROR1-positive tumor cells, including primary CLL, the mantle cell lymphoma cell line Jeko-1, and K562 cells stably transfected with ROR1 (K562 / ROR1), rather than naïve ROR1-negative K562 cells. CD4+ROR1-CAR T cells also demonstrated weak but specific cytolytic activity against ROR1-positive tumor cells.

[0021] Figure 5 (i.e.) Figures 5A-5B Together) showed the results from IFNγELISA ( Figure 5A ) and multiple cytokine detection ( Figure 5BResults of the study: Cytokine secretion in CD4+ and CD8+ ROR1-CAR T cell lines. CD4+ ROR1-CAR and CD8+ ROR1-CAR T cells were co-incubated with ROR1+ tumor cells. Interferon-γ (IFNg) levels were measured by ELISA (5A), and IFNg, TNF-α, IL-2, IL-4, IL-10, and IL-17 were measured by Luminex assay (5B). CD4+ ROR1-CAR-modified T cells specifically recognized ROR1-positive tumor cells and tumor cell lines, and produced significantly more Th1 cytokines, including IFN-γ, TNF-α, and especially IL-2, than CD8+ ROR1-CAR-modified T cells. These data indicate that, upon ROR1-CAR stimulation, CD4+ ROR1-CAR cells exhibit helper effector functions in addition to mediating direct anti-tumor responses, and can also enhance the ability of CD8+ ROR1-CAR-modified T cells to mediate cellular immune responses.

[0022] Figure 6 The results of proliferation studies are described, showing that CD4+ROR1-CAR T cells were induced to proliferate (CFSE assay) after stimulation with ROR1-positive tumor cell lines and primary tumor cells, and the percentage of proliferating cells and the number of cell divisions in the proliferating subsets were significantly higher than those of CD8+ROR1-CAR modified T cells. CD4+ROR1-CAR T cells proliferated more vigorously than CD8+ROR1-CARCTLs after stimulation with ROR1-positive tumor cells (K562 / ROR1, primary CLL, and Jeko MCL).

[0023] Figure 7 Polyclonal unselected CD4+ROR1CAR T cells contribute to tumor response by promoting the proliferation of CD8+ROR1-CAR CTLs. CD4+ROR1-CAR T cells (derived from bulk CD4+ T cells) significantly increased the proliferation of polyclonal unselected CD8+ROR1-CAR CTLs (18% in culture alone → 31.5% after co-culture with CD4+CAR T cells).

[0024] Figure 8 (i.e.) Figures 8A-8D (Together) This study presents functional analyses of CD4+ CAR T cell lines and T cells derived from flow cytometry-sorted and purified CD4+ naive, central memory, and effector memory subsets. Cytokine profiles and proliferative capacity suggest that CD4+ ROR1-CAR T cells derived from naive CD4+ T cells may be best suited to support CD8+ CTLs. Similar data were obtained in experiments comparing the function of CD4+ CAR T cell lines expressing CD19-specific CARs. Figure 8A Flow cytometry-based purification of naïve, central, and effector memory CD4+ T cells based on CD45RA, CD45RO, and CD62L expression was demonstrated. Figure 8B This study presents a proliferation analysis of sorted and purified naïve, central, and effector memory CD4+ T cells (CFSE assay) transduced with lentivirus to form ROR1-CAR T cell lines. Figure 8C This study presents an analysis of cytokine secretion from ROR1-CAR T cell lines derived from sorted and purified naïve, central, and effector memory CD4+ T cells (Luminex assay). Figure 8D This study presents an analysis of cytokine secretion from CD19-CAR T-cell lines derived from sorted and purified naïve, central, and effector memory CD4+ T cells (Luminex assay). Multiplex cytokine analysis was performed. Figure 8B Cytokine profiles obtained and cytokine profiles obtained by CFSE staining ( Figure 8C The proliferative capacity measurements showed that CD4+ ROR1-CAR-modified T cells derived from the initial subset produced the highest levels of Th1 cytokines and exhibited the most vigorous proliferation after stimulation with ROR1-positive tumor cells, suggesting that they may be most suitable for enhancing CD8+ ROR1-CAR CTLs. Cytokine secretion analysis of CD19-CAR T cell lines from sorted and purified initial, central, and effector memory CD4+ T cells (Luminex assay) indicated that the activity of CD4 T cell subsets can be generalized to many CARs.

[0025] Figure 9 This study demonstrates the co-culture of CD8+ ROR1-CAR-modified T cells with CD4+ ROR1-CAR-modified T cells (rather than untransduced control CD4+ T cells). Co-culturing CD8+ ROR1-CAR CTLs with CD4+ ROR1-CAR T cell lines derived from naive, central, and effector memory subsets determined the optimal combination of CD8+ and CD4+ T cells to maximize CD8+ ROR1-CAR CTL proliferation. Naive CD4+ ROR1-CAR T cells provided the strongest proliferation of CD8+ central memory ROR1-CAR CTLs. Co-culture increased tumor-specific proliferation of the CD8+ subset, and maximum proliferation of the CD8+ subset was observed after co-culturing with CD4+ ROR1-CAR T cells derived from naive CD4+ T cells, indicating that naive ROR1-CAR T cells...

[0026] Figure 10This study demonstrated the superior ability of the initial subset of CD4+ CAR T-cell lines to enhance the tumor-specific proliferation of central memory-derived CD8+ CAR CTLs in co-culture experiments with CD8+ CD19-CAR CTLs and CD4+ CD19-CAR T-cell lines stimulated by the CD19+ mantle cell lymphoma tumor line Jeko-1. The superior ability of the initial subset of CD4+ CAR T-cell lines to enhance the tumor-specific proliferation of central memory-derived CD8+ CAR CTLs was also confirmed in these experiments.

[0027] Figure 11 shows that CD8+ CAR T cells and CD4+ CAR T cells independently conferred direct antitumor efficacy in an immunodeficient mouse (NOD / SCID-Raji) lymphoma model. Several groups of mice (n=3) were inoculated with Raji tumor cells expressing firefly luciferase via tail vein injection and treated with a single dose of 10 x 10^6 T cells. Mice received CD19-CAR-transduced or control-mock-transduced CD8+ central memory-derived T cells (A), or CD19-CAR-transduced or control-mock-transduced CD4+ naïve-derived T cells (B). Tumor burden and distribution were analyzed using sequential bioluminescence imaging.

[0028] Figure 12 This study demonstrates the enhancing and synergistic effect of CD4+ROR1-CAR-modified T cells on the antitumor efficacy of CD8+ROR1-CAR CTLs in a mouse model of systemic mantle cell lymphoma (NSG / Jeko-1-ffLuc). The antitumor efficacy of ROR1-CAR-modified CD8+ and CD4+ T cells was also shown in the mouse model of systemic invasive mantle cell lymphoma (NSG / Jeko-1). Tumor burden analysis was performed using bioluminescence imaging after adoptive transfer of CD8+ROR1-CAR CTLs, CD4+ROR1-CAR T cells, or a combination of CD8+ and CD4+ROR1-CAR T cells. All mice received the same total dose of CAR T cells.

[0029] Figure 13 illustrates the synergistic effect of CD8+ and CD4+CD19-CAR T cells in a mouse model of systemic lymphoma (NSG / Raji). NSG mice were inoculated with Raji tumor cells transduced with firefly luciferase. Raji tumor transplantation was confirmed by bioluminescence imaging on day 6 post-inoculation (before treatment) (treatment protocol shown in Figure A; tumors by bioluminescence are shown in Figure B). Several groups of mice (n=5) were then treated with either CD8+CD19-CAR-modified T cells or a combination of CD8+ and CD4+CD19-CAR T cell products. All mice received the same total dose of T cells (10 x 10^6). Tumor burden analysis using bioluminescence imaging in mice treated with CD8+CD19-CAR T cells (as well as in mice treated with the combination of CD8+ and CD4+CD19-CAR T cell products (the black and gray bars in the middle after treatment) (Figure B) showed complete eradication of the Raji tumors. The mice were then challenged by a second inoculation with Raji tumor cells, and the frequencies of CD4+ and CD8+ CAR T cells in peripheral blood and tumor transplantation were analyzed. In mice treated with a combination of CD8+ and CD4+ CAR T-cell products, significantly higher levels of CD8+ CAR T cells were observed after tumor challenge (lower part of Figure D), and the mice completely rejected the Raji inoculation (grey bar on the right after tumor challenge, Figure B). Conversely, in mice receiving only CD8+CD19-CAR CTLs, no increase in CAR T cells was detected after tumor challenge (Figure C), and the Raji tumor cells were capable of transplantation (black bar on the right after tumor challenge, Figure B).

[0030] Detailed description of preferred implementation scheme

[0031] As used herein, "T cells" or "T lymphocytes" may be derived from any mammal, preferably primates, including monkeys, dogs, and humans. In some embodiments, the T cells are allogeneic to the recipient individual (from different donors of the same species); in some embodiments, the T cells are autologous (the donor and recipient are the same); and in some embodiments, the T cells are syngeneic (the donor and recipient are different, but are identical twins).

[0032] As used in this article, cytotoxic T lymphocytes (CTLs) refer to T lymphocytes that express CD8 on their surface (i.e., CD8+). + T cells). In some implementations, these cells are preferably antigen-stimulated "memory" T cells (T cells). M cell).

[0033] As used in this article, "central memory" T cells (or "T cells") CM ") refers to CTLs stimulated with antigens expressing CD62L and CD45RO on their surface, and which, compared to naïve cells, do not express CD45RA or have reduced CD45RA expression. In the implementation embodiment, central memory cells are positive for expression of CD62L, CCR7, CD28, CD127, CD45RO, and CD95, and have reduced CD54RA expression compared to naïve cells.

[0034] As used in this article, “effect memory” T cells (or “T cells”) EM ") refers to antigen-stimulated CTLs that, compared to central memory cells, do not express CD62L or have reduced CD62L expression on their surface, and, compared to naive cells, do not express CD45RA or have reduced CD45RA expression. In the implementation embodiment, compared to naive cells or central memory cells, effector memory cells are negative for expression of CD62L, CCR7, CD28, and CD45RA, but positive for expression of CD127."

[0035] As used herein, "naive" T cells refer to unstimulated T lymphocytes that express CD62L and CD45RA, and which, compared to central memory cells, do not express CD45RO or have reduced CD45RO expression. In some embodiments, naive CD8+ T lymphocytes are characterized by expressing naive T cell phenotypic markers including CD62L, CCR7, CD28, CD3, CD127, and CD45RA.

[0036] As used in this article, "effect" and "T" E "T cells refer to cytotoxic T lymphocytes stimulated by antigens. Compared with central memory cells, they do not express CD62L, CCR7, or CD28, or their expression of CD62L, CCR7, and CD28 is reduced, and they are positive for granzyme B and perforin."

[0037] As used herein, “enriched” and “reduced” in describing the number of cell types in a mixture refer to processes or steps that subject the cell mixture to an increase in the number of “enriched” types and a decrease in the number of “reduced” cells. Therefore, depending on the source of the original cell population undergoing the enrichment process, the mixture or composition may contain 60%, 70%, 80%, 90%, 95%, or 99% or more (number or count) of “enriched” cells and 40%, 30%, 20%, 10%, 5%, or 1% or less (number or count) of “reduced” cells.

[0038] Interleukin-15 is known and described, for example, in U.S. Patent No. 6,344,192.

[0039] As used in this article, "CAR" refers to a chimeric antigen receptor, which contains an extracellular variable domain of an antibody specific to a disease or symptom-associated antigen and an intracellular signaling domain, such as a co-stimulatory domain, of a T cell or other receptor. Detailed Implementation

[0040] Cultured CD4+ T lymphocytes in vitro significantly increased the proliferation, persistence, and antitumor responsiveness of tumor-specific CD8+ T cells both in vitro and in vivo. In some implementations, naïve CD4+ T cells possess an intrinsic programming that enables them to exhibit superior helper activity compared to CD4+ T cells derived from central and effector memory or large CD4+ T cells.

[0041] In the implementation scheme, tumor-reactive CD4+ T cells are modified with a chimeric antigen receptor (CAR) derived from a single-chain antibody specific to the orphan receptor ROR1 or CD19 molecule of tyrosine kinase. ROR1 is uniformly expressed in chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL), and when expressed in CD8+ cytotoxic T cells (CTLs), the ROR1-specific CAR derived from an anti-ROR1 monoclonal antibody (mAb) confers specific recognition of malignant rather than normal mature B cells. ROR1-CAR T cells derived from large CD4+ T cells and flow-cytometry-sorted purified naïve, central, and effector memory CD4+ T cells are obtained from peripheral blood of healthy donors and CLL patients. CD4+ CAR T cells exhibit specific but weak cytolytic activity against ROR1+ tumors, including primary CLL, MCL lines Jeko-1, and K562 cells transfected with ROR1. Multiplex cytokine analysis detected high levels of Th1 cytokines and significantly higher levels of IFNγ, TNFα, and especially IL-2 compared to CD8+CAR CTLs. CFSE staining showed significantly increased proliferation after stimulation with ROR1-positive tumor cells compared to CD8+CAR CTLs, with a significantly higher percentage of proliferating cells and a significantly higher number of cell divisions in the proliferating cell subsets. CD4+ T cells obtained from healthy donors and CLL patients acquired antitumor responsiveness after genetic modification with ROR1-specific CARs. Moreover, the ability to proliferate and produce high levels of Th1 cytokines in the absence of exogenous cytokines indicates that CD4+CAR T cells exhibit typical helper functions after CAR stimulation, meaning that in addition to conferring direct antitumor effects, they can also be used to enhance tumor-specific CD8+ CTLs.

[0042] Cytokine profiles and proliferative capacity of ROR1-CAR T cells derived from flow cytometry-sorted and purified CD4+ naïve, central, and effector memory cell subsets were obtained. CD4+ CAR T cells derived from the naïve CD45RA+CD45RO-CD62L+ subset produced the highest levels of Th1 cytokines, particularly IL-2, and proliferated in response to ROR1+ tumor cells. Indeed, in co-culture experiments, the addition of CAR-transduced CD4+ T cells, rather than untransduced CD4+ T cells, resulted in a significant increase in tumor-specific proliferation of CD8+ CAR CTLs. In some embodiments, CAR-modified CD4+ T cells derived from naïve rather than central and effector memory cell subsets or large CD4+ T cells resulted in increased proliferation of CD8+ CAR CTLs.

[0043] CD8+ central memory T cells possess an intrinsic program that allows them to persist for extended periods after administration, making them a preferred cell subset for immunotherapy. In one embodiment, ROR1-CAR or CD19CAR-modified CTLs derived from sorted and purified CD8+ central memory T cells and CD4+ naïve CAR-modified T cells enhance the proliferation of the CD8+ T cell subset. In another embodiment, tumor-specific CD4+ T cells exhibit anti-tumor responsiveness and are beneficial to CD8+ T cells both in vitro and in vivo. In one specific embodiment, tumor-specific CD4+ T cells derived from the naïve subset are used.

[0044] In another embodiment, the CD8+ and CD4+ T cells may be modified with T cell receptors (TCRs). The TCRs may be specific to any antigen, pathogen, or tumor (TCRs exist for many tumor antigens in melanoma (e.g., MART1, gp100), leukemia (e.g., WT1, minor histocompatibility antigens), and breast cancer (e.g., HER2, NY-BR1).

[0045] Detailed description

[0046] Composition

[0047] This disclosure provides an adoptive cellular immunotherapy composition comprising a genetically modified helper T lymphocyte preparation that enhances the ability of the genetically modified cytotoxic T lymphocyte preparation to mediate a cellular immune response, wherein the helper T lymphocyte preparation comprises CD4+ T cells having a chimeric antigen receptor, the chimeric antigen receptor comprising an extracellular antibody variable domain specific to the disease or condition-associated antigen and an intracellular signaling domain of a T cell receptor or other receptor.

[0048] In some embodiments, the adoptive cellular immunotherapy composition further comprises a chimeric antigen receptor-modified tumor-specific CD8+ cytotoxic T lymphocyte preparation that elicits a cellular immune response, wherein the cytotoxic T lymphocyte preparation comprises CD8+ T cells having a chimeric antigen receptor comprising an extracellular single-chain antibody specific to the disease or condition-associated antigen and an intracellular signaling domain of a T cell receptor.

[0049] In some embodiments, the adoptive cellular immunotherapy composition comprises a chimeric antigen receptor-modified tumor-specific CD8+ cytotoxic T lymphocyte formulation that elicits a cellular immune response, wherein the cytotoxic T lymphocyte formulation comprises CD8+ T cells having a chimeric antigen receptor, the chimeric antigen receptor comprising an extracellular single-chain antibody specific to the disease or condition-associated antigen and an intracellular signaling domain of a T cell receptor; the formulation is combined with antigen-reactive chimeric antigen receptor-modified naïve CD4+ T helper cells derived from CD45RO-negative, CD62L-positive CD4-positive T cells, and a pharmaceutically acceptable carrier.

[0050] In other embodiments, the adoptive cellular immunotherapy composition comprises an antigen-specific CD8+ cytotoxic T lymphocyte preparation that elicits a cellular immune response from a patient, combined with initial CD4+ T helper cells modified with an antigen-responsive chimeric antigen receptor that enhances the CD8+ immune response, wherein the helper T lymphocyte preparation comprises CD4+ T cells having a chimeric antigen receptor that comprises an extracellular antibody variable domain specific to the disease or condition-associated antigen and an intracellular signaling domain of a T cell receptor.

[0051] In yet another embodiment, the adoptive cellular immunotherapy composition comprises naïve CD4+ T helper cells modified with an antigen-reactive chimeric antigen receptor that enhances CD8+ immune responses, wherein the helper T lymphocyte preparation comprises CD4+ T cells having a chimeric antigen receptor comprising an extracellular antibody variable domain specific to a disease or symptom-associated antigen and an intracellular signaling domain of a T cell receptor.

[0052] In some embodiments, the CD4+ helper T lymphocytes are selected from naive CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ T cells, or large CD4+ T cells. In some embodiments, the CD4+ helper lymphocytes are naive CD4+ T cells, wherein the naive CD4+ T cells include CD45RO-, CD45RA+, and CD62L+ CD4+ T cells. In some embodiments, the CD8+ cytotoxic T lymphocytes are selected from naive CD8+ T cells, central memory CD8+ T cells, effector memory CD8+ T cells, or large CD8+ T cells. In some embodiments, the CD8+ cytotoxic T lymphocytes are central memory T cells, wherein the central memory T cells include CD45RO+, CD62L+, and CD8+ T cells. In other embodiments, the CD8+ cytotoxic T lymphocytes are central memory T cells, and the CD4+ helper T lymphocytes are naive CD4+ T cells.

[0053] In an optional implementation, T cells can be modified with recombinant T cell receptors. TCRs can be specific to any antigen, pathogen, or tumor. TCRs exist targeting many tumor antigens in melanoma (e.g., MART1, gp100), leukemia (e.g., WT1, minor histocompatibility antigens), and breast cancer (e.g., HER2, NY-BR1).

[0054] Selection and sorting of T lymphocyte populations

[0055] The compositions described herein provide antigen-responsive CD4+ and CD8+ T lymphocytes.

[0056] T lymphocytes can be collected using known techniques and enriched or depleted using known techniques, such as affinity binding to antibodies, flow cytometry, and / or immunomagnetic bead selection. After enrichment and / or depletion steps, the desired T lymphocytes can be expanded in vitro using known techniques (including, but not limited to, those described in U.S. Patent No. 6,040,177 to Riddell et al.) or variations thereof that are obvious to those skilled in the art.

[0057] For example, a desired T cell population or subset can be expanded by adding an initial T lymphocyte population to an in vitro culture medium, then adding feeder cells such as undividing peripheral blood mononuclear cells (PBMCs) to the culture medium (e.g., so that the resulting cell population contains at least about 5, 10, 20, or 40 or more PBMC feeder cells for each T lymphocyte in the initial cell population to be expanded); and incubating the culture (e.g., for a time sufficient to expand the number of T cells). The undividing feeder cells may include γ-ray irradiated PBMC feeder cells. In some embodiments, the PBMCs are irradiated with γ-rays in the range of about 3000-3600 rads. The order in which the T cells and feeder cells are added to the culture medium can be reversed as needed. The culture is typically cultured under conditions suitable for T lymphocyte growth, such as temperature. For the growth of human T lymphocytes, the temperature is typically at least about 25°C, preferably at least about 30°C, and more preferably at least about 37°C.

[0058] The expanded T lymphocytes include cytotoxic T lymphocytes (CTLs) and helper T lymphocytes that are specific to antigens present on human tumors or pathogens.

[0059] Optionally, the amplification method may further include the step of adding non-dividing EBV-transformed lymphoblastoid cells (LCLs) as feeder cells. LCLs may be irradiated with gamma rays in the range of approximately 6,000–10,000 rads. The LCL feeder cells may be provided in any suitable amount, such as an LCL feeder cell to naïve T lymphocyte ratio of at least approximately 10:1.

[0060] Optionally, the amplification method may further include the step of adding an anti-CD3 monoclonal antibody to a culture medium (e.g., at a concentration of at least about 0.5 ng / ml). Optionally, the amplification method may further include the step of adding IL-2 and / or IL-15 to the culture medium (e.g., where the concentration of IL-2 is at least about 10 units / ml).

[0061] After T lymphocytes are isolated, both cytotoxic and helper T lymphocytes before and after expansion can be classified into naive, memory, and effector T cell subsets.

[0062] CD8+ cells can be obtained using standard methods. In some embodiments, CD8+ cells are further classified into initial, central memory, and effector cells by identifying cell surface antigens associated with each of the initial, central memory, and effector CD8+ cells. In some embodiments, memory T cells are present in the CD62L+ and CD62L- subsets of CD8+ peripheral blood lymphocytes. After staining with anti-CD8 and anti-CD62L antibodies, PBMCs are classified into CD62L-CD8+ and CD62L+CD8+ fractions. In some embodiments, the expression of phenotypic markers of central memory TCMs includes CD45RO, CD62L, CCR7, CD28, CD3, and CD127, and they are negative for granzyme B. In some embodiments, central memory T cells are CD45RO+, CD62L+, and CD8+ T cells. In some embodiments, effector T cells are... E The cells are negative for CD62L, CCR7, CD28, and CD127, and positive for granzyme B and perforin. In some implementations, the naïve CD8+ T lymphocytes are characterized by expressing naïve T cell phenotypic markers, including CD62L, CCR7, CD28, CD3, CD127, and CD45RA.

[0063] Whether a cell or cell population is positive for a specific cell surface marker can be determined by flow cytometry using staining with a specific antibody against the surface marker and an isotype-matched control antibody. A marker-negative cell population is defined as a cell population that does not show significant staining with a specific antibody compared to the isotype control, while a positive cell population is defined as a cell population that shows uniform staining compared to the isotype control. In some embodiments, a reduction in the expression of one or more markers is defined as a decrease in mean fluorescence intensity of 1 log10 compared to a reference cell population and / or a reduction in the percentage of cells displaying the marker of at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and 100%, and any percentage between 20% and 100%. In some embodiments, a cell population that is positive for one or more markers refers to a cell population that, compared to a reference cell population, shows the marker at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and 100% of the cells, and any percentage between 50% and 100%.

[0064] CD4+ T helper cells are classified into primary, central memory, and effector cells by identifying cell populations with cell surface antigens. CD4+ lymphocytes can be obtained using standard methods. In some embodiments, primary CD4+ T lymphocytes are CD45RO-, CD45RA+, and CD62L+CD4+ T cells. In some embodiments, central memory CD4+ cells are CD62L- and CD45RO-positive. In some embodiments, effector CD4+ cells are CD62L- and CD45RO-negative.

[0065] Antigen-specific CD4+ and CD8+ cell populations can be obtained by stimulating naïve or antigen-specific T lymphocytes with the antigen. For example, antigen-specific T cell clones can be generated against cytomegalovirus antigens by isolating T cells from an infected individual and stimulating said cells in vitro with the same antigen. Naïve T cells can also be used. Any number of antigens derived from tumor cells, cancer cells, or infectious agents can be used. Examples of such antigens include HIV antigens, HCV antigens, HBV antigens, CMV antigens, parasite antigens, and tumor antigens such as tyrosine kinase orphan receptors ROR1, tEGFR, Her2, L1-CAM, CD19, CD20, CD22, mesothelin, and CEA. In some embodiments, adoptive cellular immunotherapy compositions can be used to treat diseases or conditions including solid tumors, hematologic malignancies, melanoma, or viral infections.

[0066] Modification of T lymphocyte populations

[0067] According to the present invention, in some embodiments, it is desirable to introduce functional genes into T cells intended for immunotherapy. For example, one or more introduced genes may improve the efficacy of the treatment by enhancing the viability and / or function of metastatic T cells; or they may provide genetic markers to allow selection and / or assessment of in vivo survival or migration; or they may integrate functions that improve the safety of immunotherapy, for example by making cells readily available for in vivo negative selection, as described by Lupton SD et al., Mol. and Cell Biol., 11:6 (1991); and Riddell et al., Human Gene Therapy 3:319-338 (1992); see also Lupton et al.’s patent publications PCT / US91 / 08442 and PCT / US94 / 05601, which describe the use of bifunctional selection fusion genes derived from fusion dominant positive selection markers and negative selection markers. This can be implemented based on the disclosure of this invention according to known techniques (see, for example, U.S. Patent No. 6,040,177, columns 14-17, owned by Riddell et al.) or variations thereof that are obvious to those skilled in the art.

[0068] In some embodiments, T cells are modified with a chimeric antigen receptor (CAR). In some embodiments, the CAR comprises a single-chain antibody fragment (scFv) derived from the variable heavy chain (VH) and variable light chain (VL) of a monoclonal antibody (mAb) linked to the TCR CD3+ chain that mediates T-cell activation and cytotoxicity. Fusion of the co-stimulatory domain of CD28 or 4-1BB to the CD3+ chain can also provide a co-stimulatory signal via the CAR. The CAR is specific for HLA-independent cell surface molecules, overcoming limitations of TCR recognition, including HLA restriction and low levels of HLA expression on tumor cells.

[0069] CARs with specificity for any cell surface marker can be constructed by utilizing, for example, antigen-binding fragments or variable domains of antibody molecules. The antigen-binding molecule can be linked to one or more cell signaling modules. In some embodiments, the cell signaling module includes a CD3 transmembrane domain, a CD3 intracellular signaling domain, and a CD28 transmembrane domain. In some embodiments, the intracellular signaling domain includes a CD28 transmembrane and signaling domain linked to the CD3 intracellular domain. In some embodiments, the CAR may also include a transduction marker such as tEGFR.

[0070] In one implementation, the intracellular signaling domain of CD8+ cytotoxic T cells is the same as that of CD4+ helper T cells. In other implementations, the intracellular signaling domain of CD8+ cytotoxic T cells is different from that of CD4+ helper T cells.

[0071] In some embodiments, both CD8+ T cells and CD4+ T cells are genetically modified with antibody heavy chain domains that specifically bind to pathogen-specific cell surface antigens. In some embodiments, the CAR is specific for cell surface antigens expressed in relation to pathogens, tumors, or cancer cells. In some embodiments, the CAR is specific for HIV antigens, HCV antigens, HBV antigens, CMV antigens, parasitic antigens, and tumor antigens such as tyrosine kinase orphan receptors ROR1, tEGFR, Her2, L1-CAM, CD19, CD20, CD22, mesothelin, and CEA. This article describes methods for preparing CARs, and see also Forman's US 6,410,319, Jensen et al.'s WO 2002 / 077029, 7,446,191, WO 2010 / 065818, WO 2010 / 025177, WO 2007 / 059298 and 7,514,537, and Berger C. et al.'s description in J. Clinical Investigation, 118:1294-308 (2008), all of which are incorporated herein by reference in their entirety.

[0072] In one implementation, the same or different CARs may be introduced into each CD4+ and CD8+ T lymphocyte. In another implementation, the CAR in each of these cell populations has an antigen-binding molecule that specifically binds to the same antigen. The cell signal transduction modules may be different. In one implementation, each CD4 or CD8 T lymphocyte may be classified as a naive, central memory, effector memory, or effector cell prior to transduction. In an alternative implementation, each CD4 or CD8 T lymphocyte may be classified as a naive, central memory, effector memory, or effector cell prior to transduction.

[0073] In an optional implementation, T cells may be modified with recombinant T cell receptors. TCRs may be specific to any antigen, pathogen, or tumor. Many tumor antigens exist that target melanoma (e.g., MART1, gp100), leukemia (e.g., WT1, minor histocompatibility antigens), and breast cancer (e.g., HER2, NY-BR1).

[0074] Various infection techniques have been developed for gene delivery using recombinant infectious viral particles. This represents the current preferred method for T lymphocyte transduction according to the present invention. Viral vectors already used in this manner include those derived from simian virus 40, adenovirus, adeno-associated virus (AAV), lentiviral vectors, and retroviruses. Thus, while there are numerous gene transfer and expression methods, their essential function is the introduction and expression of genetic material into mammalian cells. Some of the aforementioned techniques have been used to transduce hematopoietic cells or lymphocytes, including calcium phosphate transfection, protoplast fusion, electroporation, and infection with recombinant adenovirus, adeno-associated virus, and retroviral vectors. Primary T lymphocytes have been successfully transduced via electroporation and retroviral infection.

[0075] Retroviral vectors provide an efficient method for transferring genes into eukaryotic cells. Moreover, retroviral integration occurs in a controlled manner, enabling each cell to stably integrate one or more copies of new genetic information.

[0076] Overexpression of stimulating factors (e.g., lymphokines or cytokines) is anticipated to be toxic to the treated individual. Therefore, within the scope of this invention, gene fragments that facilitate negative selection of the T cells of this invention in vivo are included. As used, “negative selection” means that, as a result of changes in the individual’s in vivo conditions, fused cells can be removed. The negative selection phenotype may be the result of inserting a gene that confers sensitivity to an administered agent, such as a compound. Negative selection genes known in the art include, among others, the following genes: the herpes simplex virus type I thymidine kinase (HSV-I TK) gene conferring sensitivity to ganciclovir (Wigler et al., Cell 11:223, 1977), the cellular hypoxanthine phosphoribosyltransferase (HPRT) gene, the cellular adenine phosphoribosyltransferase (APRT) gene, and bacterial cytidine deaminase (Mullen et al., Proc. Natl. Acad. Sci. USA. 89:33 (1992)).

[0077] In some embodiments, it is useful to include a positive marker in T cells that enables the selection of cells with a negative selection phenotype in vitro. The positive selection marker may be a gene that, when introduced into a host cell, expresses a dominant phenotype that allows positive selection of cells carrying the gene. Such genes are known in the art and, among others, include the hygromycin-B phosphotransferase gene (hph) conferring resistance to hygromycin-B, the aminoglycoside phosphotransferase gene (neo or aph) encoding Tn5 for resistance to the antibiotic G418, the dihydrofolate reductase (DHFR) gene, the adenosine deaminase gene (ADA), and the multidrug resistance (MDR) gene.

[0078] Preferably, the positive selection marker and the negative selection element are linked such that the loss of the negative selection element necessarily accompanies the loss of the positive selection marker. Even more preferably, the positive and negative selection markers are fused, so that the loss of one necessarily leads to the loss of the other. An example of a fusion polynucleotide that produces an expression product polypeptide conferring the desired positive and negative selection characteristics described above is the hygromycin phosphotransferase-thymidine kinase fusion gene (HyTK). Expression of this gene produces a polypeptide conferring hygromycin B resistance for positive selection in vivo and ganciclovir sensitivity for negative selection in vivo. See Lupton SD et al., Mol. and Cell. Biology 11:3374-3378, 1991. Furthermore, in a preferred embodiment, the polynucleotide of the present invention encoding the chimeric receptor is in a retroviral vector containing the fusion gene, particularly those conferring hygromycin B resistance for positive selection in vivo and ganciclovir sensitivity for negative selection in vivo, such as the HyTK retroviral vector, described in Lupton, SD et al. (1991), ibid. See also SDLupton’s patent publications PCT / US91 / 08442 and PCT / US94 / 05601, which describe the use of bifunctional selection of fusion genes derived from fusion dominant positive selection markers and negative selection markers.

[0079] Preferred positive selection markers are derived from genes selected from the group consisting of hph, nco, and gpt, and preferred negative selection markers are derived from genes selected from the group consisting of cytosine deaminase, HSV-I TK, VZV TK, HPRT, APRT, and gpt. Particularly preferred markers are bifunctional selection fusion genes, wherein the positive selection marker is derived from hph or neo, and the negative selection marker is derived from cytosine deaminase or TK genes or selection markers.

[0080] T lymphocytes can be transduced using various methods well known in the art. For example, retroviral transduction can be performed as follows: On day 1 after REM stimulation as described herein, cells are given 20-30 units / ml of IL-2; on day 3, half of the culture medium is replaced with retroviral supernatant prepared according to standard methods, and then 5 μg / ml polyglobulin and 20-30 units / ml IL-2 are added to the culture medium; on day 4, the cells are washed and placed in fresh culture medium supplemented with 20-30 units / ml IL-2; on day 5, the cells are repeatedly exposed to retrovirus; on day 6, the cells are placed in selective culture medium supplemented with 30 units / ml IL-2 (e.g., containing an antibiotic corresponding to the antibiotic resistance gene provided in the retroviral vector); on day 13, live cells are separated from dead cells using the Ficoll Hypaque density gradient separation method, and then the live cells are subcloned.

[0081] CD4+ and CD8+ cells can be modified with expression vectors encoding CARs. In one embodiment, these cells are then further classified into primary, central memory, and effector cell subpopulations by sorting for a unique cell surface antigen in each of these cell populations, as described above. Furthermore, CD4+ or CD8+ cell populations can be selected based on their cytokine profile or proliferative activity. For example, CD4+ T lymphocytes that exhibit increased cytokine production such as IL-2, IL-4, IL-10, TNFα, and IFNγ upon antigen stimulation, compared to sham-transduced cells or transduced CD8+ cells, can be selected. In other embodiments, primary CD4+ T cells with increased IL-2 and / or TNFα production are selected. Similarly, CD8+ cells with increased IFNγ production are selected compared to sham-transduced CD8+ cells.

[0082] In the implementation scheme, CD4+ and CD8+ cells that proliferate in response to the antigen are selected. For example, CD4+ cells that proliferate more rapidly when stimulated with the antigen, compared to sham-transduced cells or transduced CD8+ cells, are selected.

[0083] In some embodiments, CD4+ and CD8+ cells that exhibit cytotoxicity to cells carrying the antigen are selected. In these embodiments, CD4+ cells are expected to exhibit weaker cytotoxicity compared to CD8+ cells.

[0084] This application anticipates that combinations of CD4+ and CD8+ T cells can be used in compositions. In one embodiment, CAR-transduced CD4+ cells can be combined with CD8+ antigen-reactive cells that have the same antigen specificity to the CAR. In other embodiments, CAR-transduced CD8+ cells are combined with antigen-reactive CD4+ cells. In yet another embodiment, CAR-modified CD4+ and CD8+ cells are combined.

[0085] As described herein, this application anticipates the ability to further separate CD4+ and CD8+ cells into subpopulations such as primary, central memory, and effector cell populations. As described herein, in some embodiments, primary CD4+ cells are CD45RO-, CD45RA+, and CD62L+CD4+ T cells. In some embodiments, central memory CD4+ cells are CD62L-positive and CD45RO-positive. In some embodiments, effector CD4+ cells are CD62L-negative and CD45RO-positive. Each of these cell populations can be independently modified with CAR.

[0086] As described herein, in some embodiments, memory T cells are present in the CD62L+ and CD62L- subsets of CD8+ peripheral blood lymphocytes. After staining with anti-CD8 and anti-CD62L antibodies, PBMCs are classified into CD62L-CD8+ and CD62L+CD8+ fractions. In some embodiments, the expression of phenotypic markers of central memory TCMs includes CD62L, CCR7, CD28, CD3, and CD127, which are negative for granzyme B. In some embodiments, central memory T cells are CD45RO+, CD62L+, and CD8+ T cells. In some embodiments, effector T cells... E The cells are negative for CD62L, CCR7, CD28, and CD127, and positive for granzyme B and perforin. In some implementations, the naïve CD8+ T lymphocytes are characterized by CD8+, CD62L+, CD45RO+, CCR7+, CD28+, CD127+, and CD45RO+. Each of these cell populations can be independently modified with CAR.

[0087] Each of the CD4+ and CD8+ cell subsets can be combined with one another. In one specific implementation, modified naïve CD4+ cells are combined with modified central memory CD8+ T cells to provide synergistic cytotoxicity against antigen-carrying cells such as tumor cells.

[0088] method

[0089] This application provides methods for preparing adoptive cell immunotherapy compositions, as well as uses and methods for administering cell immunotherapy to individuals suffering from diseases or conditions using these compositions.

[0090] In an embodiment, a method for preparing the composition includes obtaining modified initial CD4+ T helper cells, wherein the modified helper T lymphocyte preparation comprises CD4+ T cells having a chimeric antigen receptor comprising an extracellular antibody variable domain and an intracellular signal transduction domain specific to a disease or symptom-associated antigen.

[0091] In another embodiment, the method further includes obtaining modified CD8+ cytotoxic T cells, wherein the modified cytotoxic T lymphocyte preparation comprises CD8+ cells having a chimeric antigen receptor comprising an extracellular antibody variable domain specific to a disease or symptom-associated antigen and an intracellular signaling domain of a T cell receptor.

[0092] In another embodiment, the method includes obtaining modified CD8+ cytotoxic T cells, wherein the modified cytotoxic T lymphocyte preparation comprises CD8+ T cells having a chimeric antigen receptor comprising an extracellular antibody variable domain specific to a disease or symptom-associated antigen and an intracellular signaling domain of a T cell receptor, and further comprises combining the modified CD8+ cytotoxic T cells with an antigen-specific CD4+ helper lymphocyte preparation.

[0093] CAR-modified CD4+ and CD8+ cell preparations have been described above and in the examples. Antigen-specific T lymphocytes can be obtained from patients with diseases or conditions, or can be prepared by in vitro stimulation of T lymphocytes in the presence of antigens. CD4+ and CD8+ T lymphocyte subsets can also be isolated and combined in the preparation methods described herein.

[0094] This application also provides a cellular immunotherapy method for individuals suffering from a disease or condition, comprising administering the composition of any one of claims 1-19. In other embodiments, the method comprises administering to an individual a genetically modified cytotoxic T-lymphocyte preparation that provides a cellular immune response, wherein the cytotoxic T-lymphocyte preparation comprises CD8+ T cells having a chimeric antigen receptor, the chimeric antigen receptor comprising an extracellular antibody variable domain specific to an antigen associated with the disease or condition and an intracellular signaling domain of a T cell or other receptor; and a genetically modified helper T-lymphocyte preparation that induces direct tumor recognition and enhances the ability of the genetically modified cytotoxic T-lymphocyte preparation to mediate a cellular immune response, wherein the helper T-lymphocyte preparation comprises CD4+ T cells having a chimeric antigen receptor, the chimeric antigen receptor comprising an extracellular antibody variable domain specific to an antigen associated with the disease or condition and an intracellular signaling domain of a T cell receptor.

[0095] In another embodiment, a method of performing cellular immunotherapy on an individual suffering from a disease or condition includes administering to the individual a genetically modified helper T lymphocyte preparation, wherein the modified helper T lymphocyte preparation comprises CD4+ T cells having a chimeric antigen receptor, the chimeric antigen receptor comprising an extracellular antibody variable domain specific to an antigen associated with the disease or condition and an intracellular signaling module of a T cell receptor. In one embodiment, the method further comprises administering to the individual a genetically modified cytotoxic T lymphocyte preparation, wherein the modified cytotoxic T lymphocyte preparation comprises CD8-positive cells having a chimeric antigen receptor, the chimeric antigen receptor comprising an extracellular antibody variable domain specific to an antigen associated with the disease or condition and an intracellular signaling module of a T cell receptor.

[0096] Another embodiment describes a method for administering cellular immunotherapy to an individual suffering from a disease or condition, comprising: analyzing the presence of disease or condition-related antigens in a biological sample of the individual, and administering the adoptive cellular immunotherapy composition described herein, wherein a chimeric antigen receptor specifically binds to the antigen.

[0097] A CAR is prepared having a component that provides antigen-specific binding associated with diseases or conditions such as solid tumors, cancer, viral infections, and parasitic infections. In embodiments, the intracellular signaling module of the chimeric antigen receptor's T-cell receptor includes a transmembrane domain, a CD28 signaling domain, and a CD3 intracellular signaling domain, or other domains of a T-cell co-stimulatory molecule. In some embodiments, the intracellular signaling molecule includes a CD3 intracellular domain, a CD28 domain, a CD28 transmembrane and signaling domain linked to the CD3 intracellular domain, or other domains of a T-cell co-stimulatory molecule.

[0098] In an optional implementation, T cells may be modified with recombinant T cell receptors. TCRs can be specific to any antigen, pathogen, or tumor. TCRs exist targeting a wide range of tumor antigens in melanoma (e.g., MART1, gp100), leukemia (e.g., WT1, minor histocompatibility antigens), and breast cancer (e.g., HER2, NY-BR1).

[0099] In some embodiments, the CD4+ helper T lymphocytes are selected from naive CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ T cells, or large CD4+ T cells. In one specific embodiment, the CD4+ helper lymphocytes are naive CD4+ T cells, wherein the naive CD4+ T cells include CD45RO-, CD45RA+, and CD62L+ CD4+ T cells. In other embodiments, the CD8+ cytotoxic T lymphocytes are selected from naive CD8+ T cells, central memory CD8+ T cells, effector memory CD8+ T cells, or large CD8+ T cells. In one specific embodiment, the CD8+ cytotoxic T lymphocytes are central memory T cells, wherein the central memory T cells include CD45RO+, CD62L+, and CD8+ T cells. In one specific embodiment, the CD8+ cytotoxic T lymphocytes are central memory T cells, and the CD4+ helper T lymphocytes are naive CD4+ T cells.

[0100] In one embodiment, both CD8+ T cells and CD4+ T cells are genetically modified with a CAR containing an antibody heavy chain domain that specifically binds to pathogen- or tumor-specific cell surface antigens. In other embodiments, the intracellular signaling domain of the CD8 cytotoxic T cells is the same as that of the CD4 helper T cells. In other embodiments, the intracellular signaling domain of the CD8 cytotoxic T cells is different from that of the CD4 helper T cells.

[0101] Individuals that can typically be treated by this invention are human and other primate individuals, such as monkeys and apes used for veterinary medicinal purposes. These individuals can be male or female and can be of any suitable age, including infants, children, adolescents, young adults, and elderly individuals.

[0102] The method can be used to treat, for example, solid tumors, hematologic malignancies, melanoma, or viral or other pathogen infections. Pathogen infections include HIV, HCV, HBV, CMV, and parasitic diseases. In some embodiments, the antigen associated with the disease or condition is selected from tyrosine kinase orphan receptors ROR1, tEGFR, Her2, L1-CAM, CD19, CD20, CD22, mesothelin, CEA, and hepatitis B surface antigen.

[0103] Treatable individuals include those with cancer, including but not limited to cancers of the colon, lung, liver, breast, prostate, ovary, skin (including melanoma), bone, and brain. In some embodiments, tumor-associated antigens are known, such as those for melanoma, breast cancer, squamous cell carcinoma, colon cancer, leukemia, myeloma, and prostate cancer (in these embodiments, memory T cells can be isolated or modified by introducing the T cell receptor gene). In other embodiments, genetically modified T cells expressing a modified immune receptor can be used to target the tumor-associated protein. Examples include, but are not limited to, B-cell lymphoma, breast cancer, prostate cancer, and leukemia.

[0104] Individuals who are eligible for treatment also include those suffering from or at risk of developing infectious diseases, including but not limited to viral, retroviral, bacterial, and protozoan infections. Individuals eligible for treatment include immunodeficiency patients with viral infections, including but not limited to cytomegalovirus (CMV), Epstein-Barr virus (EBV), adenovirus, and BK polyomavirus infections in transplant recipients.

[0105] Cells prepared according to the above description can be used in methods and compositions for adoptive immunotherapy, based on known techniques or variations thereof that are obvious to those skilled in the art based on the disclosure of this application. See, for example, U.S. Patent Application Publication No. 2003 / 0170238 by Gruenberg et al., and also U.S. Patent No. 4,690,915 by Rosenberg.

[0106] In some embodiments, the cells are formulated as follows: they are first harvested from their culture medium, and then washed and concentrated in a culture medium and container system (“pharmaceutically acceptable” carrier) suitable for administration in therapeutically effective amounts. Suitable infusion media can be any isotonic medium formulation, typically physiological saline, Normosol R (Abbott), or Plasma-Lyte A (Baxter) injection, or 5% glucose solution or Ringer's lactate solution may also be used. The infusion medium may be supplemented with human serum albumin.

[0107] The therapeutically effective number of cells in the composition is at least 2 cells (e.g., 1 CD8+ central memory T cell and 1 CD4+ helper T cell subset), or more typically greater than 10. 2 Cells, up to 10 6 up to and including 10 8 Or 10 9 10 cells, and can be greater than 10 10 The number of cells depends on the intended end application of the composition and the cell types it comprises. For example, if specificity for a specific antigen is desired, the cell population should contain more than 70%, typically more than 80%, 85%, and 90-95% of such cells. For the applications described herein, cells are typically in a volume of 1 liter or less, which can be 500 ml or less, or even 250 ml or 100 ml or less. Therefore, the desired cell density is typically greater than 10. 6 Cells / ml, typically greater than 10 7 cells / ml, usually 10 8 Cells / ml or higher. Clinically relevant immune cell counts can be allocated across multiple infusions, accumulating to 10 or greater. 9 10 10 Or 10 11 Each cell.

[0108] In some embodiments, the lymphocytes of the present invention can be used to confer immunity on an individual. As used, "immunity" refers to the reduction of one or more physical symptoms associated with a response to a pathogen infection or tumor, the lymphocyte response being against said pathogen infection or tumor. The amount of cells administered is typically within the range present in a normal individual with immunity against the pathogen. Therefore, the cells are typically administered by infusion, with each infusion ranging from 2 cells to at least 10. 6 -10 10 cells / m 2 The preferred range is at least 10. 7 -10 9 cells / m 2 Clones can be administered via a single infusion or through multiple infusions over a period of time. However, because individual responses are expected to vary, the type and number of cells to be infused, as well as the number of infusions and the timeframe for multiple infusions, are determined by the attending physician and can be determined through routine examination. As illustrated herein, sufficient levels of T lymphocytes (including cytotoxic T lymphocytes and / or helper T lymphocytes) can be readily obtained using the rapid expansion method of the present invention. See, for example, U.S. Patent No. 6,040,177 to Riddell et al., column 17.

[0109] The invention is further illustrated in the following embodiments.

[0110] experiment

[0111] Example 1 - T cell transduction and CAR expression analysis

[0112] ROR1-specific CARs can be expressed in human CD8+ T cells and confer the ability to specifically recognize ROR1+ B-cell tumors rather than mature normal B cells. We constructed a ROR1-specific chimeric antigen receptor that, when expressed in T cells from healthy donors or CLL patients, confers the ability to specifically recognize primary B-CLL and mantle cell lymphoma.

[0113] Materials and methods

[0114] cell lines

[0115] As described in (25), Epstein-Barr virus (EBV)-transformed B cells (EBV-LCL) were prepared. Tumor cell lines Jeko-1 and BALL-1 were provided by Oliver Press and Dr. Jerald Radich (Fred Hutchinson Cancer Research Center). All cell lines were maintained in RPMI medium containing 10% fetal bovine serum, 0.8 mM L-glutamine, and 1% penicillin-streptomycin (LCL medium). K562 cells were obtained from the American Type Culture Collection.

[0116] K562 cells were transfected with ROR1.

[0117] For polymerase chain reaction (PCR) amplification of the ROR1 gene, total RNA was obtained from B-CLL cells (RNeasyPlusKit; QIAGEN) and reverse transcribed into cDNA using M-MLV reverse transcriptase (Invitrogen). PCR was performed using Herculase-II DNA polymerase (Stratagene) with specific primers (ROR1-F: 5-XhoIAGAGGAGGAATGCACCGGCC-3, and ROR1-R: 5-XhoI-CACAGAAGGTACTTGTTGCGATGT-3). The PCR product was cloned into the MIGR-1 retroviral vector (23), and its sequence was verified. Platinum-A cells (Cell Biolabs) containing MIGR-1 / ROR1 were transfected with Effectene transfection reagent (QIAGEN) to generate ROR1-encoded retroviruses. K562 cells were transduced and amplified by retrovirus transduction at 32°C and centrifuged at 2500 rpm for 60 min. The ROR1-positive cell subsets were then sorted and purified.

[0118] Real-time quantitative PCR

[0119] First-strand cDNAs of B-CLL, normal quiescent and activated B cells, and EBV-LCL were prepared as described in the preceding paragraphs. First-strand cDNAs from normal tissues (Human Tissue Panel I / II, Blood Fractions) were obtained from Clontech. ROR1 mRNA expression was analyzed in duplicate and normalized relative to GAPDH. Amplification was performed on an ABIpris 7900 (Applied Biosystems) using 50 μL of a reaction mixture consisting of 25 μL PowerSYBR Green PCR Master Mix (Applied Biosystems), 2.5 ng of cDNA, and 300 nM gene-specific forward and reverse primers.

[0120] ROR1-F 5-AGCGTGCGATTCAAAAGATT-3,

[0121] ROR1-R 5-GACTGGTGCCGACGATGACT-3,

[0122] GAPDH-F 5-GAAGGTGAAGGTCGGAGTC-3, and

[0123] GAPDH-R 5-GAAGATGGGTGATGGGATTTC-3.

[0124] The cycle threshold (Ct) was determined using SDS software v2.2.2 (Applied Biosystems), and gene expression levels were calculated using the comparative Ct method (2-(ΔΔCt)).

[0125] Vector construction and lentivirus preparation

[0126] The CD20-CAR (CD20R-epHIV7) and the green fluorescent protein (GFP)-encoded lentiviral vector (GFP-epHIV7) were as described previously (24). The ROR1-CAR was encoded in the same vector. A mouse mAb (clone 2A2) was prepared, cloned, and characterized in previous studies, showing specific binding to human ROR1 expressed in primary B-CLL and MCL tumor cell lines. A codon-optimized nucleotide sequence (GENEART) encoding the VL and VH chains of the scFv containing mAb 2A2 was synthesized and cloned into CD20R-epHIV7 with NheI and RsrII restriction sites to replace the CD20-specific scFv. Lentiviral viruses were prepared in 293T cells co-transfected with the lentiviral vector and packaging vectors pCHGP-2, pCMVRev2, and pCMV-G using Effectene (Qiagen). The medium was changed 16 hours after transfection, and the lentiviruses were collected 48 hours later.

[0127] Isolation of T cell clones transduced by lentivirus and CAR-transduced

[0128] PBMCs from healthy donors and B-CLL patients, as well as sorted and purified CD8+CD45RO+CD62L+ central memory T cells (TCMs) (25), were activated with anti-CD3 mAb (30 ng / mL). On days 2 and 3 post-activation, T cells were transduced in lentiviral supernatant supplemented with 1 μg / mL polybrene (Sigma-Aldrich) and 50 IU / mL recombinant human interleukin-2 (IL-2) by centrifugation at 2500 rpm for 60 min at 32 °C. T cells were expanded in RPMI (CTL medium) containing 10% human serum, 2 mM L-glutamine, and 1% penicillin-streptomycin (25). After expansion, each transduced T cell line was stained with biotin-conjugated anti-EGFR (epidermal growth factor receptor) mAb, streptavidin-PE, and anti-CD8 mAb. EGFR+CD8+ T cells were sorted, purified, and cloned using a limiting dilution method (0.5 cells / well) (25). ROR1-CAR-transduced T cells were identified by biotinylated recombinant Fc-ROR1 extracellular domain fusion protein and streptavidin-PE staining. Recombinant ROR1- protein was produced in transiently transfected 293F cells (Invitrogen), purified as described (26), and biotinylated using a BiotinTag kit (Sigma). GFP-transduced CD8+ T cells were identified by flow cytometry, sorted, purified, and cloned in a similar manner.

[0129] Chromium release and cytokine secretion assay

[0130] use 51 Cr (PerkinElmer) labeled target cells overnight, washed, and divided into triplicates at 1-2 × 10⁻⁶. 3 Cell / well ratios and varying effector cell to target cell (E:T) ratios were incubated with effector T cells. After 4 hours of incubation, the supernatant was collected for γ counting, and specific cell lysis was calculated using the standard formula (25).

[0131] result

[0132] Transduced CD8+ T cells were sorted and purified using a biotinylated anti-EGFR mAb and streptavidin-conjugated dye. ROR1-CAR expression on the surface of the sorted and purified T cells was assessed by staining with the biotinylated recombinant Fc-ROR1 extracellular domain fusion protein of scFv directly bound to ROR1-CAR, and co-staining with the streptavidin-conjugated protein. Fc-ROR1 protein-specific staining was performed on CD8+ T cells transduced with the ROR1-CAR lentiviral vector, while CD8+ T cells transduced with a control lentiviral vector encoding GFP were not stained. Figure 1 ).

[0133] We established ROR1-CAR-transduced (n=10) and control GFP-transduced CD8+ T-cell clones (n=4) using a limiting dilution method, and confirmed stable cell surface expression of CAR after multiple rounds of in vitro expansion. The growth of ROR1-CAR-transduced T-cell clones was not significantly different from that of untransduced or GFP-transduced T-cell clones (data not shown).

[0134] ROR1-CAR-transduced T-cell clones effectively lysed primary B-CLL and K562 cells stably transfected with the ROR1- gene, but not naive, ROR1-negative K562 cells, demonstrating specific recognition of ROR1. Figure 2 ).

[0135] discuss

[0136] Adoptive immunotherapy using CAR-modified T cells has been investigated in clinical trials for B-cell malignancies. The targeted surface molecules are B-cell lineage-specific and include CD19, expressed on normal B-lineage cells from progenitor B-cell stages to plasma cells, and CD20, expressed on normal B cells from pre-B-cell stages to memory B cells. Therefore, the expected effective therapeutic outcome of targeting these molecules is the reduction of normal B cells and B-cell precursors. In two independent analyses, gene expression profiling studies have identified genes preferentially or exclusively expressed by malignant rather than normal B cells, as well as ROR1, which appears as a CLL tag gene (27,28). Following inoculation with autologous tumor cells modified to express CD154, specific antibodies against ROR1 were generated in CLL patients, and lenalidomide treatment showed no significant toxicity to normal tissues, suggesting that this tumor antigen could be a suitable target for immunotherapy (29,30).

[0137] Our study demonstrates the potential to target ROR1-positive tumor cells with engineered ROR1-CAR-expressing T cells. CD8+ ROR1-CAR T cells, derived from normal donors and CLL patients, persisted for extended periods in animal models following adoptive transfer after lentiviral transduction of large PBMCs or sorted and purified TCMs (31). ROR1-CAR-transduced T cells effectively lysed primary B-CLL cells, rather than normal quiescent or activated B-cells. These T cells produced effector cytokines including TNF-α, IFNγ, and IL-2 and were able to proliferate in response to ROR1-expressing tumor cells.

[0138] Example 2 – Preparation of CD4+ CAR T cell lines and analysis of effector functions

[0139] CD4+ ROR1-CAR T cells can be generated from PBMCs of healthy donors / CLL- patients. ROR1-specific CARs can be expressed in human CD4+ T cells and confer the ability to specifically recognize ROR1+ B-cell tumors rather than mature normal B cells.

[0140] Materials and methods

[0141] cell lines

[0142] Epstein-Barr virus (EBV)-transformed B cells (EBV-LCL) were prepared as described in (25). Tumor cell lines Jeko-1 and BALL-1 were provided by Oliver Press and Dr. Jerald Radich (Fred Hutchinson Cancer Research Center). All cell lines were maintained in RPMI containing 10% fetal bovine serum, 0.8 mM L-glutamine, and 1% penicillin-streptomycin (LCL medium). K562 cells and 293T cells were obtained from the American Type Culture Collection and cultured according to its guidelines.

[0143] K562 cells were transfected with ROR1.

[0144] For polymerase chain reaction (PCR) amplification of the ROR1 gene, total RNA was obtained from B-CLL cells (RNeasyPlusKit; QIAGEN) and reverse transcribed into cDNA using M-MLV reverse transcriptase (Invitrogen). PCR was performed using Herculase-II DNA polymerase (Stratagene) with specific primers (ROR1-F: 5-XhoIAGAGGAGGAATGCACCGGCC-3, and ROR1-R: 5-XhoI-CACAGAAGGTACTTGTTGCGATGT-3). The PCR product was cloned into the MIGR-1 retroviral vector (23), and its sequence was verified. Platinum-A cells (Cell Biolabs) with MIGR-1 / ROR1 were transfected with Effectene transfection reagent (QIAGEN) to generate ROR1-encoded retroviruses. K562 cells were transduced and amplified by retrovirus transduction at 32℃ and 2500 rpm for 60 min, and then ROR1-positive cell subsets were sorted and purified.

[0145] Vector construction and lentivirus preparation

[0146] The CD20-CAR (CD20R-epHIV7) and the green fluorescent protein (GFP)-encoded lentiviral vector (GFP-epHIV7) were as previously described (24). The ROR1-CAR was encoded in the same vector. A mouse mAb (clone 2A2) had been prepared, cloned, and characterized in previous studies, showing specific binding to human ROR1 expressed in primary B-CLL and MCL tumor cell lines. A codon-optimized nucleotide sequence (GENEART) encoding the VL and VH chains of the scFv containing mAb 2A2 was synthesized and cloned into CD20R-epHIV7 with NheI and RsrII restriction sites to replace the CD20-specific scFv. Lentiviral vectors were prepared in 293T cells by co-transfection with packaging vectors pCHGP-2, pCMVRev2, and pCMV-G using Effectene (Qiagen). The medium was changed 16 h after transfection, and the lentiviruses were collected 48 h later.

[0147] Lentiviral transduction and isolation of CD4+ ROR1-CAR T cell lines

[0148] CD4+ T cells were isolated from PBMCs from healthy donors and activated with anti-CD3 mAb (30 ng / mL) (25). On days 2 and 3 post-activation, transduction was performed in lentiviral supernatant supplemented with 1 μg / mL polybrene (Sigma-Aldrich) and 50 IU / mL recombinant human interleukin-2 (IL-2) by centrifugation at 2500 rpm for 60 min at 32 °C. T cells were expanded in RPMI (CTL medium) containing 10% human serum, 2 mL M glutamine, and 1% penicillin-streptomycin (25). After expansion, each transduced T cell line was stained with biotin-conjugated anti-EGFR (epidermal growth factor receptor) mAb, streptavidin-PE, and anti-CD4 mAb. EGFR+CD4+ T cells were sorted, purified, and expanded. ROR1-CAR-transduced T cells were identified using biotinylated recombinant Fc-ROR1 extracellular domain fusion protein and streptavidin-PE staining. Recombinant ROR1 protein was produced in transiently transfected 293 cells (Invitrogen), purified as described (26), and biotinylated using a BiotinTag kit (Sigma). GFP-transduced CD4+ T cells were identified by flow cytometry, sorted, purified, and cloned in a similar manner.

[0149] Chromium release and cytokine secretion assay

[0150] use 51 Cr (PerkinElmer) labeled target cells overnight, washed, and divided into triplicates at 1-2 × 10⁻⁶. 3Cells / wells and effector cells to target cells (E:T) ratios were incubated with effector T cells. After 4 hours of incubation, the supernatant was collected for γ counting, and specific cell lysis was calculated using the standard formula (25). For cytokine secretion analysis, target cells and effector cells were seeded in three replicate wells at a 2:1 E / T ratio, and after 24 hours of incubation, interferon INFγ, tumor necrosis factor (TNF-α), and IL-2 were measured in the removed supernatant using a multiplex cytokine immunoassay (Luminex).

[0151] CFSE proliferation assay

[0152] T cells were labeled with 0.2 μM carboxyfluorescein succinimide (CFSE; Invitrogen), washed, and seeded at a 2:1 ratio with stimulator cells in CTL medium containing 10 U / mL recombinant human IL-2. After 72 hours of incubation, cells were labeled with anti-CD4 mAb and propidium iodide (PI) to exclude dead cells from the analysis. Samples were analyzed by flow cytometry, and cell division of viable CD4+ T cells was assessed by CFSE dilution.

[0153] Co-culture detection

[0154] ROR1-CAR-transfected CD4+ T cells and ROR1-CAR-transduced CD8+ cytotoxic T lymphocytes were co-cultured at ratios of 2:1, 1:1, and 1:2, labeled with CFSE. The co-cultures were then stimulated with K562 / ROR1 cells and control K562 cells, and cell proliferation was measured by CFSE staining dilution assay after 5 days of incubation. For flow cytometry analysis, samples were stained with conjugated anti-CD8 and anti-CD4 mAbs to differentiate between CD8+ and CD4+ subsets.

[0155] result

[0156] CD4 was prepared from PBMCs of healthy donors and CLL patients. + ROR1-CAR T cells

[0157] We have shown that the carcinoembryonic tyrosine kinase receptor ROR1 is homogeneously expressed on both CLL and MCL, and that ROR1-CAR is generated by anti-ROR1 mAb when it is present on CD8. + When expressed in T cells, it confers the ability to specifically recognize malignant rather than mature normal B cells (32). In this invention, we prepared CD4 + ROR1-CAR T cells were used to analyze direct tumor recognition and their enhancement of CD8. +The ability to generate ROR1-CAR CTLs. Using a lentiviral vector encoding ROR1-CAR, it is easily generated from large peripheral CD4 cells from healthy donors (n=4) and CLL patients (n=4). + T cell preparation of CAR-modified CD4 + T cells. In this vector, we encoded a truncated EGFR (epidermal growth factor receptor, tEGFR) domain downstream of ROR1-CAR and a self-splicing 2A element, as a transduction marker and for the enrichment of transgenic T cells using anti-EGFR mAb. Figure 3 Using the tEGFR biomarker, we determined the frequency of CAR-modified T cells on day 12 following a single transduction with a lentivirus encoding ROR1-CAR (MOI=3), and found that compared to CD8+ cells from the same individual... + CAR T cell lines, in CD4 + ROR1-CAR consistently exhibits higher transduction efficiency in CD4. To demonstrate that ROR1-CAR in CD4... + On the surface of T cells, we utilized biotinylated recombinant Fc-ROR1 extracellular domain fusion protein, which directly binds to the scFv of ROR1-CAR and specifically stains CD4+ transduced with ROR1-CAR lentivirus. + T cells instead of untransduced control CD4 + T cells ( Figure 3 We enriched CD4+ expressing transgenes using tEGFR markers. + T cells were used, and a CAR-positive T cell subset was expanded by stimulation with anti-CD3 mAb. More than 3 logs of CD4 were achieved at the end of the 14-day stimulation cycle. + CAR T cells, which are associated with CD8 + The amplification observed in CAR CTLs was comparable. Following amplification, we confirmed the presence of ROR1-CAR on CD4. + Stable expression of CAR T cells on the surface (data not shown), and analysis of the recognition of ROR1-positive tumor cells.

[0158] CD4 + ROR1-CAR T cells specifically recognize ROR1-positive tumors.

[0159] We analyzed CD4 + ROR1-CAR T cell effector function against ROR1-positive primary tumor cells and tumor cell lines. We analyzed CD4+ using a chromium release assay (CRA). +CAR T cells conferred direct cytotoxicity, and weak but specific cell lysis of ROR1-positive target cells was detected at the end of the standard 4-hour incubation (Figure 4). We extended the CRA to 10 hours and observed a further increase in specific cell lysis; however, the overall cytolytic activity of CD4+ CAR T cells was still lower than that of CD8+ CAR T cells. + ROR1-CAR CTL( Figure 2 4). CD4 from healthy donors and CLL patients was measured by IFN-γ ELISA. + ROR1-CAR T cells specifically recognized primary CLL cells, ROR1-positive tumor cell lines Jeko-1 (MCL) and BALL-1 (B-ALL), as well as K562 cells stably transfected with the ROR1-gene (K562 / ROR1), but did not recognize naively ROR1-negative K562 cells, demonstrating specific recognition of ROR1 on the cell surface of target cells. Figure 5A Multiple cytokine analysis revealed the production of [a substance] compared to CD8. + CAR CTLs showed significantly higher levels of other Th1 cytokines such as TNF-α and IL-2, and produced IL-4, IL-10, and IL-17. Figure 5B ).

[0160] Next, we evaluated CD4 counts after stimulation with ROR1-positive tumor cells using CFSE staining. + CAR T cell proliferation was achieved using strict culture conditions without the addition of exogenous cytokines to remove any potential nonspecific stimuli. CD4 + CAR T cells showed marked and specific proliferation in response to ROR1-positive tumor cells. The percentage of T cells that proliferated after induction and the number of cell divisions in the proliferating subsets were correlated with CD4+. + The average value is significantly higher than CD8. + CAR T cells ( Figure 6 In summary, our data indicate that CD4 from healthy donors and CLL patients... + T cells, after genetic modification with ROR1-specific CAR, acquired anti-tumor reactivity. Furthermore, their ability to proliferate and produce high levels of Th1 cytokines under conditions without exogenous cytokines suggests that CAR stimulation enhances CD4+ cytokine production. + CAR T cells exhibit typical helper functions, not only conferring direct anti-tumor effects but also enhancing CD8+. + CAR CTL.

[0161] CAR-modified but untransduced CD4 + T cells are CD8+ CAR CTL provides assistance

[0162] To analyze CD4 + Can CAR T cells be used for CD8? + CAR CTLs provide assistance; we use CAR-transduced and control-untransduced polyclonal CD4 cells established from healthy donors and CLL patients. + and CD8 + T cell lines were co-cultured. As a helpful data readout, we defined the difference between CD8 cells cultured alone and those cultured alone. + T cells, in CD4 + Tumor-specific CD8 in the presence of T cells + Improvement in effector function. We compared CAR-transduced or untransduced control CD4 cells. + T cells and CD8 + CAR CTLs were stimulated with ROR1-positive tumor cells at different CD4:CD8 ratios (2:1, 1:1, 1:2) and their proliferation was measured using a CFSE dye dilution method. We found that CAR-transduced, rather than untransduced, CD4+ CTLs... + T cells added to CD8 + CAR CTL, compared to CD8 alone + CAR CTL significantly increased CD8 + Subpopulation-specific proliferation ( Figure 7 When at least an equal amount of CD4 + The most significant increase in proliferation was observed when CAR T cells (CD4:CD8 ratio of 2:1 or 1:1) were added to the co-culture. The addition of untransduced CD4+ cells further enhanced proliferation. + and untransduced CD8 + The T-cell combination served as an additional control in CD8. + No nonspecific proliferation was induced in the subpopulation (data not shown).

[0163] discuss

[0164] In two independent analyses, gene expression profiling studies identified genes preferentially or exclusively expressed by malignant rather than normal B cells, as well as ROR1, which appears as a CLL tag gene (27,28). Our study demonstrates the potential to target ROR1-positive malignant cells with engineered ROR1-CAR-expressing T cells. CD8 and CD4+ ROR1-CAR T cells can be derived from large PBMCs or normal donors after lentiviral transduction of sorted and purified T cells. CD8+ ROR1-CAR-transduced T cells effectively lysed primary B-CLL cells rather than normal quiescent or activated B-cells. CD4+ ROR1-CAR-transduced T cells weakly lysed primary B-CLL cells rather than normal quiescent or activated B-cells. These T cells produced effector cytokines including TNF-α, IFNγ, IL-2, IL-4, and IL-10. CAR-transduced CD4+ T cells produced significantly higher levels of cytokines than transduced CD8+ cells. Both cell types were able to proliferate in response to ROR1-expressing tumor cells. Furthermore, CD4+ ROR1-CAR T cells proliferated 2-3 times more than CD8+ ROR1-CAR CTLs. These results indicate that transduced CD4+ helper T cells exhibit typical helper function, meaning they can be used to enhance CD8+ CAR CTLs.

[0165] Example 3 - Effector Function of CD4+ ROR1-CAR T Cells Derived from Primary, Central, and Effector Memory Cell Subpopulations

[0166] Effector functions of CD4T cells derived from primary, central, and effector memory cell subsets and then modified with ROR1 CAR were compared.

[0167] Materials and methods

[0168] Sorting and purification of primary, central, and effector memory CD4 cells

[0169] CD4+ T cells were isolated from PBMCs of healthy donors using negative magnetic bead selection (Miltenyi CD4 sorting kit), resulting in untouched CD4+ T cells. The CD4+ fractions were labeled with conjugated anti-CD45RA, anti-CD45RO, and anti-CD62L mAbs, purified by flow cytometry using a FACSAria flow cytometer (BD Biosciences), and purified based on the expression of these defined markers: naïve (CD45RA+CD45RO-CD62L+), central memory (CD45RA-CD45RO+CD62L+), and effector memory (CD45RA-CD45RO+CD62L-) CD4+ T cells.

[0170] CFSE proliferation assay

[0171] T cells were labeled with 0.2 μM carboxyfluorescein succinimide (CFSE; Invitrogen), washed, and seeded with stimulator cells at a 2:1 ratio in CTL medium containing 10 U / mL recombinant human IL-2. After 72 hours of incubation, cells were labeled with anti-CD8 or CD4 mAb and propidium iodide (PI) to exclude dead cells from the analysis. Samples were analyzed by flow cytometry, and cell division of viable CD8+ and CD4+ T cells was assessed using the CFSE dilution method.

[0172] Cytokine detection

[0173] To analyze cytokine secretion, target cells and effector cells were seeded in triplicate wells at an E / T ratio of 2:1. After incubation for 24 hours, the supernatant was removed and interferon INFγ, tumor necrosis factor (TNF-α), and IL-2 were measured using a multiplex cytokine immunoassay (Luminex).

[0174] result

[0175] We purified CD4+ from peripheral blood of three healthy donors using flow cytometry based on the expression of CD45RA, CD45RO, and CD62L. + N, central (CM) and effect memory (EM) CD4 + T cells ( Figure 8A We compared their effector functions after modification with ROR1-CAR. Similar high transduction efficiencies were obtained in CAR T cell lines derived from each of the three cell subpopulations. After enrichment of transgenic T cells, multiparameter flow cytometry showed increased transduction efficiency in CD4+ cells. + The expression of CD45RO and the absence of CD45RA in the NCAR T cell line are consistent with the phenotype of activation after lentiviral transduction. + The N, CM, and EM CAR T cell lines maintained differential CD62L expression, confirming that the initial flow cytometry-based purification was completed with high purity.

[0176] Then, we analyzed CD4 derived from N, CM, and EM cell subsets. + Tumor recognition, cytokine secretion, and proliferation of CAR T cells were observed and compared with CAR T cell lines derived from large CD4+ T cells. Specific recognition of ROR1-positive tumor cells was observed in each cell line using IFN-γ ELISA. Multiplex cytokine analysis revealed the presence of CD4+ cells derived from N cell subsets. +CAR T cells produced the highest levels of Th1 cytokines to date, especially IL-2 ( Figure 8C Furthermore, CFSE dye dilution assays showed that they exhibited the most vigorous proliferation in response to stimulation by ROR1-positive tumor cells. Figure 8B ).

[0177] discuss

[0178] Our study demonstrates the potential for targeting ROR1-positive malignant cells with modified T cells expressing ROR1-CAR. CD8 and CD4+ ROR1-CAR T cells can be derived from normal donors after lentiviral transduction of any large PBMC, as well as from sorted and purified T cells from defined naive or memory T cell subsets. Effector cytokines produced by CD4+ naive, central memory, and effector T cells include TNFα, IFNγ, IL-2, IL-4, and IL-10. Following CAR signal transduction, CD4+ CAR-transduced cells derived from the naive cell subset produced significantly higher levels of TNFα and IL-2 than CD4+ CAR T cells derived from central and effector memory cell subsets. All CD4 cell types responded to ROR1 / K562 and proliferated; however, in CD4+ CAR-transduced cells derived from the naive cell subset, the percentage of induced proliferating T cells and the number of divisions of the proliferating cell subset were significantly higher. Both cytokine profiles and proliferation capacity suggest that naïve CD4+ROR1-CAR T cells may be best suited to enhance CD8+ROR1-CAR CTLs.

[0179] Example 4 - Naïve CD4+ T cells are more beneficial than memory CD4+ T cells

[0180] Initial, central memory, and effector transduced CD4+ T cells were co-cultured with transduced CD8+ cytotoxic T lymphocytes, and the proliferative response of the cells to K562 / ROR1 cell stimulation was measured.

[0181] Materials and methods

[0182] Co-cultivation

[0183] CD4+ T cells transduced with ROR1-CAR derived from naïve, central, and effector memory cells, and CD8+ cytotoxic T lymphocytes transduced with ROR1-CAR derived from naïve and central memory CD8+ T cells were labeled with CFSE, and CD4+ and CD8+ CAR-T cell lines were co-cultured at a 1:1 ratio. The co-cultures were then stimulated with K562 / ROR1 cells and control K562 cells, and cell proliferation was assessed by CFSE dye dilution assay after 5 days of incubation. For flow cytometry analysis, samples were stained with conjugated anti-CD8 and anti-CD4 mAbs to differentiate between CD8+ and CD4+ subsets.

[0184] result

[0185] CD4 + Initial CAR T cells enhance CD8 + CAR CTLs have excellent effects function.

[0186] We compared CD4 + The helper function of N, CM, and EM CAR T cell lines was investigated to determine whether CD4 was involved. + The favorable cytokine profile and proliferative potential of NCAR T cells can also translate into resistance to CD8+. + The strongest assistive effect of CAR CTL. Previous work has demonstrated that N, CM, and EM CD8 + There are intrinsic differences among T cells that influence their potential utility in adoptive immunotherapy. Our group recently revealed that CD8 cells derived from CM rather than EM after adoptive transfer... + T cells can persist for a long time, making them a preferred choice for CD8+ immunotherapy. + T cell subsets (33,34). Other groups have suggested CD8... + NT cells may also possess desirable properties for T cell therapy (35,36). Therefore, we prepared CD8 from sorted and purified N and CM T cells. + CAR CTL to determine CD8+ and CD4 + The optimal combination of CAR T cell subsets. In lentiviral transduction and enrichment of CAR-transduced CD8 cells with tEGFR markers. + After T cells, we confirmed CD8 + Tumor reactivity of N and CM CAR CTLs (data not shown), and as before with CD4 + CAR T cells were co-cultured. As expected, compared to CD4... + Co-culture of CM or EMCAR T cells, or CD8 cells alone + CAR CTL, CD8 + N and CM CAR CTL with CD4 + Co-culture of N CAR T cells led to CD8 + Significantly elevated tumor-specific proliferation in subgroups ( Figure 9 Of all the combinations, CD4 + NCAR T cells and CD8 + After co-culturing with CMCAR CTLs, the maximum CD8+ production was observed in response to stimulation by ROR1-positive tumor cells. + CARCTL proliferation ( Figure 9In summary, our data indicate that N, CM, and EM CD4 + T cells exhibit intrinsic differences in cytokine profiles and proliferative potential, CD4 + NT cells produce higher levels of IL-2 and exhibit superior proliferation. Our data show that sorted and purified NT cells, rather than CM, EM, or large CD4 cells, produce higher levels of IL-2. + T cells may be best suited to enhancing CD8. + The effector function of CTL complements the previous CD8 + The function of T cells, namely CD8 derived from CM + T cells have desirable properties for adoptive immunotherapy.

[0187] discuss

[0188] In summary, these data indicate that ROR1-CAR-modified CD4 + and CD8 + Adoptive transfer of T cells conferred an effective anti-tumor response in an in vivo model of aggressive systemic lymphoma, providing CD4+. + CAR T cells for CD8 + Evidence of the beneficial and synergistic effects of CAR CTL antitumor efficacy. Our data illustrate how intrinsic cellular characterization can reveal the effects on tumor-specific CD8+ and CD4+ cells. + The rational design of T cell products can improve the effectiveness of cancer immunotherapy.

[0189] Example 5 - Mouse tumor model of systemic mantle cell lymphoma (NSG / Jeko-1-ffLuc)

[0190] We tested ROR1-CAR-modified CD8 in an in vivo model of aggressive systemic mantle cell lymphoma. + CTLs provide CD4-assisted antitumor effects.

[0191] Materials and methods

[0192] 5x10 cells that have been stably transfected with firefly luciferase (Jeko-1 / ffLuc) 5 Jeko-1 cells were transplanted via tail vein into sublethal doses of irradiated NOD / SCID / γ-ray cells. - / - (NSG) mice were used to assess tumor burden and distribution using bioluminescence imaging. We confirmed the development of consistent grafts (take rate = 100%) and rapidly progressing disseminated lymphoma in NSG mice under these conditions. Following tumor transplantation, groups of three mice received CD8+ via tail vein injection. + CARCTL (Group 1), CD4+ CAR T cells (group 2), CD8+ and CD4 + Combinations of ROR1-CAR-transduced T cells (group 3), untransduced control T cells (groups 4, 5, and 6), or untreated group (group 7). In all cases, the total number of transduced T cells was 10 x 10^6. 6 Two days after adoptive transfer, we obtained ocular blood from mice and confirmed the presence of ROR1-CAR-transduced or untransduced T cells in the peripheral blood.

[0193] result

[0194] On day 6 after T-cell metastasis, we performed bioluminescence imaging to assess tumor burden. After receiving CD8+ and CD4+ treatment... + The combination of ROR1-CAR T cells showed the strongest anti-tumor effect in mice, with a >2 log reduction in bioluminescent signal compared to the control group. Figure 10 We are still accepting CD8. + or CD4 + A strong anti-tumor effect was observed in mice with ROR1-CAR-modified T cells, with a >1 log reduction in bioluminescence signal compared to the control group. Figure 10 Importantly, the application of CD8 + / CD4 + The tumor burden reduction after CAR T-cell combination therapy was greater than that after CD8 administration. + CAR CTL and CD4 + The total tumor burden reduction of CAR T cells indicates that CD4 + CAR T cells and CD8 + CAR CTL produced a synergistic effect.

[0195] discuss

[0196] In summary, these data indicate that ROR1-CAR-modified CD4 + and CD8 + Adoptive transfer of T cells conferred an effective anti-tumor response in an in vivo model of aggressive systemic lymphoma and provided CD4+. + CAR T cells for CD8 + Evidence suggests that CAR CTLs possess beneficial and synergistic antitumor efficacy. Our data illustrate how intrinsic cellular characterization can reveal the effects of tumor-specific CD8+ and CD4+. + The rational design of T cell products can improve the effectiveness of cancer immunotherapy.

[0197] Example 6 - CD19CAR T cells exhibited the same synergistic effect

[0198] In co-cultures of aggressive, systemic mantle cell lymphoma models in vitro and in vivo, we detected CD8+ providing CD4+-CD19 modification. + The beneficial effects of CTL's anti-tumor efficacy.

[0199] Materials and methods

[0200] The preparation of CD19CAR T cells can be described as in US 2008 / 0131415, which is incorporated herein by reference.

[0201] Co-culture detection

[0202] CD19-CAR-transduced CD4+ T cells and CD19-CAR-transduced CD8+ cytotoxic T lymphocytes were labeled with CFSE and co-cultured at ratios of 2:1, 1:1, and 1:2. The co-cultures were then stimulated with K562 / ROR1 cells and control K562 cells, and cell proliferation was measured by CFSE dye dilution assay after 5 days of incubation. For flow cytometry analysis, samples were stained with conjugated anti-CD8 and anti-CD4 mAbs to distinguish between CD8+ and CD4+ subsets.

[0203] in vivo model

[0204] 5x10 cells that have been stably transfected with firefly luciferase (Jeko-1 / ffLuc) 5 Jeko-1 cells were transplanted via tail vein into sublethal doses of irradiated NOD / SCID / γ-ray cells. - / - (NSG) mice were used to assess tumor burden and distribution using bioluminescence imaging. We confirmed the development of consistent grafts (acceptance rate = 100%) and rapidly invasive disseminated lymphoma in NSG mice under these conditions. Following tumor transplantation, groups of three mice received CD8+ via tail vein injection. + CD19CARCTL (Group 1), CD4 + CD19CAR T cells (group 2), CD8+ and CD4 + Combinations of CD19CAR-transduced T cells (Group 3), untransduced control T cells (Groups 4, 5, and 6), or untreated group (Group 7). In all cases, the total number of transduced T cells was 10 x 10^6. 6 Two days after the adoption, we obtained ocular blood from the mice.

[0205] result

[0206] Figure 10This study demonstrated that, in co-culture experiments with CD8+CD19-CAR CTLs and CD4+CD19-CAR T-cell lines stimulated with the CD19+ mantle cell lymphoma tumor line Jeko-1, the CD4+CAR T-cell line derived from the initial subset exhibited superior ability to enhance tumor-specific proliferation of central memory-derived CD8+CAR CTLs, although the enhancement of tumor-specific proliferation of central memory-derived CD8+CAR CTLs by CD4+CAR T-cell lines derived from either the central or effector memory cell subsets was much less pronounced.

[0207] Figure 11 shows that CD8+ CAR T cells and CD4+ CAR T cells independently conferred direct antitumor efficacy in an immunodeficient mouse (NOD / SCID-Raji) lymphoma model. Mice received CD19-CAR-transduced or control-mock-transduced CD8+ central memory-derived T cells (A), or CD19-CAR-transduced or control-mock-transduced CD4+ naïve-derived T cells (B).

[0208] Figure 12 This study demonstrates the enhanced and synergistic effect of CD4+ ROR1-CAR-modified T cells on the antitumor efficacy of CD8+ ROR1-CAR CTLs in a mouse model of systemic mantle cell lymphoma (NSG / Jeko-1-ffLuc). In the mouse model of systemic invasive mantle cell lymphoma (NSG / Jeko-1), the antitumor efficacy of ROR1-CAR-modified CD8+ and CD4+ T cells was enhanced compared to either the isolated cell population or untransduced cells.

[0209] Figure 13 illustrates the synergistic effect of CD8+ and CD4+CD19-CAR T cells in a mouse model of systemic lymphoma (NSG / Raji). Bioluminescence imaging confirmed the transplantation of Raji tumors on day 6 post-inoculation (before treatment) (treatment protocol shown in Figure A, tumors as shown by bioluminescence in Figure B). Bioluminescence imaging analysis of tumor burden in mice treated with CD8+CD19-CAR T cells in the same batch, and in mice treated with a combination of CD8+ and CD4+CD19-CAR T-cell products (the black and gray bars in the middle after treatment, Figure B), showed complete eradication of the Raji tumors. The mice were then challenged with a second inoculation with Raji tumor cells, and the frequencies of CD4+ and CD8+ CAR T cells in peripheral blood and tumor transplantation were analyzed. In mice treated with a combination of CD8+ and CD4+ CAR T-cell products, tumor stimulation resulted in significantly elevated levels of CD8+ CAR T-cells (lower part of Figure D), and complete rejection of Raji inoculum (grey bar on the right after tumor stimulation, Figure B). Conversely, in mice receiving CD8+CD19-CAR CTLs alone, no increase in CAR T-cells was detected after tumor stimulation (Figure C), and Raji tumor cells were able to be transplanted (black bar on the right after tumor stimulation, Figure B).

[0210] discuss

[0211] In summary, these data indicate that using alternative CAR constructs, CD19-transduced cells, and CD19-CAR-modified CD4 cells... + and CD8 + T cells conferred an effective anti-tumor response in an in vivo model of aggressive systemic lymphoma and provided CD4+. + CART cells for CD8 + Evidence of the beneficial and synergistic effects of CAR CTL in anti-tumor efficacy.

[0212] The invention has been described above, but this should not be construed as limiting the invention. The invention is defined by the appended claims and their equivalents, which are included therein. All references and documents cited herein are incorporated by reference.

[0213] References

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Claims

1. An adoptive cellular immunotherapy composition comprising chimeric antigen receptor modified CD4+ T lymphocytes and chimeric antigen receptor modified CD8+ T lymphocytes, wherein: a) the chimeric antigen receptor modified CD4+ T lymphocytes of the composition consist of CD4+ helper T lymphocytes from an enriched population of naive CD4+ helper T lymphocytes from an individual that contain a chimeric antigen receptor comprising an extracellular antibody variable domain specific for an antigen associated with a disease or condition and an intracellular signaling domain; and b) the chimeric antigen receptor modified CD8+ T lymphocytes of the composition consist of CD8+ cytotoxic T lymphocytes from an enriched population of central memory CD8+ T cells from an individual that contain a chimeric antigen receptor comprising an extracellular antibody variable domain specific for an antigen associated with a disease or condition and an intracellular signaling domain.

2. The adoptive cellular immunotherapy composition of claim 1, wherein the chimeric antigen receptor modified CD4+ helper T lymphocytes are derived from CD45RO negative, CD62L positive CD4 positive T cells.

3. The adoptive cellular immunotherapy composition of claim 1, wherein the enriched population of central memory CD8+ T cells comprises CD45RO+, CD62L+, CD8+ T cells.

4. The adoptive cellular immunotherapy composition of any one of claims 1-3, wherein the disease or condition is a hematological malignancy.

5. The adoptive cellular immunotherapy composition of any one of claims 1-3, wherein the antigen associated with a disease or condition is a tumor associated cell surface antigen.

6. The adoptive cellular immunotherapy composition of any one of claims 1-3, wherein the antigen associated with a disease or condition is selected from the group consisting of tyrosine kinase orphan receptor ROR1, CD19, CD20 and CD22.

7. The adoptive cellular immunotherapy composition of any one of claims 1-3, wherein the chimeric antigen receptor comprises a single chain antibody derived chimeric antigen receptor.

8. The adoptive cellular immunotherapy composition of any one of claims 1-3, wherein the intracellular signaling module of the T cell receptor of the chimeric antigen receptor comprises a transmembrane domain, a CD28 signaling domain and a CD3 intracellular signaling domain or other domain of a T cell costimulatory molecule.

9. The adoptive cellular immunotherapy composition of any one of claims 1-3, wherein the intracellular signaling domain comprises a CD28 transmembrane and signaling domain linked to a CD3 intracellular domain.

10. The adoptive cellular immunotherapy composition of any one of claims 1-3, wherein the intracellular signaling domain of the enriched population of central memory CD8+ T cells of the chimeric antigen receptor modified CD8+ cytotoxic T lymphocytes is the same as the intracellular signaling domain of the chimeric antigen receptor modified CD4+ helper T lymphocytes. ​ ​ 11. The adoptive cellular immunotherapy composition of any one of claims 1-3, wherein the endodomain of the enriched population of central memory CD8+ T cells of the chimeric antigen receptor modified CD8+ cytotoxic T lymphocytes is different from the endodomain of the chimeric antigen receptor modified CD4+ helper T lymphocytes.

12. The adoptive cellular immunotherapy composition of any one of claims 1-3, wherein both the enriched population of central memory CD8+ T cells of the chimeric antigen receptor modified CD8+ cytotoxic T lymphocytes and the chimeric antigen receptor modified CD4+ helper T lymphocytes are genetically modified with an antibody heavy chain domain that specifically binds to a tumor specific cell surface antigen, wherein the tumor is associated with a hematological malignancy.

13. The adoptive cellular immunotherapy composition of any one of claims 1-3, wherein the endodomain of the chimeric antigen receptor comprises a CD28 costimulatory domain and a CD3 endodomain, or a 4-1 BB costimulatory domain and a CD3 endodomain.

14. The adoptive cellular immunotherapy composition of any one of claims 1-3, wherein the chimeric antigen receptor modified CD4+ helper T lymphocytes are capable of eliciting direct tumor recognition and enhancing the ability of the chimeric antigen receptor modified CD8+ cytotoxic T lymphocytes to mediate cellular immune responses.

15. Use of the adoptive cellular immunotherapy composition of any one of claims 1-14 for the manufacture of a medicament for the treatment of a hematological malignancy.

16. The use of claim 15, wherein the hematological malignancy is selected from the group consisting of B-cell lymphoma, leukemia, and myeloma.

17. The use of claim 15 or 16, wherein the chimeric antigen receptor modified CD4+ helper T lymphocytes are capable of eliciting direct tumor recognition and enhancing the ability of the chimeric antigen receptor modified CD8+ cytotoxic T lymphocytes to mediate cellular immune responses.

18. The use of claim 15, wherein the hematological malignancy is chronic lymphocytic leukemia or mantle cell lymphoma.

19. The use of claim 15, wherein a biological sample is analyzed for the presence of the antigen associated with the hematological malignancy and the chimeric antigen receptor specifically binds to the antigen.

20. The use of claim 15, wherein the antigen associated with the hematological malignancy is selected from the group consisting of tyrosine kinase orphan receptor ROR1, CD19, CD20, and CD22.

21. The use of claim 15, wherein both the enriched population of central memory CD8+ T cells of the chimeric antigen receptor modified CD8+ cytotoxic T lymphocytes and the chimeric antigen receptor modified CD4+ helper T lymphocytes are genetically modified with an antibody heavy chain domain that specifically binds to a tumor specific cell surface antigen, wherein the tumor is associated with a hematological malignancy.

22. A method of making the adoptive cellular immunotherapy composition of any one of claims 1-14, comprising: (a) separately expanding an enriched population of central memory CD8+ T lymphocytes and an enriched population of naive CD4+ T helper lymphocytes from an individual in vitro; (b) separately modifying the expanded CD8+ T lymphocytes and the expanded CD4+ helper T lymphocytes by introducing a chimeric antigen receptor comprising an extracellular antibody variable domain specific for an antigen associated with a disease or condition and an intracellular signaling domain; and (c) optionally combining the modified CD8+ T lymphocytes and the modified CD4+ T lymphocytes to produce the adoptive immunotherapy composition.

23. The method of claim 22, wherein the chimeric antigen receptor modified CD4+ helper T lymphocytes are capable of eliciting direct tumor recognition and enhancing the ability of the chimeric antigen receptor modified CD8+ cytotoxic T lymphocytes to mediate cellular immune responses.

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