JPWO2022153295A5 - - Google Patents

Description

The present invention is directed to the field of immunotherapy. Specifically, the present invention provides improved cell compositions and methods for adoptive cell therapy useful in the treatment of cancer.

Adoptive cell transfer (ACT, also known as adoptive cell therapy) is one of the most promising strategies in cancer immunotherapy and has proven effective against various types of cancer, including melanoma, cervical cancer, lymphoma, leukemia, cholangiocarcinoma, and neuroblastoma.

ACT is a cellular therapy that attempts to enhance the anti-tumor activity of immune cells. It is based on immune cells extracted from the subject, usually treated ex vivo, expanded on a large scale, and then either returned to the patient (autologous therapy) or transplanted into another individual (allogeneic therapy). A variety of immune cells can be used in ACT, including T lymphocytes (T cells), natural killer (NK) cells, dendritic cells, and stem cells.

Tumor infiltrating lymphocytes (TILs) are tumor-specific T cells isolated from patient tumor biopsies and expanded ex vivo to produce ACT compositions. For this purpose, it has been suggested to expand TILs ex vivo using high concentrations of IL-2, anti-CD3 antibodies and irradiated alloreactive feeder cells, supplemented with IL-2 (but not anti-CD3) on day 5. The cells obtained after 8-15 days of expansion are then returned to the patient with supportive IL-2 administration. However, the production of TIL compositions for ACT is technically challenging and the use of IL-2 and feeder cells introduces undesirable mutations. Furthermore, this approach is limited by the availability of tumor samples and the small number of initial TILs that are usually obtained from each sample.

Another source that can be used for the production of ACT compositions is peripheral blood mononuclear cells (PBMCs), from which lymphocyte populations can be isolated, expanded, and/or manipulated by various procedures to enhance their efficacy. Unlike T cells, which need to be autologous or histocompatible with the subject to be treated in order to exert T cell receptor (TCR)-mediated major histocompatibility complex (MHC)-II-dependent cytotoxicity, other lymphocyte populations, such as NK cells (characterized by expression of CD56 or CD16 and lack of TCR-CD3 complex) and NK-T cells, act in a non-MHCII-restricted manner. However, such populations are usually characterized by limited efficacy and require large doses to demonstrate clinical efficacy.

For example, lymphokine-activated killer (LAK) cells, which are composed of various lymphocyte types, including natural killer (NK) cell precursors and T lymphocytes, are produced by culturing PBMCs in the presence of cytokines, usually IL-2. Such heterogeneous cell compositions exhibit HLA-independent lysis of tumor cells in vitro after incubation with IL-2. Although LAK cells are highly effective in mediating tumor regression in certain individuals, this therapeutic approach can be difficult to implement due to the large number of cells required to mediate cancer regression. Furthermore, the high doses of IL-2 required mediate toxic side effects, the most common of which is capillary permeability leak syndrome, which causes significant fluid retention.

Cytokine-induced killer (CIK) cells are non-MHC-restricted cytotoxic antitumor cells expanded in vitro from circulating precursors. CIK cells share characteristics of both T and NK cells. The expansion protocol for CIK cell production starting from PBMC, lymphodepleted or umbilical cord blood requires the sequential addition of 1000 U/mL human recombinant human interferon-gamma (rIFN-γ) on day 0, followed by the addition of 50 ng/mL of a monoclonal antibody against CD3 (OKT3) and 500 IU/mL of IL-2 on day +1. IL-2 and fresh complete X-VIVO medium (no anti-CD3 antibody) are added every 5 days for 14-21 days. At the end of this method, the production of two major cell populations has been reported. One population contains CD3, CD8, NKG2D, and CD56 positive cells (usually referred to as CIK), and the other population contains CD3, CD8, and NKG2D positive cells and CD56 negative cells, and has been reported to lack cytotoxic activity (Introna et al, Int. J. Mol. Sci. 2018, 19, 358).

Despite the relative safety profile of CIK, its use in clinical practice has led to extreme heterogeneity regarding efficacy. Results from 11 clinical trials investigating the antitumor effect of CIK cells in 426 treated patients showed that of 384 (90.1%) who reported responses, only 24 (5.6%) had a complete response, 27 (6.3%) had a partial response (PR), and 40 (9.4%) had a minor response. Meanwhile, 161 (37.8%) patients had stable disease and 129 (30.3%) had progressive disease, indicating that the overall efficacy of CIK cell therapy is moderate (Introna et al., ibid.).

Protocols for the isolation and/or ex vivo expansion of various cell compositions for ACT are disclosed. Typically, such protocols often include an initial stimulation in the presence of cytokines and/or other stimuli such as TCR/CD3 activators, and expansion or propagation in the presence of different combinations of such cytokines and activators. Furthermore, such protocols may include additional steps of isolating or enriching the desired cell population, for example, by apheresis or flow cytometry or bead-based methods targeting the corresponding surface markers, either before or after expansion.

For example, US2020138861 discloses an adoptive transfer procedure for treating inflammatory bowel disease, which involves isolating CD3-negative and CD56-positive NK cells from the peripheral blood of a donor. US10149863 discloses a method for preparing isolated mammalian NKT-like cells, which involves culturing isolated mononuclear cells in vitro, adding culture cytokines that stimulate T cell proliferation and activation, and selecting the NKT-like cells using a cell sorting technique that uses NKT-like cell surface markers.

US2012258085 relates to a method for obtaining expanded and activated NK cells with the phenotype CD3 − CD56 + and NK-like T cells with the phenotype CD3 ⁺ CD56 ⁺ , comprising providing a cell sample of peripheral blood from a tumor-bearing subject, incubating and expanding the cells until at least 35% of the expanded cell population comprises activated NK cells and NK- ^like T ^cells , and harvesting the expanded cells. In particular, US2012258085 discloses a method in which PBMCs are first cultured in the presence of a monoclonal anti-CD3 antibody (OKT-3, 10 ng/mL) and 500 U/mL IL-2, and after 5 days, the culture is replenished with fresh medium containing 5% human serum and IL-2 but without OKT-3, and this is repeated every 2-3 days until the end of the culture.

Due to the limited efficacy of many ACT compositions, the use of genetically modified cells has been proposed. Examples of such include T cells expanded from PBMCs (or other lymphocyte populations such as NK cells) that express a cloned recombinant TCRαβ chain that recognizes an epitope derived from a shared tumor-associated antigen (TAA) or express a chimeric antigen receptor (CAR) composed of a signaling domain of the TCRζ chain and an immunoglobulin variable region that recognizes a tumor antigen fused to a costimulatory molecule (such as CD28 or CD137/4-1BB). For example, US2020345779 and US2020223920 disclose CAR polypeptides that can be expressed in various cell populations such as αβT cells, γδT cells, NK cells, NKT cells, B cells, innate lymphoid cells (ILCs), CIK cells, cytotoxic T lymphocytes (CTLs), LAK cells, and regulatory T cells.

Despite recent developments in cancer management, there remains a need for further tools, improved methods and cell compositions that can be used in cancer immunotherapy. In particular, non-responsiveness to primary or secondary treatments is highly prevalent among cancer patients and many existing treatments are characterized by significant adverse effects. Therefore, the development of therapeutic approaches that increase efficacy and/or safety would be highly beneficial. Furthermore, it would be beneficial to have cell compositions for ACT that exhibit improved survival and/or enhanced tumor binding or retention.

The present invention is directed to the field of immunotherapy. In particular, the present invention provides improved cell compositions and methods for adoptive cell therapy (ACT), useful in the treatment of cancer. More particularly, embodiments of the present invention employ the use of cell compositions (referred to herein as superactivated killer cells (SAK)) that contain a high percentage of activated CD8 ⁺ cells, particularly CD8 ⁺ NKG2D ⁺ Granzyme B ⁺ cells, and that are characterized by enhanced cytotoxicity. The present invention further relates to methods for producing SAK compositions from peripheral blood mononuclear cells (PBMCs), and their use in cancer management. According to advantageous embodiments, the present invention further relates to genetically modified SAK compositions that exhibit improved survival and/or enhanced tumor binding and retention.

The present invention is based, in part, on the development of a SAK composition capable of exerting highly potent antitumor cytotoxic activity against a variety of tumor cells. Surprisingly, SAK cells produced from PBMCs by the unique culture method disclosed herein were significantly more effective at eradicating tumor cells than other PBMC-derived effector cell compositions, including cytokine-induced killer cells (CIK) and interleukin-2 (IL-2)-expanded T cell compositions produced according to previously known ACT protocols. Furthermore, the produced SAK cells were found to be highly effective in inhibiting tumor development in vivo in a human lymphoma (Raji) engrafted mouse model.

As disclosed herein, the generated SAK population is characterized by high NKG2D levels detected on the surface of the entire CD8 ⁺ cell fraction and has been found to be particularly effective against tumors expressing NKG2D ligands. As further disclosed herein, the SAK population is characterized by high expression levels of FasL, granzyme B and perforin, but its antitumor cytotoxic activity is found to be largely insensitive to the use of anti-FasL neutralizing antibodies, despite being mediated primarily by granzyme B and perforin and requiring cell-cell contact. This is in contrast to previously known ACT cell populations, whose cytotoxic activity was reported to be substantially FAS-mediated. Furthermore, the cells have been demonstrated to display a favorable cell surface receptor profile, including abundant expression of receptors known to positively affect survival, proliferation and/or activity of effector immune cells, and minimal expression of inhibitory checkpoint molecules known to be associated with exhaustion and/or activation-induced cell death. As further demonstrated herein, SAK cells have been found to possess both MHC-dependent and non-MHC-dependent cytotoxic activity against a variety of tumor cells. Moreover, adoptive transfer of SAK cells has been shown to improve safety, significantly reducing the risk of causing graft-versus-host disease (GVHD), and maintains high levels of activity after cryopreservation. The present invention is also based, in part, on the production of genetically modified SAK compositions with enhanced antitumor activity.

Thus, in embodiments of the present invention, a cell composition for ACT is provided that is characterized by improved therapeutic properties. In other embodiments, the present invention relates to an improved method for producing a cell composition for ACT. According to some embodiments, the cell composition of the present invention can be advantageously produced directly from a PBMC sample by the ex vivo expansion protocol disclosed herein, without a preceding step of apheresis or other purification steps, and without the need to isolate tumor-specific cells from a tumor biopsy. In some embodiments, the cell composition of the present invention is produced by a method in which the cells of the sample are expanded by incubation or culture in the presence of IL-2 and a CD3 activator (e.g., a CD3-specific stimulating antibody) for at least 9 days, typically 10-16 days (e.g., 10-12 days). According to advantageous embodiments disclosed herein, the cells are expanded in the constant presence of an effective amount of IL-2 and a CD3 activator, such that the cells are periodically replenished with an effective amount of IL-2 and a CD3 activator throughout the expansion period (usually every 2-4 days).

In one aspect, an ACT cell composition is provided that comprises in vitro expanded PBMCs (e.g., at least 5 x 10 ⁶ to 10 x 10 ⁸ viable cells), 70-85% of which are CD3 ⁺ CD8 ⁺ cells expressing NKG2D and granzyme B, at least 70% are CD56 ^- cells, and up to 5% are CD3 ^- cells, and that exhibits significant cytotoxic activity against tumor cells in vitro and in vivo.

In one embodiment, 7-12% of the cells are CD3 ⁺ CD4 ⁺ cells, 20-28% are CD3 ⁺ CD56 ⁺ cells, and 10-14% are CD3 ⁺ CD8 ⁺ CD56 ⁺ cells. In another embodiment, the amount of CD3 ⁺ CD8 ⁺ cells in the composition is 6-8 times the amount of CD3 + CD4 ⁺ cells in the composition. In an exemplary embodiment, the cell composition is characterized by a ratio of CD3 ⁺ CD8 ⁺ cells to CD3 ⁺ CD4 ⁺ cells of 7-8. In another embodiment, the composition comprises less than 3% CD3 ^- CD56 ⁺ cells. In another embodiment, at least 40% of the CD3 ⁺ CD8 ⁺ cells are characterized by surface expression of at least one marker selected from the group consisting of perforin, Fas ligand (FasL), CXCR4, and CXCR2 ^. In other embodiments, the CD3 ⁺ CD8 ⁺ cells are further characterized as DNAM-1 ⁺ , CTLA-4 ⁻ cells. In other embodiments, at least 90% of the CD3 ⁺ CD8 ⁺ cells are characterized by surface expression of CXCR3 and at least 30% of the CD3 ⁺ CD8 ⁺ cells are characterized by surface expression of CXCR6. In other embodiments, no more than 50% of the CD3 ⁺ CD8 ⁺ cells are characterized by surface expression of multiple immune checkpoint molecules selected from the group consisting of CTLA-4, PD-1, and TIGIT.

In other embodiments, the cell composition exhibits non-MHC restricted cytotoxic activity mediated by granzyme B and/or perforin against tumor cells. In other embodiments, the cell composition exhibits non-MHC restricted cytotoxic activity mediated by granzyme B and/or perforin against hematopoietic tumor cells and solid tumor cells. In other embodiments, the cytotoxic activity is not substantially mediated by FAS-FasL interactions. In other embodiments, the cell composition is capable of inhibiting or preventing the development of tumors in non-histocompatible subjects in vivo without substantially inducing GVHD.

In another embodiment, the cell composition is capable of specifically eradicating hematopoietic tumor cells in vitro within 24 hours at a ratio of 0.25:1. In another embodiment, the cell composition is capable of significantly specifically eradicating hematopoietic tumor cells when incubated in vitro with hematopoietic tumor cells at a ratio of 0.25:1 for 24 hours and/or is capable of specifically eradicating hematopoietic tumor cells such that 80-90% of tumor cells are eradicated when incubated in vitro for 24 hours at a ratio of 1:1 composition cells to tumor cells. In another embodiment, the tumor cells express at least one NKG2D ligand. In another embodiment, the tumor cells express multiple NKG2D ligands selected from the group consisting of MICA, MICB, HLAE, ULBP1, ULBP256, and ULBP3. In another embodiment, the cell composition comprises between ^5x106 and ^10x109 viable cells.

In another embodiment, the cell composition is prepared by a method comprising expanding PBMCs in the presence of an effective amount of IL-2 and a CD3 activator for at least 9 days, and replenishing the effective amount of IL-2 and a CD3 activator every 48-96 hours. In another embodiment, the incubation is performed in the absence of other cytokines or antibodies.

In another embodiment, the cells of the cell composition are genetically modified. In another embodiment, the cells are genetically modified to express at least one of a chimeric antigen receptor (CAR), a cytokine, a chemokine, a cytokine or chemokine receptor, or any combination thereof. In another embodiment, the cell composition is genetically modified to express a CAR for an antigen selected from the group consisting of BCMA, CD47, PDL-1, mesothelin, EpCAM, CD34, CD44, PSCA, MUC16, CD276, CD123, CD19, CD20, and EFFRvIII. In another embodiment, the cell composition expresses one of a CD19-specific CAR and a CD123-specific CAR. In another embodiment, the cells are genetically modified to express a modified CXCR4 receptor comprising a mutation or truncation of the C-terminal tail domain. In another embodiment, the cell composition expresses one of exogenous IL-2, exogenous IL-15, exogenous IL-21, or any combination thereof. In other embodiments, the cell composition is modified to express (i) a CD19-specific CAR or a CD123-specific CAR, (ii) a modified CXCR4 receptor comprising a mutation or truncation of the C-terminal tail domain, and (iii) at least one cytokine selected from the group consisting of IL-2, IL-15, and IL-21.

In another aspect, a method for producing a cell composition for ACT, comprising:
a. providing a PBMC sample;
b. A method is provided which comprises expanding PBMCs in the constant presence of effective amounts of IL-2 and a CD3 activator, and in the absence of other cytokines or antibodies, for at least 9 days, with replenishing effective amounts of IL-2 and a CD3 activator every 48-96 hours, thereby increasing the number of viable cells in the culture by at least 20 fold, and obtaining a cell composition comprising at least 5 x ¹⁰ to 10 x ¹⁰ viable cells.

In another embodiment, the expansion is carried out in the constant presence of IL-2 and a CD3 activator as the only exogenously added cell stimulatory factors. In another embodiment, step b comprises expanding the cells in the presence of 500-1500 IU/mL IL-2 and 10-60 ng/mL anti-CD3 antibody, which are replenished every 48-96 hours for 10-16 days. In another embodiment, the cells are not subjected to further enrichment, stimulation or expansion steps. In another embodiment, the expansion is carried out to increase the number of viable cells by at least 30-fold. In another embodiment, the expansion is carried out to produce a cell composition comprising 5×10 ⁶ to 10×10 ⁹ viable mononuclear cells, of which 70-85% are CD3 ⁺ CD8 ⁺ cells expressing NKG2D and granzyme B. In another embodiment, the expansion is carried out to produce a cell composition capable of specifically eradicating hematopoietic tumor cells when incubated in vitro with hematopoietic tumor cells at a ratio of 0.25:1 for 24 hours. In other embodiments, the resulting cell composition is capable of specifically eradicating hematopoietic malignant cells in vitro at a ratio of 0.25:1 within 24 hours. In other embodiments, step (b) further comprises genetically modifying the cells. In other embodiments, the method comprises genetically modifying the cells to express at least one of a CAR, a cytokine, a chemokine, a receptor for a cytokine or chemokine, or any combination thereof. In other embodiments, the method comprises modifying the cells to express (i) a CD19-specific CAR or a CD123-specific CAR, (ii) a modified CXCR4 receptor comprising a mutation or truncation of the C-terminal tail domain, and (iii) at least one cytokine selected from the group consisting of IL-2, IL-15, and IL-21. In other embodiments, the method further comprises cryopreserving the resulting cell composition.

In another embodiment, a cell composition prepared by this method is provided.
a. providing a PBMC sample;
b. expanding the PBMCs for at least 9 days in the constant presence of an effective amount of IL-2 and a CD3 activator, and in the absence of other cytokines or antibodies, whereby the number of viable cells in the culture is increased by at least 20 fold by replenishing the effective amount of IL-2 and the CD3 activator every 2-4 days, thereby obtaining a cell composition comprising at least 5 x ¹⁰⁶ to 10 x ¹⁰⁸ viable cells;
and c. recovering the resulting cell composition.

In another aspect, a cell composition is provided that includes leukocytes genetically modified to express (i) a CD19-specific CAR or a CD123-specific CAR, (ii) a modified CXCR4 receptor that includes a mutation or truncation of the C-terminal tail domain, and (iii) at least one cytokine selected from the group consisting of IL-2, IL-15, and IL-21.

In another embodiment, 70-85% of the cells are CD3 ⁺ CD8 ⁺ cells that express NKG2D and granzyme B, at least 70% are CD56 ⁻ cells, and up to 5% are CD3 ⁻ cells. In another embodiment, the cell composition is prepared by a method comprising expanding PBMCs in the constant presence of an effective amount of IL-2 and a CD3 activator for at least 9 days, supplemented with an effective amount of IL-2 and a CD3 activator for 48-96 hours.

In another embodiment, a cell composition of the present invention is provided for treating a tumor in a subject in need of such treatment. In another embodiment, an ACT cell composition for treating a tumor in a subject in need of such treatment is provided, the ACT cell composition comprising at least 5×10 ⁶ to 10×10 ⁸ viable in vitro expanded PBMCs, 70-85% of which are CD3 ⁺ CD8 ⁺ cells expressing NKG2D and granzyme B, at least 70% are CD56 ⁻ cells, and up to 5% are CD3 ⁻ cells, and exhibits significant cytotoxic activity against tumor cells in vitro and in vivo. In another embodiment, a cell composition for use in treating a tumor in a subject in need of such treatment is provided, the cell composition comprising leukocytes genetically modified to express (i) a CD19-specific CAR or a CD123-specific CAR, (ii) a modified CXCR4 receptor comprising a mutation or truncation of the C-terminal tail domain, and (iii) at least one cytokine selected from the group consisting of IL-2, IL-15, and IL-21. In other embodiments, there is provided a cell composition prepared by the methods disclosed herein for use in treating a tumor in a subject in need thereof.

In various embodiments, the tumor is a hematopoietic tumor or a solid tumor. In other embodiments, the tumor is selected from the group consisting of leukemia, multiple myeloma, prostate tumor, or mesothelioma. In other embodiments, the tumor is characterized by expression of at least one NKG2D ligand. In other embodiments, the tumor expresses multiple NKG2D ligands selected from the group consisting of MICA, MICB, HLAE, ULBP1, ULBP256, and ULBP3. In other embodiments, the tumor expresses MICA, MICB, HLAE, ULBP1, ULBP256, and ULBP3. In other embodiments, the tumor is characterized by expression of at least one chemokine selected from the group consisting of CXCR6 ligand, CXCR4 ligand, CXCR2 ligand, and CXCR3 ligand.

In another embodiment, the tumor is characterized by expression of at least one ligand of an inhibitory immune checkpoint molecule selected from the group consisting of CTLA-4, PD-1, and TIGIT. In another embodiment, the tumor is resistant to treatment with at least one checkpoint molecule inhibitor. In another embodiment, the tumor is resistant to treatment with at least one CTLA-4 specific blocking antibody. In another embodiment, the subject has not received a treatment regimen with a checkpoint molecule inhibitor. In another embodiment, the use further comprises administering to the subject at least one checkpoint molecule inhibitor against Lag-3, Tim-3, or a combination thereof.

In other embodiments, the cell composition has undergone cryopreservation and thawing. In various embodiments, the cell composition is autologous, histocompatible allogeneic, or non-histocompatible allogeneic to the subject. In certain embodiments, the cell composition is non-histocompatible allogeneic to the subject. In other embodiments, the tumor is characterized by downregulation of MHC I expression and/or activity.

In another aspect, a method of treating a tumor in a subject in need of such treatment is provided comprising contacting tumor cells with a therapeutically effective amount of a cell composition of the invention, thereby treating the tumor in the subject. In another embodiment, a method of treating a tumor in a subject in need of such treatment is provided comprising contacting tumor cells with a therapeutically effective amount of an ACT cell composition comprising at least 5×10 ⁶ to 10×10 ⁸ viable in vitro expanded PBMCs, 70-85% of which are CD3 ⁺ CD8 ⁺ cells that express NKG2D and granzyme B, at least 70% of which are CD56 ⁻ cells, and up to 5% of which are CD3 ⁻ cells, and the composition exhibits significant cytotoxic activity against tumor cells in vitro and in vivo, thereby treating the tumor in the subject. In another embodiment, a method of treating a tumor in a subject in need of tumor treatment is provided comprising contacting tumor cells with a therapeutically effective amount of a cell composition comprising: (i) a CD19-specific CAR or a CD123-specific CAR, (ii) a modified CXCR4 receptor comprising a mutation or truncation of the C-terminal tail domain, and (iii) a leukocyte genetically modified to express at least one cytokine selected from the group consisting of IL-2, IL-15, and IL-21, for use in treating a tumor in a subject in need of tumor treatment, thereby treating the tumor in the subject. In another aspect, a method of treating a tumor in a subject in need of tumor treatment is provided comprising contacting tumor cells with a therapeutically effective amount of a cell composition prepared by the methods disclosed herein, thereby treating the tumor in the subject.

In various embodiments, the cell composition is autologous, histocompatible allogeneic, or non-histocompatible allogeneic to the subject. In certain embodiments, the cell composition is non-histocompatible allogeneic to the subject. In other embodiments, the contacting occurs in vivo. In other embodiments, the contacting occurs ex vivo. In other embodiments, the cell composition has undergone cryopreservation and thawing prior to contacting with the tumor cells. In other embodiments, the tumor is a hematopoietic tumor. In other embodiments, the tumor is a solid tumor. In other embodiments, the tumor is selected from the group consisting of leukemia, multiple myeloma, prostate tumor, or mesothelioma. In other embodiments, the tumor is characterized by expression of at least one NKG2D ligand. In other embodiments, the tumor expresses a plurality of NKG2D ligands selected from the group consisting of MICA, MICB, HLAE, ULBP1, ULBP256, and ULBP3. In other embodiments, the tumor expresses MICA, MICB, HLAE, ULBP1, ULBP256, and ULBP3.

In another embodiment, the tumor is characterized by expression of at least one chemokine selected from the group consisting of CXCR6 ligand, CXCR4 ligand, CXCR2 ligand, and CXCR3 ligand. In another embodiment, the tumor is characterized by expression of at least one ligand of an inhibitory immune checkpoint molecule selected from the group consisting of CTLA-4, PD-1, and TIGIT. In another embodiment, the tumor is resistant to treatment with at least one checkpoint molecule inhibitor. In another embodiment, the tumor is resistant to treatment with at least one CTLA-4 specific blocking antibody. In another embodiment, the subject has not received a treatment regimen with a checkpoint molecule inhibitor. In another embodiment, the use further comprises administering to the subject at least one checkpoint molecule inhibitor against Lag-3, Tim-3, or a combination thereof. In another embodiment, the tumor is characterized by downregulation of MHCI expression and/or activity.

Other objects, features and advantages of the present invention will become apparent from the following description and drawings.

Figures 1A-1D: Characterization of superactivated killer (SAK) cells at various time points during expansion (days 0, 4, 6, 8 and 11). Figure 1A: Total viable cell count ( ^x106 ). Figures 1B-1D: Flow cytometry analysis of percentages of CD3 ⁺ CD8 ⁺ , CD3 ⁺ CD4 ⁺ and CD3 ⁺ CD56 ⁺ cells, respectively. Ibid. Figures 2A-2B: Cytotoxic activity of SAK cells at various target:effector cell ratios against hematopoietic tumor cells. Figure 2A: Effect on leukemia (THP-1) cells. Figure 2B: Effect on multiple myeloma (RPMI8266) cells. The percentage of viable tumor cells (CFSE ⁺ PI ^- ) after 24 hours of incubation was assessed by FACS. Figures 3A-3C: Cytotoxic activity of SAK cells at various target cell:effector cell ratios against hematopoietic and solid tumor cells. Figure 3A: Effect on leukemia MV4-11 cells. Figure 3B: Effect on prostate cancer PC3 cells. Figure 3C: Effect on mesothelioma cancer H28 cells. Figures 4A-4B: At various target:effector cell ratios, SAK cells exhibit superior cytotoxic activity against leukemic MV4-11 cells compared to other effector cells. Figure 4A: Effect compared to cytokine-induced killer cells ("CIK IFN"). Figure 4B: Effect compared to activated lymphocyte composition ("T cell IL-2"). FIG. 5: Expression of NKG2D receptor on cytotoxic CD8 ⁺ SAK cells. Figures 6A-6C: Expression of effector molecules by SAK cells. Figure 6A: Expression of FasL. Figure 6B: Expression of granzyme B. FIG. 6C: Perforin expression. FIG. 7: Perforin secretion by SAK cells after 24 h incubation with leukemic MV4-11 cells. Figures 8A-5C: Evaluation of cytotoxicity in the presence of various inhibitors. SAK cells were incubated with MV4-11 cells at various ratios of target cells:effector cells in the presence of various inhibitors. Figure 8A: Granzyme B inhibitor (DCI). Figure 8B: Anti-FasL antibody. Figure 8C: Perforin inhibitor. Figures 9A-9B: Chemokine receptor expression and function on CD8 ⁺ SAK cells at day 11 of expansion. Figure 9A: Expression of CXCR2, CXCR3 (top panels, left and right, respectively), CXCR4 and CXCR6 (bottom panels, left and right, respectively). Black lines indicate staining with isotype control antibodies (IC) conjugated to the respective fluorescent markers (FITC, APC or PE). Figure 9B: Transwell migration of SAK cells towards the chemokines IL-8, SDF-1 and IP-10. Figures 10A-10C: Shows inhibition of tumor cell engraftment in NSG mice following CD19 ⁺ SAK cell transplantation compared to controls, measured as the percentage of CD19 ⁺ Raji cells. Figure 10A: Engraftment in the spleen. Figure 10B: Engraftment in the bone marrow (BM). Figure 10C: Engraftment in the blood. Figures 11A-11B: Cytotoxic immune responses induced by CD19CAR SAK cells at various target:effector ratios. Figure 11A: Cytotoxic activity of SAK cells and CD19CAR SAK cells against CD19 ⁺ tumor cells (Toledo). Figure 11B: Cytotoxic activity of SAK cells and CD19CAR SAK cells against ^CD19- tumor cells (U937). Figures 12A-12C: SAK cells induce much less graft-versus-host disease (GVHD) in NSG mice compared to PBMCs. Irradiated NSG mice were administered SAK cells or PBMCs at a dose of ^5x106 cells. Figure 12A: Survival rate. Figure 12B: Body weight change. Figure 12C: Mean GVHD scoring ("Mean Scoring"). Figures 13A-13B: Cytotoxic activity of SAK cells after freeze-thaw cycles (denoted as "frozen SAK") compared to fresh, non-frozen SAK cells (denoted as "SAK") against THP-1 or MV411 tumor cells at various SAK-tumor cell ratios. Figure 13A: Activity against THP-1 cells. Figure 13B: Activity against MV411. Figure 14: Levels of the following immune checkpoint molecules on the surface of CD8 ⁺ SAK cells: Tim-3 (top panel, left), DNAM-1 (top panel, right), PD-1 (center panel, left), TIGIT (center panel, right), Lag-3 (bottom panel, left) or CTLA-4 (bottom panel, right). APC or PE-labeled non-specific antibodies were used as isotype controls (IC) .

The present invention relates to improved cell compositions and methods for adoptive cell therapy (ACT), useful in the treatment of cancer. More specifically, embodiments of the invention employ the use of cell compositions, referred to herein as superactivated killer cells (SAK), characterized by enhanced cytotoxicity (e.g., against non-histocompatible tumor cells). The present invention further relates to methods for producing SAK compositions and their use in cancer management. According to advantageous embodiments, the present invention further relates to genetically modified SAK compositions that exhibit improved survival and/or enhanced tumor binding and retention.

The present invention is based, in part, on the development of a SAK composition capable of exerting highly potent antitumor cytotoxic activity against a variety of tumor cells without the need for MHC recognition. Surprisingly, SAK cells generated from peripheral blood mononuclear cells (PBMCs) by the unique culture method disclosed herein were significantly more effective in eradicating tumor cells than other PBMC-derived effector cell compositions, including cytokine-induced killer cells (CIK) and interleukin-2 (IL-2) expanded T cell compositions generated according to previously known ACT protocols. Furthermore, the generated SAK cells were found to be highly effective in inhibiting tumor development in vivo, with improved safety, a significantly reduced risk of causing graft-versus-host disease (GVHD), and compatible with cryopreservation. Furthermore, the cells were demonstrated to exhibit a favorable profile of cell surface receptors, including immune checkpoint molecules and chemokine receptors. In particular, SAK cells have been found to be characterized by high expression of activating receptors, including DNAM-1, and minimal expression of inhibitory checkpoint molecules (e.g., CTLA-4). The present invention is also based, in part, on the development of genetically modified SAK cell compositions suitable for improved therapeutic approaches with enhanced and/or prolonged tumor-specific activity.

Thus, in embodiments of the invention, a cell composition for ACT is provided that is characterized by improved therapeutic properties. In other embodiments, the invention relates to an improved method for producing a cell composition for ACT. According to some embodiments, the cell composition of the invention can be advantageously produced directly from a PBMC sample by the ex vivo expansion protocol disclosed herein, without a preceding step of apheresis or other purification steps, and without the need to isolate tumor-specific cells from a tumor biopsy. In some embodiments, incubation or culture in the presence of IL-2 and a CD3 activator (e.g., a CD3-specific stimulating antibody) is carried out for at least 9 days, typically 9-16 days (more typically 10-12 days), to expand the cells of the sample (e.g., ^0.25x106 to ^5x106 PBMCs). It is to be understood that the anti-CD3 antibody can be conveniently used in the form of a free suspension and does not need to be plate-bound or presented by allogeneic feeder cells.

In particular, the cells are advantageously expanded in the constant presence of effective amounts of IL-2 and CD3 activator, so as to be maintained in sufficient amounts throughout the entire expansion period (e.g., 500-1500 IU/mL IL-2 and 20-40 ng/mL anti-CD3 antibody for at least 9 days). Thus, according to the present invention, the constant presence of stimulatory factors is maintained by periodically supplementing the culture medium with IL-2 and CD3 activator every 1-5 days (usually every 2-4 days). For example, PBMCs can be cultured at a concentration of about 1 x 106 cells/mL in the presence of an initial concentration of about 1000 IU/mL recombinant human IL-2 (rhIL-2) and about 30 ng/mL anti-human CD3 antibody (e.g., OKT-3), which are replenished every 2-4 days during culture (e.g., days 0, 4, 6 and ⁸ ).

According to further advantageous embodiments, the expansion is carried out in the presence of IL-2 and a CD3 activator as the only cell stimulatory factor. Thus, in some embodiments, the expansion is carried out in the absence of exogenously added additional cell stimulatory factors, such as cytokines (e.g., IFN-γ) and/or feeder cells. In other embodiments, the method is carried out to increase the number of viable cells in the culture by at least 20-fold, typically by an average of at least 30-fold (e.g., 30-55-fold). In other embodiments, the method increases the number of viable cells by 25-55-fold within 11 days of culture. In other embodiments, the method is carried out to produce at least 1.5×10 ⁸ , typically 2×10 ⁸ to 4×10 ⁸ viable cells, most typically 5×10 ⁶ to 10×10 ⁸ viable cells, up to about 10×10 ⁹ viable cells.

In one aspect, an ACT cell composition is provided comprising in vitro expanded PBMCs, 70-85% of which are CD3 ⁺ CD8 ⁺ cells that express NKG2D and granzyme B, at least 70% of which are CD56 ^- cells, and up to 5% of which are CD3 ^- cells, and which exhibit significant cytotoxic activity against tumor cells in vitro and in vivo.

In another aspect, a method for producing a cell composition for ACT, comprising:
a. providing a PBMC sample;
b. A method is provided which comprises expanding PBMCs in the constant presence of effective amounts of IL-2 and a CD3 activator, and in the absence of other cytokines or antibodies, for at least 9 days, with replenishing effective amounts of IL-2 and a CD3 activator every 48-96 hours, thereby increasing the number of viable cells in the culture by at least 20 fold, and obtaining a cell composition comprising at least 5 x ¹⁰ to 10 x ¹⁰ viable cells.

In another embodiment, a cell composition prepared by this method is provided.
a. providing a PBMC sample;
b. expanding the PBMCs for at least 9 days in the constant presence of an effective amount of IL-2 and a CD3 activator, and in the absence of other cytokines or antibodies, whereby the number of viable cells in the culture is increased by at least 20 fold by replenishing the effective amount of IL-2 and the CD3 activator every 2-4 days, thereby obtaining a cell composition comprising at least 5 x ¹⁰⁶ to 10 x ¹⁰⁸ viable cells;
and c. recovering the resulting cell composition.

In another aspect, a cell composition is provided that includes leukocytes genetically modified to express (i) a CD19-specific CAR or a CD123-specific CAR, (ii) a modified CXCR4 receptor that includes a mutation or truncation of the C-terminal tail domain, and (iii) at least one cytokine selected from the group consisting of IL-2, IL-15, and IL-21.

In another embodiment, a cell composition of the present invention is provided for use in treating a tumor in a subject in need of such treatment. In another embodiment, an ACT cell composition is provided for use in treating a tumor in a subject in need of such treatment, comprising at least 5×10 ⁶ to 10×10 ⁸ viable in vitro expanded PBMCs, 70-85% of which are CD3 ⁺ CD8 ⁺ cells expressing NKG2D and granzyme B, at least 70% are CD56 ⁻ cells, and up to 5% are CD3 ⁻ cells, and which exhibit significant cytotoxic activity against tumor cells in vitro and in vivo. In another embodiment, a cell composition is provided for use in treating a tumor in a subject in need of such treatment, comprising leukocytes genetically modified to express (i) a CD19-specific CAR or a CD123-specific CAR, (ii) a modified CXCR4 receptor comprising a mutation or truncation of the C-terminal tail domain, and (iii) at least one cytokine selected from the group consisting of IL-2, IL-15, and IL-21. In other embodiments, there is provided a cell composition prepared by the methods disclosed herein for use in treating a tumor in a subject in need thereof.

In another aspect, a method of treating a tumor in a subject in need of such treatment is provided comprising contacting tumor cells with a therapeutically effective amount of a cell composition of the invention, thereby treating the tumor in the subject. In another embodiment, a method of treating a tumor in a subject in need of such treatment is provided comprising contacting tumor cells with a therapeutically effective amount of an ACT cell composition comprising at least 5×10 ⁶ to 10×10 ⁸ viable in vitro expanded PBMCs, 70-85% of which are CD3 ⁺ CD8 ⁺ cells that express NKG2D and granzyme B, at least 70% of which are CD56 ⁻ cells, and up to 5% of which are CD3 ⁻ cells, and the composition exhibits significant cytotoxic activity against tumor cells in vitro and in vivo, thereby treating the tumor in the subject. In another embodiment, a method of treating a tumor in a subject in need of such treatment is provided, comprising contacting tumor cells with a therapeutically effective amount of a cell composition comprising leukocytes genetically modified to express (i) a CD19-specific CAR or a CD123-specific CAR, (ii) a modified CXCR4 receptor comprising a mutation or truncation of the C-terminal tail domain, and (iii) at least one cytokine selected from the group consisting of IL-2, IL-15, and IL-21, for use in treating a tumor in a subject in need of such treatment, thereby treating the tumor in the subject.

These and other aspects and embodiments are described in further detail and illustrated below.

Preparation Methods In some embodiments, the present invention relates to methods for producing the cell compositions of the present invention. In some embodiments, a method for producing a cell composition for ACT comprises the steps of: a. providing a PBMC sample;
b. expanding the PBMCs for at least 9 days in the constant presence of an effective amount of IL-2 and a CD3 activator, and in the absence of other cytokines or antibodies, whereby the number of viable cells in the culture is increased by at least 20 fold by replenishing the effective amount of IL-2 and the CD3 activator every 2-4 days, thereby obtaining a cell composition comprising at least 5 x ¹⁰⁶ to 10 x ¹⁰⁸ viable cells;
and c. recovering the resulting cell composition.

In another embodiment, the method comprises:
a. providing a PBMC sample;
b. Expanding the PBMCs in the constant presence of an effective amount of IL-2 and a CD3 activator, and in the absence of other cytokines or antibodies, for at least 9 days, wherein the effective amount of IL-2 and the CD3 activator are replenished every 48-96 hours, thereby increasing the number of viable cells in the culture by at least 20 fold and obtaining a cell composition comprising at least 5 x ¹⁰ to 10 x ¹⁰ viable cells.

As used herein, the term "PBMC sample" or "peripheral blood mononuclear cell sample" or "unfractionated PBMC" refers to total PBMCs, i.e., a population of viable white blood cells with round nuclei that are not substantially enriched in any given subpopulation. Typically, the PBMC sample according to the invention is obtained from peripheral blood, has not been subjected to a selection process, and contains only adherent PBMCs (consisting essentially of more than 90% monocytes) or non-adherent PBMCs (including T cells, B cells, natural killer (NK) cells, NKT cells, and DC precursors).

Typically, PBMCs can be obtained by centrifugation (e.g., from buffy coats), density gradient centrifugation (e.g., via Ficoll-Hypaque), or other methods known in the art (e.g., extraction or partial purification from peripheral blood). For example, PBMCs can be extracted from whole blood using Ficoll, a hydrophilic polysaccharide that separates blood layers, with the PBMCs forming a ring of cells below the plasma layer. Additionally, PBMCs can be extracted from whole blood by hypotonic lysis, which preferentially lyses red blood cells. Such procedures are known to those skilled in the art.

As used herein, the term "expansion" refers to an increase in cell number of a cell population by cell replication. In particular, PBMC expansion as disclosed herein refers to an in vitro or ex vivo culture method that includes polyclonal activation and expansion of a cell population within a PBMC to produce an ACT cell composition according to the present invention. Typically, expansion is performed in a specific cGMP-grade environment and cGMP-grade medium to produce, for example, a clinical grade ACT composition for the treatment of a human subject. In some embodiments, expansion is performed for 9-16 days or 11-16 days, e.g., 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, or 16 days.

More specifically, the medium is supplemented with interleukin-2 (IL-2) and a CD3 activator during the expansion method. In some embodiments, the cell composition is prepared by the method described above, and an effective amount of IL-2 and a CD3 activator is administered every 48 to 96 hours, e.g., every 48 hours, 49 hours, 50 hours, 51 hours, 52 hours, 53 hours, 54 hours, 55 hours, 56 hours, 57 hours, 58 hours, 59 hours, 60 hours, 61 hours, 62 hours, 63 hours, 64 hours, 65 hours, 66 hours, 67 hours, etc. , every 68 hours, 69 hours, 70 hours, 71 hours, 72 hours, 73 hours, 74 hours, 75 hours, 76 hours, 77 hours, 78 hours, 79 hours, 80 hours, 81 hours, 82 hours, 83 hours, 84 hours, 85 hours, 86 hours, 87 hours, 88 hours, 89 hours, 90 hours, 91 hours, 92 hours, 93 hours, 94 hours, 95 hours, or 96 hours.

The cytokine IL-2 acts as a growth factor for lymphocytes in culture and is necessary, for example, to prevent cell death of activated T cells. The activity of IL-2 is mediated by binding to and signaling through the IL-2 receptor (IL-2R), a complex composed of three chains designated alpha (CD25), beta (CD122), and gamma (CD132). Dimeric IL-2R is expressed by memory CD8 ⁺ T cells and NK cells, whereas regulatory and activated T cells express high levels of trimeric IL-2R. As used herein, "interleukin 2" or "IL-2" refers to mammalian IL-2, preferably human IL-2 (e.g., recombinantly produced human IL-2). In some embodiments, polypeptides (e.g., IL-2 fusion proteins, conjugates, and variants) recognized in the art as biological equivalents of human IL-2 and capable of selectively binding to the IL-2 receptor and exerting equivalent activity in supporting the expansion and proliferation of human lymphocytes can also be used. IL-2 can be produced recombinantly (e.g., by the methods disclosed herein) and is commercially available from a variety of sources. For example, recombinant human IL-2 (rhIL-2) can be purchased from R&D Systems. Proleukin® (aldesleukin) is a highly purified rhIL-2 protein that has been approved for clinical use in cancer immunotherapy.

The CD3 complex binds to the T cell receptor (TCR) chain to form a TCR-CD3 complex on the surface of T cells. Thus, although CD3 is considered a T cell marker, certain other cell populations, such as NK-T cells, are also characterized by CD3 surface expression (CD3 ⁺ cells), whereas other cell types, such as NK cells, B cells, and myeloid cells, are not typically characterized by a lack of CD3 surface expression (CD3 ⁻ cells). In mammals, the TCR-CD3 complex is composed of the CD3 γ chain, the CD3 δ chain, and two CD3 ε chains, which form a complex with the TCR chain and the CD3-zeta (ζ chain). As used herein, a “CD3 activator” is an agent capable of inducing or mediating polyclonal stimulation and proliferation by binding to the CD3 molecule (typically a human CD3 molecule) on the surface of lymphocytes. Examples of CD3 activators include stimulatory CD3-specific antibodies, antigen-binding portions thereof, and conjugates thereof. Typically, the CD3 activator is a stimulatory anti-CD3 monoclonal antibody (mAb), typically directed against a CD3 signaling domain, such as CD3ε. In a particular embodiment, a preferred CD3 activator for use in embodiments of the invention is OKT3, a murine monoclonal antibody against the human CD3ε chain. Other examples of antibodies that cross-link the T cell receptor/CD3 complex include the HIT3a and UCHT1 anti-CD3 mAbs. Further examples of CD3 activators are CD3 ligands and CD3-binding mitogens. Such antibodies and agents are commercially available from a variety of sources. For example, OKT3 and other anti-CD3 mAbs are available from Biolegend and eBioscience.

According to an embodiment of the invention, PBMCs are expanded in the absence of other cytokines or antibodies. In other words, no cytokines or antibodies other than IL-2 and CD3 activators are supplemented (exogenously added) to the culture. Thus, the expansion method of the present invention can be distinguished from other expansion protocols used to produce other types of cell compositions (e.g., CIK cells, particularly by expansion of (e.g., PBMCs) in the presence of IFN-γ). However, it should be understood that various factors, including cytokines, may be produced by the cultured cells and secreted into the medium during the expansion method.

Furthermore, the expansion method of the present invention is carried out in the constant presence of IL-2 and CD3 activator, which are continuously or intermittently replenished so that IL-2 and CD3 activator are maintained in effective amounts throughout the expansion method. Thus, the expansion method of the present invention is distinguishable from other expansion protocols used to produce other types of cell compositions (e.g., in the production of CIK cells, IL-2 and OKT3 are added on the second day of expansion, but OKT3 is not subsequently supplemented to the culture).

In other embodiments, the expansion is carried out in the constant presence of IL-2 and CD3 activators as the only exogenously added cell stimulatory factors. Thus, other cell stimulatory factors used to induce lymphocyte activation and proliferation during the in vitro or ex vivo expansion method, such as antibodies against costimulatory molecules (e.g., CD28), antigens (e.g., MHC-antigen complexes), etc. (which are of exogenous source to the cultured cells), are not artificially introduced (supplemented). It should be understood that certain cell stimulatory factors may be endogenously produced by the cultured cells during the expansion method. It should be further understood that various factors contained in standard tissue culture media (e.g., complete media such as RPMI) are not considered exogenously added cell stimulatory factors according to these embodiments. For example, expansion can be conveniently carried out in the presence of tissue culture serum, e.g., fetal calf serum (FCS) at a final concentration of 5-15%, typically 7-12% or 8-13%, more typically about 10%.

As disclosed herein, expansion is advantageously performed to increase the number of viable cells in culture by at least 20-fold (or in some embodiments, 30-60-fold), thereby obtaining a cell composition comprising 5×10 ⁶ to 10×10 ⁸ viable cells (or in other embodiments, up to 10×10 ⁹ viable cells). As used herein, the term "viable cells" refers to cells that are not undergoing necrosis or are not in an early or late apoptotic state. Assays for determining cell viability are known in the art, such as using propidium iodide (PI) staining detectable by flow cytometry. Thus, in one embodiment, viable cells are cells that do not exhibit propidium iodide uptake and do not express phosphatidylserine. Necrosis can be further identified using light, fluorescence or electron microscopy techniques, or by uptake of the dye trypan blue.

Additionally, the production methods of the present invention include a step of harvesting the cell composition obtained by the expansion step. For example, the cells can be harvested and subjected to centrifugation or other washing steps to remove residual antibodies and cytokines. In some embodiments, the cells can be resuspended in an appropriate diluent or medium (e.g., PBS) prior to contacting with the tumor cells or administering to the subject.

In some embodiments, the cells have not been subjected to further steps of enrichment (e.g., apheresis prior to the expansion step, lymphoapheresis, or immunocapture of leukocytes), stimulation (e.g., antigen-specific) and/or expansion (e.g., rapid expansion protocols in the presence of feeder cells, or expansion in the presence of other/additional cytokines).

In another aspect, there is provided a method for producing a cell composition of the invention, comprising the steps of:
a) providing a PBMC sample;
b) expanding the PBMCs in the constant presence of effective amounts of IL-2 and a CD3 activator for at least 9 days, whereby effective amounts of IL-2 and a CD3 activator are replenished every 2-4 days;
and c) recovering the resulting cell composition.

In some embodiments, expansion is performed to obtain the cell compositions of the invention disclosed herein. In other embodiments, expansion is performed in the constant presence of IL-2 and CD3 activator as the only exogenously added cell stimulators (in the absence of other cytokines and antibodies).

In other embodiments, the method further comprises genetically modifying the cells to obtain the cell composition disclosed herein. In some embodiments, the resulting cell composition is advantageously suitable for cryopreservation. Thus, in contrast to certain other cell compositions for ACT, the cell compositions of the present invention can be frozen after expansion and thawed prior to administration to a subject without substantial loss of potency. Thus, in other embodiments, the method further comprises freezing the resulting cell composition. For example, the cryopreservation protocol can be performed using liquid nitrogen and suspending the cells in an appropriate medium (e.g., including, but not limited to, 90% FCS + 10% dimethyl sulfoxide (DMSO)). If necessary, commercially available systems or excipients (e.g., CryoSure® USP grade cryoprotectant) can be used.

In other embodiments, step b comprises expanding the cells in the presence of 500-1500 IU/mL IL-2 and 10-60 ng/mL anti-CD3 antibody, which are replenished every 2-4 days for 10-16 days. In other embodiments, the cells are not subjected to further enrichment, stimulation or expansion steps (e.g., before or after step b). In other embodiments, expansion is performed to increase the number of viable cells by at least 30-fold. In other embodiments, expansion is performed to produce a cell composition comprising at least 5×10 ⁶ to 10×10 ⁸ (e.g., 5×10 ⁶ to 10×10 ⁹ ) viable mononuclear cells, of which 70-80% (or 70-85% in other embodiments) are CD3 ⁺ CD8 ⁺ cells expressing NKG2D and granzyme B. In other embodiments, expansion is performed to produce a cell composition capable of specifically eradicating hematopoietic tumor cells when incubated in vitro with tumor cells at a ratio of 0.25:1 for 24 hours. In other embodiments, step b further comprises genetically modifying the cells, e.g., to produce a genetically modified cell composition as disclosed herein. It may be advantageous to modify (e.g., transduce or transfect) the cells prior to subjecting them to CD3 stimulatory factors and IL-2 supplementation.

Cell Compositions In another embodiment, a cell composition prepared by the methods disclosed herein is provided. In another embodiment, a cell composition comprising SAK cells as disclosed herein is provided. In another embodiment, the invention relates to an adoptive cell transfer (ACT) composition as disclosed herein. In another embodiment, an ACT cell composition is provided, the ACT cell composition comprising 5×10 ⁶ to 10×10 ⁸ viable in vitro expanded PBMCs, 70-80% of which are CD3 ⁺ CD8 ⁺ cells expressing NKG2D and granzyme B, at least 70% of which are CD56 ⁻ cells, and up to 5% of which are CD3 ⁻ cells, and which exhibit significant cytotoxic activity against non-histocompatible tumor cells in vitro and in vivo. In another embodiment, an ACT cell composition is provided comprising at least 5 x ¹⁰ to 10 x ¹⁰ viable in vitro expanded PBMCs, 70 to 85% of which are CD3 ⁺ CD8 ⁺ cells expressing NKG2D and granzyme B, at least 70% of which are CD56 ^- cells, and up to 5% of which are CD3 ^- cells, and which exhibits significant cytotoxic activity against tumor cells in vitro and in vivo.

Unless otherwise specified, the term "adoptive transfer" as used herein refers to a form of passive immunotherapy in which a pre-sensitized immunological agent (e.g., cells or serum) is transferred to a recipient. The terms "adoptive transfer immunotherapy", "adoptive cell therapy" and "adoptive cellular immunotherapy" are used interchangeably herein to refer to a therapeutic or prophylactic regimen or modality in which effector immunocompetent cells, such as the cell composition of the present invention, are administered (adoptive transfer) to a subject in need thereof to alleviate or ameliorate the onset or symptoms of cancer. Thus, the ACT composition of the present invention is produced under sterile and appropriate (e.g., cGMP grade) conditions and comprises an effective amount (e.g., at least 5×10 ⁶ cells, up to about 10×10 ⁹ cells) to be administered to a human subject suffering from a neoplastic disorder as part of an anti-tumor regimen.

As used herein, the term "in vitro expanded PBMCs" refers to mononuclear cells derived from peripheral blood that have undergone an in vitro or ex vivo expansion process to result in the cell compositions disclosed herein. Such cells and compositions are therefore distinct from leukocyte compositions obtained or derived from other sources, such as tumor-derived lymphocytes (TILs, obtained from tumor biopsies rather than from peripheral blood), leukocyte cell lines, and leukocytes differentiated in vitro from pluripotent stem cells.

T lymphocytes (T cells) are one of several different cell types involved in the immune response and are characterized by surface expression of CD3 (CD3 ⁺ cells). T cell activity is controlled by antigens that are presented to the T cell in association with major histocompatibility complex (MHC) molecules. The T cell receptor (TCR) then binds to the MHC-antigen complex. Once the antigen is complexed with MHC, the MHC-antigen complex binds to a specific TCR on the T cell, thereby altering the activity of the T cell. Proper activation of T lymphocytes by antigen-presenting cells requires not only stimulation of the TCR but also the combined and coordinated engagement of its co-receptors.

Helper T cells (TH cells) assist other white blood cells in immunological processes such as maturation of B cells into plasma cells and memory B cells, and activation of cytotoxic T cells and macrophages. These cells are also known as CD4 ⁺ T cells (or CD3 ⁺ CD4 ⁺ cells) because they express the CD4 glycoprotein on their surface. Helper T cells become activated when peptide antigens are presented to them by MHC class II molecules expressed on the surface of antigen-presenting cells (APCs). Once activated, they divide rapidly and secrete small proteins called cytokines that regulate or support a vigorous immune response.

Cytotoxic T cells (TC cells, or CTLs) destroy virus-infected and tumor cells and also play a role in transplant rejection. These cells are also known as CD8 ⁺ T cells (or CD3 ⁺ CD8 ⁺ cells) because they express the CD8 glycoprotein on their surface. These cells recognize their targets by binding to antigens associated with MHC class I molecules, which are present on the surface of all nucleated cells.

Natural killer cells (NK cells) are a subset of peripheral blood lymphocytes defined by the expression of CD56, CD16 and/or CD57 and the absence of T cell receptor-CD3 complexes. NK cells are characterized by their ability to bind and kill cells that fail to express "self" MHC/HLA antigens by activation of specific cytolytic enzymes, to kill tumor cells or other diseased cells that express ligands for NK activating receptors, and to release cytokines that stimulate or inhibit the immune response.

Natural killer T cells (NKT cells or NK-T cells) are CD1d-restricted T cells that express the TCR. Unlike conventional T cells, which detect peptide antigens presented by conventional MHC molecules, NK-T cells recognize lipid antigens presented by the non-classical MHC molecule, CD1d. Invariant or type I NK-T cells express a very limited TCR repertoire, whereas non-classical or non-invariant type II NKT cells show more heterogeneous TCRαβ utilization. Adaptive or invariant (type I) NKT cells can be identified by expression of at least one or more of the following markers: TCR Va24-Ja18, Vb11, CD1d, CD3, CD4, CD8, aGalCer, CD161, and CD56.

Neural cell adhesion molecule (NCAM), also known as CD56, is a member of the immunoglobulin superfamily involved in both so-called homophilic and heterophilic interactions. There are three major isoforms of CD56 (NCAM-120, NCAM-140, and NCAM-180), all produced by alternative splicing from a single gene, which differ in the length of the intracellular domain. CD56 expression is most closely associated with NK cells, which constitute the majority of CD56 ⁺ cells isolated from peripheral blood. However, CD56 has also been detected on other lymphoid cells, including, but not limited to, gamma delta (γδ) T cells and dendritic cells (DCs).

As used herein, a cell is considered "positive" for a cell surface marker if it expresses the cell surface marker on the cell surface in an amount sufficient to be detected using methods known to one of skill in the art (e.g., contacting the cell with an antibody that specifically binds to the cell surface marker, followed by flow cytometric analysis of the contacted cells to determine whether the antibody specifically binds to the cell). It should be understood that while a cell may express messenger RNA for the cell surface marker, a cell must express the marker of interest on its surface to be considered positive for the compositions and methods described herein. Similarly, a cell is considered "negative" for a cell surface marker if it does not express the cell surface marker on the cell surface in an amount sufficient to be detected using methods known to one of skill in the art (e.g., contacting the cell with an antibody that specifically binds to the cell surface marker, followed by flow cytometric analysis of the contacted cells to determine whether the antibody specifically binds to the cell).

In various embodiments, the cell compositions of the invention are characterized by high relative CD8 ⁺ cell levels and low relative CD4 ⁺ cell levels. In some embodiments, the compositions are characterized by a high ratio of CD3 ⁺ CD8 ⁺ cells to CD3 ⁺ CD4 ⁺ cells (at least 6:1, e.g., 6.5-8.5:1, typically 7-8:1). In other words, the amount of CD3 ⁺ CD8 ⁺ cells in the composition is 6-8 times, typically 7-8 times, the amount of CD3 ⁺ CD4 ⁺ cells in the composition. In other embodiments, the cell compositions are characterized by high NK-T cell levels. In other embodiments, the cells are characterized by a high ratio of NK-T cells to T cells. In some embodiments, the cell compositions are characterized by an incidence of CD3 ⁺ CD8 ⁺ CD56 ⁺ cells relative to CD3 ⁺ CD8 ⁺ cells of 15-30%, typically 19-25% or 20-22%. In yet other embodiments, the cell compositions are characterized by a high ratio of CD8 ⁺ cells to CD56 ⁺ cells (e.g., 2-4). In various embodiments, such high levels and ratios may be associated with an enhancement relative to the original PBMC sample, or an enhancement relative to other ACT compositions, such as CIK cell preparations, as described herein. Each possibility represents a separate embodiment of the present invention.

For example, in some embodiments, the cell compositions of the invention are characterized by the presence of an average of at least 60%, typically 75% (e.g., 70-80%, 70-85%, 73-76% or 73-78%) cytotoxic CD3 ⁺ CD8 ⁺ cells. In other embodiments, the cell compositions are characterized by the presence of 70-85% CD3 ⁺ CD8 ⁺ cells. In other embodiments, the cell compositions are characterized by the presence of up to 15% CD3 ⁺ CD4 ⁺ cells (e.g., 7-12%, 11-13%, 9-12% or 8-11%), typically up to 10% CD3 ⁺ CD4 ⁺ cells. In other embodiments, on average at least 15% of the CD3 ⁺ CD8 ⁺ cells are CD8 ⁺ CD56 ⁺ cells (e.g., 15-30% or 17-20%). In another embodiment, the cell composition is characterized by at least 9%, typically 10-14%, CD3 ⁺ CD8 ⁺ CD56 ⁺ cells. In another embodiment, the cell composition is characterized by the presence of 7-17% CD3 ⁺ CD8 ⁺ CD56 ⁺ cells. In yet another embodiment, the cell composition comprises about 8-35%, typically 20-28% or 22-26%, CD3 ^{+ CD56 + cells. In another embodiment, the cell composition comprises 15-30% CD3 + CD56 + cells .}

In some embodiments, the cell composition is characterized by high expression of activation markers and chemokine receptors. In some embodiments, the cells express at least one, and typically more than one, receptor selected from the group consisting of NKG2D, Granzyme B, Perforin, FASL, CXCR4, and CXCR2, each possibility representing a separate embodiment of the invention. For example, as shown herein, it has been found that substantially all CD8 ⁺ cells of an expanded cell composition express the activation markers NKG2D and Granzyme B (see Examples 4 and 5 below). Thus, in its embodiments, the invention provides improved cell compositions in which the CD8 ⁺ cells are also typically characterized by expression of NKG2D and Granzyme B.

Natural killer group 2 member D (NKG2D) is a transmembrane protein that belongs to the NKG2 family of C-type lectin-like receptors. In humans, NKG2D is expressed by NK cells, γδ T cells, and CD8 ⁺ αβ T cells and acts as an activating receptor that promotes effector cell function against target cells expressing NKG2D ligands. Such ligands are inducible self-proteins that belong to the MHC class I chain-related protein (MIC) or UL-16 binding protein (ULBP) family (e.g., MICA, MICB, HLAE, ULBP1, ULBP256, and ULBP3), which are completely absent or present only at low levels on the surface of normal cells and overexpressed in infected, malignant, transformed, senescent, and stressed cells. Its expression is regulated in various steps (by various stress pathways, most notably by the DNA damage response). NKG2D is associated with DNAX-activating protein 10 (DAP10), which promotes and stabilizes its surface membrane expression. NKG2D lacks signaling motifs in its cytoplasmic domain, and signaling occurs upon phosphorylation-mediated ligation of DAP10, which recruits downstream signaling effector molecules and ultimately leads to cytotoxicity.

According to further embodiments, the CD8 ⁺ cells can further express perforin and/or FASL. In other embodiments, the CD8 ⁺ cells can further express CXCR4 and/or CXCR2. In other embodiments, the CD8 ⁺ cells can further express CXCR3 and/or CXCR6. Each possibility represents a separate embodiment of the invention. In other embodiments, at least 40% of the CD3 ⁺ CD8 ⁺ cells are characterized by surface expression of at least one marker selected from the group consisting of perforin, FasL, CXCR4, and CXCR2. In other embodiments, at least 90% of the CD3 ⁺ CD8 ⁺ cells are characterized by surface expression of CXCR3 and at least 30% of the CD3 ⁺ CD8 ⁺ cells are characterized by surface expression of CXCR6.

CXC chemokine receptors are integral membrane proteins that specifically bind and respond to cytokines of the CXC chemokine family. Structurally, such receptors are characterized as G protein-linked receptors, known as seven-transmembrane (7-TM) proteins, because they span the cell membrane seven times.

CXCR4 (also known as fusin) is the receptor for the chemokine known as CXCL12 (or SDF-1) as its canonical ligand, a molecule with strong chemotactic activity for lymphocytes. CXCR4 has a wide cellular distribution and is expressed on various hematopoietic cell types (e.g., neutrophils, monocytes, T and B cells, dendritic cells, Langerhans cells and macrophages). CXCR4 activation can promote both cell migration and cell proliferation, and CXCL12 is also known to be important in hematopoietic stem cell homing to the bone marrow and in hematopoietic stem cell quiescence. Several additional ligands for CXCR4 have been disclosed, including the damage-associated molecular pattern molecule high mobility group box 1 protein (HMGB1), macrophage migration inhibitory factor (MIF), a cytokine involved in the regulation of innate immunity, extracellular ubiquitin (eUb) and beta defensin-3 (HBD3).

CXCR2, also known as interleukin 8 receptor beta (IL8RB), is expressed on the surface of neutrophils and certain other cell types. CXCR2 binds with high affinity to IL-8 (also known as CXCL8), a chemokine produced by macrophages and other cell types, including epithelial cells, airway smooth muscle cells, and endothelial cells, which is the primary cytokine involved in the recruitment of neutrophils to sites of injury or infection. Other ligands for CXCR2 include, for example, CXCL2, CXCL3, and CXCL5.

CXCR3 and its ligands, CXCL9, CXCL10, and CXCL11, are important immunochemoattractants during interferon-induced inflammatory responses. CXCR3 (GPR9/CD183) is an interferon-inducible chemokine receptor expressed in a variety of cell types, but is preferentially expressed in monocytes, Th1 T cells, CD8 ⁺ T cells, NKT cells, NK cells, dendritic cells, and some cancer cells. There are three isoforms of CXCR3 in humans: CXCR3-A, CXCR3-B, and chemokine receptor 3 alternative (CXCR3-alt). CXCR3-A binds to the CXC chemokines CXCL9 (MIG), CXCL10 (IP-10), and CXCL11 (I-TAC), whereas CXCR3-B can bind to CXCL9, CXCL10, and CXCL11 as well as CXCL4.

CXCR6 (also known as STRL33 and TYMSTR) is a chemokine receptor expressed, for example, on naive CD8 ⁺ cells, CD3 ⁻ CD16 ^−/low CD56 ⁺ and CD3 ⁻ CD16 ^low CD56 ⁻ NK cells, NKT cells, activated CD4 ⁺ and CD8 ⁺ T cells, and 30-40% of γδ T cells. CXCR6 binds only to CXCL16 and plays a role in various immune methods, such as T cell recruitment in graft-versus-host disease, maintenance of liver-resident CD8 T cells after infection, and modulating lung disease severity. Its ligands are chemotactic cytokines belonging to the α-chemokine subfamily that play important roles in tumor cell proliferation, migration, invasion, and metastasis. In particular, it has been suggested that activation of CXCR6 is important for the survival and proliferation of cytotoxic T cells in the tumor microenvironment, thereby attenuating T cell exhaustion.

Additionally, the cell compositions of the present invention are further characterized by surface expression of DNAM-1, hi other embodiments, at least 90%, 93%, 95%, 97%, and typically substantially all (up to 100%) of the CD3 ⁺ CD8 ⁺ cells are characterized by surface expression of DNAM-1.

DNAX accessory molecule-1 (DNAM-1, CD226) is a transmembrane nectin-like glycoprotein of the immunoglobulin superfamily that acts as an activating receptor in NK and T cells. Its ligands, CD112 (also known as poliovirus receptor (PVR)) and CD155 (also known as nectin-2), are also members of the nectin family, and the DNAM-1-CD112/CD155 axis is primarily known for its key role in NK cell-mediated tumor cell killing. It has been proposed that the DNAM-1/PVR axis is involved in NK cell-mediated lysis of allogeneically activated T cells, whereas NKG2D appears as the primary receptor in the autologous setting.

In other embodiments, the cell compositions of the present invention are characterized by reduced expression of inhibitory immune checkpoint molecules.

Immune checkpoint molecules represent an immune evasion mechanism that prevents the immune system from attacking one's own body by inducing self-tolerance. Normally, immune checkpoint receptors are present on T cells and interact with immune checkpoint ligands expressed on antigen-presenting cells. However, some cancers can stimulate immune checkpoint targets to protect themselves from attack. T cells recognize antigens presented on MHC molecules and become activated to trigger an immune response, while the interaction of immune checkpoint receptors with ligands in parallel controls T cell activation.

Immune checkpoint receptors include costimulatory receptors and inhibitory receptors, and T cell activation and immune responses are controlled by the balance between these two receptors. As used herein, the term "inhibitory immune checkpoint molecule" specifically encompasses inhibitory immune checkpoint receptors that are expressed on immune effector cells such as T cells and that can mediate downregulation or inhibition of antitumor immune responses upon binding with their respective ligands. Examples include, but are not limited to, CTLA-4, PD-1, VISTA, B7-H3, B7-H4, HVEM, KIR family receptors, TIM-1, TIM-3, LAG-3, and TIGIT.

According to certain embodiments, the CD3 ⁺ CD8 ⁺ cells of the ACT compositions of the invention are characterized by reduced surface expression of one or more immune checkpoint molecules selected from the group consisting of CTLA-4, PD-1, TIGIT, Lag-3, and Tim-3. In various embodiments, such reduced surface levels may be associated with a reduction relative to the original PBMC sample, or a reduction relative to other ACT compositions, such as CIK cell preparations, as shown herein. Each possibility represents a separate embodiment of the present invention.

In other embodiments, the CD3 ⁺ CD8 ⁺ cells are characterized by a substantial lack of surface expression of CTLA-4. In other embodiments, at least 95%, 97%, and up to about 100% of the CD3 ⁺ CD8 ⁺ cells are CTLA-4 ⁻ cells. Cytotoxic T-lymphocyte antigen 4 (CTLA-4/CD152) is a co-receptor constitutively expressed on regulatory T cells and is upregulated on conventional CD4 ⁺ and CD8 ⁺ T cells following activation, an event particularly prominent in cancer. CTLA-4 binds to CD80 and CD86 on APCs with higher affinity and avidity than the activating receptor CD28, and is therefore able to outcompete CD28 for its ligand and promote T-cell inhibition.

In other embodiments, no more than 25%, 20%, 15%, or 10% of the CD3 ⁺ CD8 ⁺ cells are characterized by surface expression of PD-1. In other embodiments, about 15% of the CD3 ⁺ CD8 ⁺ cells are characterized by surface expression of PD-1. Programmed cell death protein 1 (PD1) is expressed by all T cells during activation, as well as regulatory T cells, B cells, natural killer cells, and some myeloid cell populations. PD1 often shows high and sustained expression levels upon persistent antigen encounter, which may occur in the setting of chronic infection or cancer. In such settings, PD1 may limit protective immunity. PD1 has two ligands, PD-L1 and PD-L2, and expression of PD-L1 in the tumor microenvironment is commonly associated with tumor immune evasion.

In other embodiments, no more than 30%, 25%, 22%, 20%, or 18% of the CD3 ⁺ CD8 ⁺ cells are characterized by surface expression of TIGIT. In other embodiments, about 20-22% of the CD3 ⁺ CD8 ⁺ cells are characterized by surface expression of TIGIT. T cell immunoglobulin with ITIM domain (TIGIT) is an inhibitory receptor expressed on NK cells and T cells, including CD4 ⁺ T cells, CD8 ⁺ T cells, and ^Tregs . TIGIT expression is normally low on naive cells, but both T cells and NK cells have been shown to upregulate TIGIT upon activation. TIGIT has three ligands, CD155 (poliovirus receptor: PVR, NECL-5), CD112, and CD113, all of which belong to the nectin and NECL families of molecules.

In other embodiments, up to 55%, 53%, 51%, 50%, or 48% of the CD3 ⁺ CD8 ⁺ cells are characterized by surface expression of Lag-3. In other embodiments, about 50-51% of the CD3 ⁺ CD8 ⁺ cells are characterized by surface expression of Lag-3. Lymphocyte activation gene 3 (LAG3, also known as CD223) is a member of the immunoglobulin superfamily expressed on activated T cells, TILs, _Tregs , NK cells, B cells, and plasmacytoid DCs. LAG3 interacts with MHC-II to inhibit binding of the same MHC molecule to TCR and CD4, directly interfering with TCR signaling in immune responses. Additional LAG3 ligands include FGL-1, α-synuclein fibrils (α-syn), the lectin galectin-3 (Gal-3), and lymph node sinusoidal endothelial cell C-type lectin (LSECtin). LAG3, like other immune checkpoint molecules, has been reported to negatively regulate T cell proliferation, activation, and homeostasis, and play a role in Treg suppressive function. For example, in T cells, LAG3 suppresses the production and proliferation of cytokines and granzymes, while promoting differentiation into regulatory T cells.

In other embodiments, up to 55%, 54%, 52%, 50%, or 48% of the CD3 ⁺ CD8 ⁺ cells are characterized by surface expression of Tim-3. In other embodiments, about 50-54% of the CD3 ⁺ CD8 ⁺ cells are characterized by surface expression of Tim-3. T-cell immunoglobulin and mucin domain-containing protein 3 (TIM3), a member of the TIM family, is expressed by interferon-γ (IFNγ)-producing CD4 ⁺ and CD8 ⁺ T cells, as well as by many other cell types, including regulatory T cells, myeloid cells, NK cells, and mast cells. TIM3 has been reported to have several different ligands, such as galectin 9, phosphatidylserine, carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), and high mobility group protein B1 (HMGB1). TIM3 is part of a module that contains multiple co-inhibitory receptors (checkpoint receptors), which are co-expressed and co-regulated in dysfunctional or "exhausted" T cells in chronic viral infections and cancer.

In other embodiments, the cell composition exerts cytotoxic activity against tumor cells. In some embodiments, the tumor cells are of hematopoietic origin. The phrase "tumor cells of hematopoietic origin" refers to malignant cells derived from blood cells or their precursors. In other embodiments, the tumor cells are of lymphoid origin. In other embodiments, the tumor cells are of myeloid origin. In certain embodiments, the tumor cells are leukemia cells, e.g., acute myeloid leukemia (AML) cells. In other specific embodiments, the tumor cells are multiple myeloma cells.

According to exemplary embodiments, the cell composition is capable of specifically eradicating (killing) hematopoietic tumor cells when incubated in vitro with the hematopoietic tumor cells at a ratio of at least 0.25:1 (respectively) for 24 hours. According to further exemplary embodiments, the cell composition is capable of specifically eradicating at least 50%, typically 60-99%, more typically about 80-90% of the hematopoietic tumor cells when incubated in vitro with the hematopoietic tumor cells at a ratio of 1:1 for 24 hours. In other embodiments, the tumor cells express at least one, typically multiple NKG2D ligands (e.g., but not limited to, MICA, MICB, HLAE, ULBP1, ULBP256, and ULBP3). In other embodiments, the tumor cells express MICA, MICB, HLAE, ULBP1, ULBP256, and ULBP3. In other embodiments, as shown herein, the cell composition can specifically eradicate hematopoietic tumor cells to a greater extent than the CIK composition under the same conditions (e.g., 6-10 fold greater at a 1:1 effector to tumor cell ratio, and up to about 15 fold greater at a 0.5:1 effector to tumor cell ratio). In other embodiments, the tumor cells are solid tumor cells. In certain embodiments, the cells are prostate tumor cells or mesothelioma cells. Each possibility represents a separate embodiment of the present invention.

As used herein, the term "specific eradication" of tumor cells refers to targeted tumor cell death that is selective (or preferential) for the target tumor cells. Typically, eradication is exerted by cytotoxic activity induced by the tumor cells (e.g., upon contact of the cell composition with the tumor cells under conditions disclosed herein).

The terms "cytotoxicity" and "cytotoxic activity" as used herein refer in particular to cell-mediated cytotoxicity or cytolysis, i.e., the cell-killing activity of immune cells, particularly effector immune cells. Prominent cytotoxic effector cells of the immune system include NK cells, cytotoxic T cells (e.g., CD8 ⁺ T cells) and NK-T cells. Cytotoxic cells (e.g., CTL or NK cells) can kill target cells by apoptosis or lysis of the target cell using one or more different mechanisms, including, but not limited to, release of one or more cytotoxins stored in cytolytic granules or expression of Fas ligand (FasL). Examples of cytotoxins include, for example, perforin, granzymes and granulysin. Currently known granzymes are granzymes A, B, H, K and M. Such cytotoxic or cytolytic activity can be measured and quantified using standard techniques, for example, by assays using selective labeling of live cells. For example, but without limitation, succinimidyl ester diacetate (CFSE) and/or propidium iodide based assays, or other suitable assays known in the art, may be used.

In other embodiments, the cytotoxic activity is mediated by granzyme B and/or perforin. In other embodiments, the cytotoxic activity is not substantially mediated by FAS-FasL interactions.

Perforin is a pore-forming cytolytic protein that translocates to target cells in a Ca ^+2- dependent manner. Upon releasing cytolytic granules in close proximity to the cell to be killed, perforin creates pores in the plasma membrane of the target cell through which granzymes and related molecules can enter and induce apoptosis. Granzyme B (also known as granzyme 2 and cytotoxic T lymphocyte-associated serine esterase 1) is a particularly important serine protease that rapidly induces apoptosis of target cells in cell-mediated immune responses by cleaving and activating caspases. The FAS receptor, also known as Fas, FasR, apoptosis antigen 1 (APO-1 or APT), cluster of differentiation 95 (CD95) or tumor necrosis factor receptor superfamily member 6 (TNFRSF6), is a cell surface death receptor that triggers programmed cell death (apoptosis) upon binding to its ligand, Fas ligand (FasL). Expression of FasL by immune effector cells, and subsequent FAS-FasL interaction upon contact with FAS-expressing target tumor cells, can induce tumor cell apoptosis in a perforin-independent manner. Such perforin-independent cytotoxic activity can be examined in a ^Ca2+ -independent system in which polymerization of perforin monomers is not possible, yet cell-mediated cytolysis still occurs.

As used herein, cytotoxic activity mediated by a particular agent or pathway (e.g., perforin, granzyme B, or FAS-FasL) refers to activity that is triggered by and/or substantially dependent on the involvement or activity of the agent. Such activity requires that the agent is activated after expression in the corresponding effector cell. Thus, in the presence of a particular pharmacological inhibitor or antibody to the agent or pathway, the cytotoxic activity is significantly reduced (typically at least 50%, more typically at least 60%, 70%, 80%, 90%, and up to 100% inhibition) compared to the activity under the same conditions in the absence of the inhibitor or antibody, indicating that the activity is mediated by the agent (e.g., perforin or granzyme B). Similarly, the absence of a significant reduction (statistically significant, or in some embodiments, a reduction of 5%, 10%, 20%, or 30% or less) indicates that the activity is not mediated by the agent (e.g., FasL). For example, the cytotoxicity assays disclosed herein can be performed in the presence of 3,4-dichloroisocoumarin (DCI) or other commercially available granzyme B inhibitors, concanamycin A (CMA) or other commercially available perforin inhibitors, or anti-FasL (or anti-FAS) blocking antibodies available from Sigma or other suppliers. Non-limiting examples of such assays are described in the Examples below.

As shown herein, the cell compositions of the present invention have been found to have both MHC-restricted and non-MHC-restricted cytotoxic activity against a variety of tumor cells. Thus, in one embodiment, the cytotoxic activity is MHC-dependent (i.e., requires the presence or activity of an MHC molecule on the cell). In another embodiment, the cytotoxic activity is non-MHC-dependent. In another embodiment, the cytotoxic activity is substantially MHC I-independent. In another embodiment, the cytotoxic activity is substantially MHC II-independent.

In other embodiments, the cell compositions of the present invention can exert tumor-specific cytotoxic activity without substantial cytotoxic activity against non-tumor cells. In other embodiments, the cell compositions of the present invention do not substantially induce graft-versus-host (GVH) reactions or graft-versus-host disease (GVHD). Thus, in some embodiments, the cell compositions of the present invention need not be histocompatible with the subject being treated. Thus, autologous, histocompatible allogeneic, and non-histocompatible allogeneic PBMC samples can be used to prepare the compositions of the present invention.

As used herein, the term "graft-versus-host disease" or "GVHD" refers to an inflammatory disease resulting from an attack by transplanted leukocytes against the tissues of the transplant recipient. GVHD typically occurs in an immunocompromised host when the graft is recognized as non-self by immunocompetent cells of the graft and encompasses acute and/or chronic GVHD. In the context of ACT, the term GVHD specifically refers to the pathological process and clinical symptoms resulting from damage induced by adoptively transferred immune cells to non-tumor cells, tissues and/or organs of the recipient subject. GVHD often manifests as inflammation-mediated damage to epithelial tissues, particularly the skin, liver and gastrointestinal mucosa.

The term "histocompatibility" refers to the similarity of tissues between different individuals. The level of histocompatibility represents how well the patient and donor are matched. The major histocompatibility determinants are the human leukocyte antigens (HLA). HLA typing is performed between a potential donor and a potential recipient to determine how well their HLAs match. As used herein, the term "histocompatibility" refers to an embodiment in which all six HLA antigens (two A antigens, two B antigens, and two DR antigens) are identical between the donor and the recipient.

As further shown herein, the cell compositions of the present invention exhibit significant cytotoxic activity against tumor cells (including non-histocompatible tumor cells) in vivo. For example, as shown in Example 8 herein, the compositions of the present invention exhibited significant cytotoxic activity against lymphoid tumor cells, inhibiting tumor development by at least 78% to 97%. Thus, in some embodiments, the present invention relates to cell compositions that can inhibit the development of hematopoietic tumors in the spleen by at least 50%, at least 60%, at least 70%, or at least 80%, 90%, 95%, or 97%, each possibility representing a separate embodiment of the present invention. Furthermore, advantageous compositions of the present invention (compositions of the present invention) exhibit significantly higher cytotoxic activity against non-histocompatible tumor cells in vivo than that exerted by PBMCs (generally considered to be substantially ineffective in eradicating tumor cells in vivo) or various PBMC-derived compositions such as CIK and LAK.

In another embodiment, an ACT cell composition is provided that comprises ^5x106-10x109 viable in vitro expanded PBMCs, of which 70-85% are CD3 ⁺ CD8 ⁺ cells expressing NKG2D, DNAM-1 ⁺ and granzyme B, at least 65% are ^CD56- cells, and up to 8% are ^CD3- cells, and that exhibit significant cytotoxic activity against tumor cells in vitro and in vivo. In another embodiment, 5-15% of the viable cells are CD3 ^{+ CD4 +} cells, 15-30% are CD3 ⁺ CD56 ⁺ cells, and 7-17% are CD3 ⁺ CD8 ⁺ CD56 ⁺ cells. In another embodiment, the cell composition is characterized by a ratio of CD3 ⁺ CD8+ cells to CD3 ⁺ CD4 ⁺ cells of 7-8, and/or comprises less than ⁵ % CD3 ^- CD56 ⁺ cells.

Genetically Modified Cells In other embodiments, the cell compositions of the invention are genetically modified. In one embodiment, the cells are modified to express at least one receptor that confers tumor specificity, such as a recombinant T cell receptor (TCR) or a chimeric antigen receptor (CAR). In other embodiments, cell compositions are provided that include a recombinant construct of the invention, or a unique combination of recombinant constructs disclosed below.

CARs combine a binding site (i.e., "binding moiety") of a molecule that binds strongly to the target antigen with the cytoplasmic domain ("signaling moiety") of a conventional immune receptor that is responsible for initiating signal transduction leading to lymphocyte activation. Most commonly, the binding moiety used is derived from the structure of a Fab (antigen-binding) fragment of a monoclonal antibody (mAb) that has high affinity for the target antigen. Since Fab is the product of two genes, the corresponding sequences are usually linked via a short linker fragment, which allows the heavy chain to fold onto a peptide derived from the light chain in its native conformation, forming a single-chain fragment variable (scFv) region. Many of the original known CAR systems were also called "T-bodies" because they bind antibody fragments to T cells. Other possible antigen-binding moieties include signaling moieties of hormone or cytokine molecules, extracellular domains of membrane receptors, and peptides derived from library screening (e.g., phage display). Antigen targets suitable for the CARs used in the compositions of the invention are the disease-specific antigens disclosed herein.

The term "antibody" is meant to encompass both intact molecules and fragments thereof that contain an antigen-binding site. The antibodies disclosed by the present invention may be fully synthetic, in which case the polypeptide chains of the antibody are synthesized and, optionally, optimized to bind to the polypeptides disclosed herein as receptors. Such antibodies may be chimeric or humanized, and may be fully tetrameric in structure, or dimeric, containing only a single heavy chain and a single light chain.

Methods for producing monoclonal and polyclonal antibodies are well known in the art. Antibodies can be produced by any one of several known methods, including inducing in vivo production of antibody molecules, screening immunoglobulin libraries, or producing monoclonal antibody molecules by continuous cell lines in culture. Such methods include, but are not limited to, hybridoma technology, human B-cell hybridoma technology, and Epstein-Barr Virus (EBV) hybridoma technology. In addition to traditional methods for producing antibodies in vivo, antibodies can be produced in vitro using phage display technology by methods well known in the art (e.g., Current Protocols in Immunology, Colligan et al (Eds.), John Wiley & Sons, Inc. (1992-2000), Chapter 17, Section 17.1). Single-chain Fvs are prepared by constructing a structural gene that includes DNA sequences encoding the heavy and light chain variable domains linked by an oligonucleotide encoding a peptide linker. The structural gene is inserted into an expression vector, which is then introduced into a host cell such as E. coli. The recombinant host cell synthesizes a single polypeptide chain with a linker peptide bridging the two variable domains. Ample guidance for producing single-chain Fvs is provided in the literature in the art.

With respect to the cytoplasmic domain, the CAR can be designed to include a signaling domain of a costimulatory molecule, e.g., CD80, CD86, CD40, CD83, 4-1BB (CD137), CD28, and/or CD3ζ signaling domain, alone or in combination with other desired cytoplasmic domains useful in the context of the CAR. In other embodiments, the CAR includes a signaling domain of a costimulatory molecule selected from the group consisting of 4-1BB, CD28, CD3ζ, or a combination thereof. In an exemplary embodiment, the CAR-modified cells of the invention can be produced by introducing a viral vector, such as a lentiviral vector, into a cell that includes a desired CAR, e.g., a CAR that includes an anti-CD19 binding domain, a transmembrane domain, and a cytoplasmic signaling domain. Alternatively, a vector that is stably maintained in the cell without being integrated into the genome is used. In other embodiments, the CAR-modified cells of the invention can be produced by transducing or transfecting a gene encoding such a CAR molecule into the cell.

In certain embodiments, the cells are genetically modified to express a CAR, e.g., a CD19-specific CAR or a CD123-specific CAR. The B-lymphocyte antigen CD19, also known as the CD19 molecule (cluster of differentiation 19), B-lymphocyte surface antigen B4, T-cell surface antigen Leu-12 and CVID3, is a transmembrane protein that in humans is encoded by the gene CD19. In humans, CD19 is expressed on all B-lineage cells. CD19 plays two major roles in human B cells. On the one hand, it acts as an adaptor protein that recruits cytoplasmic signaling proteins to the membrane, and on the other hand, it acts within the CD19/CD21 complex to lower the threshold of the B-cell receptor signaling pathway. As it is present on all B cells, it is a biomarker for B-lymphocyte development, lymphoma diagnosis, and can be used as a target for leukemia immunotherapy. The interleukin-3 receptor (CD123) is a molecule present on cells that helps signal interleukin-3, a soluble cytokine important in the immune system. The gene encoding the receptor is located in the pseudoautosomal regions of the X and Y chromosomes. This receptor belongs to the type I cytokine receptor family and is a heterodimer consisting of a unique α chain paired with a common β (βc or CD131) subunit. The gene for the α subunit is 40 kilobases long and has 12 exons.

In one exemplary embodiment, the CAR comprises an scFv of an anti-CD19 antibody linked to 4-1BB and CD3ζ signaling domains. In other exemplary embodiments, the CAR can further comprise additional domains, such as, but not limited to, a hinge region and/or a transmembrane region from CD8. In other exemplary embodiments, the CAR can comprise an scFv of an anti-CD19 antibody linked to CD28 and CD3ζ signaling domains. Non-limiting examples of specific CAR molecules and constructs encoding same that can be used in accordance with embodiments of the present invention are described in Examples 8 and 9 below.

In other embodiments, CARs can be used against other targets, such as BCMA, CD47, PDL-1, mesothelin, EpCAM, CD34, CD44, PSCA, MUC16, CD276, CD123, CD19, CD20, and EFFRvIII. In still other embodiments, a variety of other markers can be used as CAR targets, such as AFP, Axl, B7-H6, BCMA, biotin, CD10, CD117, CD123, CD133, CD138, CD19, CD20, CD200, CD22, CD276, CD30, CD33, CD34, CD38, CD44, CD5, CD70, CD79A, CD80, CD86, CD99, CEA, CLD Examples of receptors include, but are not limited to, N18.2, c-Met, CS1, DLL3, DR5, EGFR, EGFRvIII, EpCAM, EphA2, ErbB2, ERBB3, FAP, fibroblast activation protein (FAP), FRα, GD2, GPC3, IL1RAP, ITGB7, LeY, mesothelin, MUC1, MUC16, PD-1, PD-L1, PMEL, PSCA, PSMA, and VEGFR2. Sequences for a variety of receptors are available and can be used to generate nucleic acid constructs for genetic modification. For example, CAR constructs against CD19 or CD123 for use in modifying T cell compositions are disclosed, but are not limited to, in US20140271635 and US20140322212, respectively.

In other embodiments, constructs encoding recombinant TCRs against tumor associated antigens (TAA) or other targets useful, for example, in the treatment of cancer, can be used. Non-limiting examples of well-known TAAs are MART-1, gp100 _209-217 , gp100 _154-163 , CSPG4, NY-ESO, MAGE-A1, and tyrosinase. Such TCR constructs are known in the art and can be obtained or produced by recombinant methods.

In other embodiments, the cells are modified to express at least one cytokine, chemokine, or receptor thereof. In one embodiment, the cells are genetically modified to express a modified chemokine receptor. In one embodiment, the chemokine receptor is modified to enhance tumor localization or retention. As used herein, enhanced tumor localization or retention refers to the ability of adoptively transferred effector cells to migrate, penetrate, and/or proliferate in the tumor microenvironment, and is manifested as an increase in the relative number of identifiable effector cells in the tumor after adoptive transfer, compared to levels in the absence of such transgenic receptor. For example, (e.g., when the tumor to be treated originates from or resides in bone marrow), the modified chemokine receptor advantageously enhances bone marrow retention of the transgenic cells. In certain embodiments, the modified chemokine receptor is a modified CXCR4 receptor that includes a mutation or truncation of the C-terminal tail domain (e.g., resulting in enhanced tumor localization and/or retention as described herein). For example, WHIM mutations in the CXCR4 gene (identified in patients with WHIM syndrome) are associated with C-terminal truncations of the encoded receptor (e.g., removal of 15-20 C-terminal amino acids (aa)). In certain embodiments, modified CXCR4 is characterized by a 19 C-terminal aa truncation, for example, due to an R334X mutation (also referred to herein as a WHIM R334x mutation, which is a CXCR4 containing a truncation mutation after residue 333, exemplified by SEQ ID NO: 8 below). By way of non-limiting example, such modified cells are particularly useful for the treatment of hematopoietic tumors and metastases localized to the bone marrow.

In other embodiments, the cells are genetically modified to express at least one cytokine of the IL-2 family. For example, the cells can be modified to exogenously express (e.g., constitutively or upregulated) IL-2, IL-15, and/or IL-21. Each possibility represents a separate embodiment of the invention. In other embodiments, the cell composition expresses any one of exogenous IL-2, exogenous IL-15, exogenous IL-21, or any combination thereof. The term "exogenous" when used with reference to a protein, gene, nucleic acid, or polynucleotide in a cell or organism means a protein, gene, nucleic acid, or polynucleotide that has been introduced into the cell or organism by artificial or natural means. An exogenous nucleic acid may be from a different organism or cell, or may be one or more additional copies of a nucleic acid that is naturally present in the organism or cell. As a non-limiting example, an exogenous nucleic acid is a nucleic acid that is at a different chromosomal location than in a native cell, or that is adjacent to a different nucleic acid sequence than that found in nature. The exogenous nucleic acid can also be an extrachromosomal nucleic acid, such as an episomal vector.

IL-15 is a cytokine structurally and functionally similar to IL-2 mentioned above. Like IL-2, IL-15 binds to and signals through a complex composed of the IL-2/IL-15 receptor beta chain (CD122) and the common gamma chain (gamma-C, CD132). IL-15 is encoded by the IL15 gene and induces the proliferation of NK cells. IL-21 is encoded by the human IL21 gene and induces the proliferation of immune system members such as NK cells and cytotoxic T cells.

In some embodiments, the cells are characterized by multiple modifications. According to non-limiting, advantageous embodiments, disclosed herein are cell compositions modified to express (i) a CD19-specific CAR or a CD123-specific CAR, (ii) a modified CXCR4 receptor comprising a mutation or truncation in the C-terminal tail domain, and (iii) at least one cytokine selected from the group consisting of IL-2, IL-15, and IL-21. As disclosed herein, in some embodiments, such modified SAK cell compositions are particularly useful for treating tumors localized in the bone marrow, such as hematopoietic tumors and leukemias. In some embodiments, the cells are modified by transfection, transduction, or infection with a nucleic acid construct encoding (i) a CD19-specific CAR or a CD123-specific CAR, (ii) a modified CXCR4 receptor comprising a mutation or truncation in the C-terminal tail domain, and (iii) at least one cytokine selected from the group consisting of IL-2, IL-15, and IL-21. In other embodiments, the construct is a viral vector. In other embodiments, the construct further comprises at least one translational regulatory sequence, e.g., an internal ribosome entry site (IRES) sequence, or a self-cleaving sequence, such as a 2A peptide sequence. In exemplary embodiments, the translational regulatory sequence is located between the nucleic acid sequences encoding elements (i) and (ii) and/or between elements (ii) and (iii). In other specific embodiments, the elements are constitutively expressed.

In another embodiment, a cell composition is provided that includes leukocytes genetically modified to express (i) a CD19-specific CAR or a CD123-specific CAR, (ii) a modified CXCR4 receptor comprising a mutation or truncation of the C-terminal tail domain, and (iii) at least one cytokine selected from the group consisting of IL-2, IL-15, and IL-21.

In another embodiment, a nucleic acid construct is provided that encodes (i) a CD19-specific CAR or a CD123-specific CAR, (ii) a modified CXCR4 receptor comprising a mutation or truncation of the C-terminal tail domain, and (iii) at least one cytokine selected from the group consisting of IL-2, IL-15, and IL-21. In yet another embodiment, a nucleic acid construct of the invention disclosed herein is provided.

In particular, the preparation of nucleic acid constructs, including expression constructs or vectors used to deliver and express a desired gene product, is known in the art. An isolated nucleic acid sequence can be obtained from its natural source as an entire gene (i.e., a complete gene) or a portion thereof. Nucleic acid molecules can also be produced by recombinant DNA techniques (e.g., polymerase chain reaction (PCR) amplification, cloning) or chemical synthesis (see, e.g., Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York; Ausubel, et al., 1989, Chapters 2 and 4). Nucleic acid sequences include naturally occurring nucleic acid sequences and their homologs, including, but not limited to, naturally occurring allelic variants and modified nucleic acid sequences in which nucleotides have been inserted, deleted, substituted, and/or inverted in a manner that does not substantially interfere with the ability of the nucleic acid molecule to encode a functional peptide. A polynucleotide or oligonucleotide sequence can be deduced from the genetic code of a protein, taking into account the degeneracy of the code and the exception to the classical base pairing at the third position of a codon due to the so-called "Wobble rule". Polynucleotides containing approximately nucleotides can result in the same or equivalent protein. Using recombinant production methods, a selected host cell, for example a microorganism such as E. coli or yeast, is transformed with a hybrid virus or plasmid DNA vector containing a specific DNA sequence encoding a polypeptide or polypeptide analog, and the polypeptide is synthesized within the host upon transcription and translation of the DNA sequence.

Exemplary sequences of cytokines, chemokine receptors, CARs and other genetic elements that can be cloned into constructs according to embodiments of the invention are described in Examples 8 and 9 below. For example, an exemplary nucleic acid sequence for human IL-2 is set forth in SEQ ID NO:3, an exemplary nucleic acid sequence for human IL-15 is set forth in SEQ ID NO:4, an exemplary nucleic acid sequence for wild-type CXCR4 is set forth in SEQ ID NO:7, an exemplary nucleic acid sequence for modified C'-truncated CXCR4 is set forth in SEQ ID NO:8, an exemplary nucleic acid sequence for a CD19-specific CAR is set forth in SEQ ID NO:9, and an exemplary nucleic acid sequence for a CD123-specific CAR is set forth in SEQ ID NO:1. In other embodiments, functionally equivalent variants and homologues of these sequences that retain at least 90% sequence identity, e.g., 93%, 95%, 97%, or 99% homology, can be used.

As known in the art, the construct may also include other control sequences or selectable markers. The nucleic acid construct (also referred to herein as an "expression vector") may include additional sequences that render the vector suitable for replication and integration in prokaryotes, eukaryotes, or possibly both (e.g., shuttle vectors). Additionally, a typical cloning vector may also include transcription and translation initiation sequences, transcription and translation terminators, and a polyadenylation signal.

In addition to the elements already described, the expression vectors of the invention can typically contain other specialized elements intended to enhance the level of expression of the cloned nucleic acid or to facilitate identification of cells harboring the recombinant DNA. For example, many animal viruses contain DNA sequences that promote extrachromosomal replication of the viral genome in permissive cell types. Plasmids carrying such viral replicons will replicate episomally as long as the appropriate factors are provided by genes carried on the plasmid or in the genome of the host cell.

A vector may or may not contain a eukaryotic replicon. If a eukaryotic replicon is present, the vector is amplifiable in a eukaryotic cell using an appropriate selectable marker. If the vector does not contain a eukaryotic replicon, episomal amplification is not possible. Instead, the recombinant DNA is integrated into the genome of the engineered cell, and the promoter directs expression of the desired nucleic acid.

Examples of mammalian expression vectors include, but are not limited to, pcDNA3, pcDNA3.1(+/-), pGL3, pZeoSV2(+/-), pSecTag2, pDisplay, pEF/myc/cyto, pCMV/myc/cyto, pCR3.1, pSinRep5, DH26S, DHBB, pNMT1, pNMT41, and pNMT81 (available from Invitrogen), pCI (available from Promega), pMbac, pPbac, pBK-RSV and pBK-CMV (available from Strategene), pTRES (available from Clontech) and derivatives thereof, which can serve as vector backbones for constructs useful in the embodiments described herein.

Expression vectors containing regulatory elements of eukaryotic viruses such as retroviruses can also be used. SV40 vectors include, for example, pSVT7 and pMT2. Vectors derived from bovine papilloma virus include pBV-1MTHA, and Epstein-Barr virus include pHEBO and p2O5. Other examples of vectors include pMSG, pAV009/A ⁺ , pMTO10/A ⁺ , pMAMneo-5, baculovirus pDSVE, and any other vector that allows expression of a protein under the direction of the SV40 early promoter, SV40 late promoter, metallothionein promoter, mouse mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown to be effective for expression in eukaryotic cells. These can serve as vector backbones for the constructs of the present invention.

As mentioned above, viruses are very specialized infectious agents that have evolved to often evade the host's defense mechanisms. Typically, viruses infect and grow in specific types of cells. The targeting specificity of viral vectors exploits their natural specificity to specifically target a given cell type, thereby introducing a recombinant gene into the infected cell. Thus, the type of vector used by the present invention depends on the type of cell to be transformed. The ability to select an appropriate vector depending on the cell type to be transformed is well within the capabilities of one of skill in the art, and therefore a general discussion of selection considerations is not provided herein.

Vectors derived from retroviruses include, for example, lentiviral vectors. "Lentiviral vector" and "recombinant lentiviral vector" are derived from a subset of retroviral vectors known as lentiviruses. By lentiviral vector is meant a nucleic acid construct that carries and, in certain embodiments, directs the expression of a nucleic acid molecule of interest. Lentiviral vectors include at least one transcriptional promoter/enhancer or locus-defining element or other elements that control gene expression by other means such as alternative splicing, nuclear RNA transport, post-translational modification of messengers, or post-transcriptional modification of proteins. Such vector constructs should also include packaging signals, long terminal repeats (LTRS) or portions thereof, and plus-strand and minus-strand primer binding sites appropriate for the lentiviral vector used (if these are not already present in the retroviral vector). Optionally, the recombinant lentiviral vector can also include a signal directing polyadenylation, a selectable marker such as Neo, TK, hygromycin, phleomycin, histidinol, or DHFR, and one or more restriction sites and a translation termination sequence. By way of example, such vectors typically include a 5'LTR, a tRNA binding site, a packaging signal, an origin of second strand DNA synthesis, and a 3'LTR or a portion thereof.

"Lentiviral vector particle" may be utilized within the present invention and refers to a lentivirus carrying at least one gene of interest. Retroviruses may also include a selectable marker. Recombinant lentiviruses can reverse transcribe their genetic material (RNA) into DNA upon infection and integrate this genetic material into the DNA of a host cell. Lentiviral vector particles may have a lentiviral envelope, a non-lentiviral envelope (e.g., an amphotropic or VSV-G envelope), or a chimeric envelope.

In some embodiments, the modified cell composition is an inventive SAK cell composition or an ACT cell composition as disclosed herein, for example, the ACT cell composition comprises at least 5×10 ⁶ to 10×10 ⁸ viable in vitro expanded PBMCs, of which 70-85% are CD3 ⁺ CD8 ⁺ cells expressing NKG2D and granzyme B, at least 70% are CD56 ⁻ cells, and up to 5% are CD3 ⁻ cells, the composition exhibits significant cytotoxic activity against tumor cells in vitro and in vivo, and is amenable to genetic modification as disclosed herein.

In other embodiments, other leukocyte preparations or populations (e.g., but not limited to, T cells, NK cells, NK-T cells, and DCs, and various ACT compositions known in the art (e.g., TIL, CIK, and LAK)) can be used for modification. However, it should be understood that the use of leukocytes that have not been modified by the improved constructs of the present invention disclosed herein should be distinguished from the teachings and principles of the present invention. According to certain embodiments, the use of such alternative leukocyte compositions is expressly excluded.

Therapeutic Uses In another aspect, the cell compositions of the present invention can be used to treat cancer. In another aspect, a method of treating a tumor in a subject in need of such treatment is provided, comprising contacting tumor cells with a therapeutically effective amount of the cell composition of the present invention, thereby treating the tumor in the subject. In another embodiment, an ACT composition for use in treating a tumor in a subject in need of such treatment is provided, the ACT composition comprising at least 5×10 ⁶ to 10×10 ⁸ viable in vitro expanded peripheral blood mononuclear cells (PBMCs), 70-85% of which are CD3 ⁺ CD8 ⁺ cells expressing NKG2D and granzyme B, at least 70% of which are CD56 ⁻ cells, and up to 5% of which are CD3 ⁻ cells, and which exhibit significant cytotoxic activity against tumor cells in vitro and in vivo. In another embodiment, there is provided a cell composition for use in treating a tumor in a subject in need thereof comprising: (i) a CD19-specific CAR or a CD123-specific CAR, (ii) a modified CXCR4 receptor comprising a mutation or truncation of the C-terminal tail domain, and (iii) a leukocyte genetically modified to express at least one cytokine selected from the group consisting of IL-2, IL-15, and IL-21.

As used herein, the terms "tumor" and "cancer" are used interchangeably and may refer to both solid and non-solid malignant tumors. In another embodiment, the tumor is a hematopoietic tumor. Non-limiting examples of lymphoid hematopoietic tumors include, for example, leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hairy cell lymphoma, and Burkitt's lymphoma. Non-limiting examples of myeloid hematopoietic tumors include, for example, acute and chronic myeloid leukemia, myelodysplastic syndromes and promyelocytic leukemia, tumors of mesenchymal origin such as fibrosarcoma and rhabdomyosarcoma. In another embodiment, the tumor is a solid tumor. Non-limiting examples of solid tumors include, for example, lung tumors, gastric tumors, ovarian tumors, breast tumors, colorectal tumors, breast tumors, pancreatic tumors, and renal tumors. In various embodiments, the tumor is selected from the group consisting of leukemia, multiple myeloma, prostate cancer, mesothelioma, lung cancer, and pancreatic cancer, with each possibility representing a separate embodiment of the present invention. In certain embodiments, the tumor is selected from the group consisting of leukemia, multiple myeloma, prostate cancer, and mesothelioma. In certain embodiments, the tumor is AML.

In other embodiments, the tumor is characterized by expression of at least one NKG2D ligand. In some embodiments, the NKG2D ligand is selected from the group consisting of MICA, MICB, HLAE, ULBP1, ULBP256, and ULBP3. In other embodiments, the tumor expresses multiple NKG2D ligands selected from the group consisting of MICA, MICB, HLAE, ULBP1, ULBP256, and ULBP3. In other embodiments, the tumor expresses MICA, MICB, HLAE, ULBP1, ULBP256, and ULBP3. Each possibility represents a separate embodiment of the present invention.

Advantageously, expression of markers such as NKG2D ligands or checkpoint molecule ligands by a tumor of interest can be confirmed by obtaining tumor cells (e.g., from a solid tumor biopsy or from a bodily fluid such as blood or urine) and examining the expression of the markers on the surface of the tumor cells by a suitable assay (e.g., including but not limited to, immunostaining and flow cytometry). Non-limiting examples of such assays are provided herein.

In other embodiments, the tumor is characterized by expression of at least one inhibitory immune checkpoint molecule and/or its ligand. In some embodiments, the tumor can express at least one ligand of CTLA-4, PD-1, TIGIT, Lag-3, Tim-3, and combinations thereof, each possibility representing a separate embodiment of the invention. In other embodiments, the tumor is characterized by expression of at least one ligand of an inhibitory immune checkpoint molecule selected from the group consisting of CTLA-4, PD-1, and TIGIT. In other embodiments, the tumor is characterized by expression of ligands of multiple inhibitory immune checkpoint molecules selected from the group consisting of CTLA-4, PD-1, and TIGIT. In other embodiments, the tumor is characterized by expression of at least one CTLA-4 ligand (e.g., CD80 and/or CD86).

In other embodiments, the tumor is resistant to treatment with at least one checkpoint molecule inhibitor. In other embodiments, the tumor is resistant to treatment with at least one inhibitor (e.g., a blocking antibody) of at least one checkpoint molecule selected from the group consisting of CTLA-4, PD-1, TIGIT, Lag-3, Tim-3, and ligands and combinations thereof, each possibility representing a separate embodiment of the invention.

According to an exemplary embodiment, the tumor is resistant to treatment with at least one CTLA4-specific inhibitor (e.g., a blocking antibody) selected from the group consisting of ipilimumab (YERVOY®) and tremelimumab. In other embodiments, the tumor is resistant to treatment with at least one PD1-specific inhibitor (e.g., a blocking antibody against PD-1 or PD-L1) selected from the group consisting of atezolizumab (TECENTRIQ®), durvalumab (IMFINZI®), avelumab (BAVENCIO®), nivolumab (OPDIVO®), and pembrolizumab (KEYTRUDA®). In other embodiments, the tumor is resistant to treatment with at least one TIGIT-specific inhibitor selected from the group consisting of tiragolumab (MTIG7192A), osipellimab (BGB-A1217), vibostolimab (MK-7684), domvanalimab (AB-154), BMS-986207, EOS-448, ASP-8374, COM-902, etigilimab (MPH-313), IBI-939, AGEN-1307, CASC-674, anti-PVR antibody (NB-6253), and PH-804. In other embodiments, the tumor is resistant to treatment with at least one LAG3-specific inhibitor selected from the group consisting of PRS-332, P13B02-3, LBL-007, eftilagimod alfa (IMP321), LAG525 (IMP701), MK-4280, REGN3767, leratolimab (BMS-986016), BI 754111, FS118, tebotelimab (MGD013), TSR-033, INCAGN2385, Sym022, and XmAb22841. In other embodiments, the tumor is resistant to treatment with at least one TIM3-specific inhibitor, e.g., ICAGN02390, Sym023, LY3321367, MGB453, TSR022, BGBA425, and BMS986258.

In other embodiments, the tumor is resistant to treatment with multiple checkpoint inhibitors or treatment with inhibitors of multiple checkpoint molecules. For example, but not limited to, the tumor may be resistant to treatment with a dual inhibitor that targets both TIM3 and PD1, such as R07121661, or combination therapy with nivolumab and ipilimumab.

In other embodiments, the subject is receiving a therapeutic regimen with one or more checkpoint molecule inhibitors. In other embodiments, the subject is not receiving a therapeutic regimen with a checkpoint molecule inhibitor. For example, YERVOY™ (ipilimumab) is a human CTLA-4 blocking antibody for intravenous infusion indicated for the treatment of unresectable or metastatic melanoma. OPDIVO® (nivolumab) is a PD-1 blocking antibody indicated for the treatment of unresectable or metastatic melanoma. KEYTRUDA® (pembrolizumab) is a PD-1 blocking antibody indicated for the treatment of melanoma, NSCLC, squamous cell carcinoma of the head and neck, classical Hodgkin lymphoma (cHL), primary mediastinal large B-cell lymphoma, urothelial carcinoma, microsatellite instability high or mismatch repair deficient cancers, microsatellite instability high or mismatch repair deficient colorectal cancer, gastric cancer, esophageal cancer, cervical cancer, hepatocellular carcinoma (HCC), Merkel cell carcinoma (MCC), renal cell carcinoma (RCC), endometrial cancer, cancers with high tumor mutation burden, cutaneous squamous cell carcinoma, and triple-negative breast cancer. TECENTRIQ® (atezolizumab) is a programmed death-ligand 1 (PD-L1) blocking antibody indicated for the treatment of locally advanced or metastatic urothelial carcinoma, metastatic NSCLC, metastatic non-squamous NSCLC without EGFR or ALK genomic tumor aberrations, small cell lung cancer (SCLC), extensive stage small cell lung cancer (ES-SCLC), unresectable or metastatic HCC, and melanoma. IMFINZI® (durvalumab) is a PD-L1 blocking antibody indicated for the treatment of adult patients with unresectable stage III NSCLC whose disease has not progressed after concurrent platinum-based chemotherapy and radiation therapy, in combination with etoposide and either carboplatin or cisplatin, as first-line treatment of adult patients with ES-SCLC. BAVENCIO® (avelumab) is a PD-L1 blocking antibody indicated for the treatment of MCC, UC, and RCC.

In other embodiments, the above-mentioned method or use further comprises administration (concurrently or sequentially in combination with the cell composition of the present invention) of at least one checkpoint molecule inhibitor disclosed herein. For example (e.g., where the tumor is characterized by surface expression of at least one ligand of Lag-3, Tim-3, or TIGIT), the above-mentioned method or use further comprises administration of at least one checkpoint molecule inhibitor against each of Lag-3, Tim-3, or TIGIT. In other embodiments, the above-mentioned method or use further comprises administration of at least one checkpoint molecule inhibitor against Lag-3, Tim-3, or a combination thereof. In yet other embodiments, the cell composition of the present invention can be used as the only therapeutic agent. For example, but not limited to, the composition of the present invention can be used as a monotherapy in the treatment of tumors characterized by surface expression of at least one ligand of CTLA-4 or PD-1. Each possibility represents a separate embodiment of the present invention.

In other embodiments, the tumor is characterized by expression of at least one chemokine selected from the group consisting of CXCR6 ligand, CXCR4 ligand, CXCR2 ligand, and CXCR3 ligand. In other embodiments, the tumor is characterized by expression of at least one chemokine selected from the group consisting of CXCR6 ligand, CXCR4 ligand, and CXCR3 ligand. In other embodiments, the tumor is characterized by expression of multiple chemokines selected from the group consisting of CXCR6 ligand, CXCR4 ligand, CXCR2 ligand, and CXCR3 ligand. In other embodiments, the tumor is characterized by expression of multiple chemokines selected from the group consisting of CXCR6 ligand, CXCR4 ligand, and CXCR3 ligand. In certain embodiments, the tumor is characterized by expression of a CXCR4 ligand (e.g., CXCL12). In other certain embodiments, the tumor is characterized by expression of a CXCR6 ligand (e.g., CXCL16). For example, CXCL16-expressing tumors can include, but are not limited to, non-small cell lung cancer (NSCLC) tumors, gastric tumors, ovarian tumors, breast tumors, colorectal tumors, breast tumors, pancreatic tumors, and renal tumors, with each possibility representing a separate embodiment of the present invention.

In other embodiments, the contacting is performed in vivo. Thus, the methods of the invention can include administering to a subject a therapeutically effective amount of a cell composition of the invention (e.g., an ACT cell composition), the composition comprising at least 5×10 ⁶ to 10×10 ⁸ viable in vitro expanded PBMCs, 70-85% of which are CD3 ⁺ CD8 ⁺ cells expressing NKG2D and granzyme B, at least 70% of which are CD56 ⁻ cells, and up to 5% of which are CD3 ⁻ cells, and the composition exhibits significant cytotoxic activity against tumor cells in vitro and in vivo. In other embodiments, the contacting is performed ex vivo. In other embodiments, the cells are administered to the subject simultaneously with administration of IL-2. In still other embodiments (e.g., when improved genetically modified cells are used), the cells can be advantageously used without co-administration of IL-2, thereby increasing safety and reducing side effects.

By "effective amount" or "therapeutically effective amount" is meant an amount sufficient to exert a beneficial result in the methods of the invention. More specifically, a therapeutically effective amount means an amount of active ingredient (e.g., effector cells) effective to prevent, reduce or ameliorate symptoms of a disorder (e.g., cancer) or to prolong the survival of the subject being treated. For example, an ACT composition of the invention for administration to a subject comprises an effective amount of at least ^5x106 to ^10x108 , typically up to about ^10x109 , viable cells expanded from PBMCs as disclosed herein. An effective amount of a cell composition for contacting tumor cells ex vivo can comprise, for example, at least about 0.25 to 1 effector cell for each tumor cell in culture.

In the context of an in vitro cell culture method, an effective amount of a cell stimulator, such as IL-2 and a CD3 activator, is an amount sufficient to exert the beneficial phenotypic modulation disclosed herein, including, but not limited to, cell proliferation, upregulation of activation markers (e.g., DNAM-1, NKG2D), and enhanced cytotoxicity. For example, an effective amount of IL-2 can be, for example, 500-1500, 600-1300, 750-1500, or 500-1200 IU/mL for recombinant human IL-2, and an effective amount of an anti-CD3 antibody can be, for example, but not limited to, 10-60, 20-40, 10-40, or 20-60 ng/mL for OKT3.

Determination of a therapeutically effective amount is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein. For any preparation used in the methods of the invention, the dose or therapeutically effective amount can be estimated initially from in vitro and cell culture assays. For example, a dose can be formulated in animal models to achieve a desired concentration or potency. Such information can be used to more accurately determine useful doses in humans.

Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures, or in experimental animals. The data obtained from such in vitro assays, cell culture assays, and animal studies can be used in formulating a range of dosages for use in humans. Dosages may vary depending on the dosage form employed and the route of administration utilized. The exact formulation, route of administration, and dosage can be chosen by the individual physician in view of the patient's condition (see, e.g., Fingl, E. et al. (1975), "The Pharmacological Basis of Therapeutics," Ch. 1, p.1.).

In other embodiments, the tumor is characterized by downregulation of MHCI expression and/or activity. As shown herein, the cell compositions of the present invention have been found to have both MHC-dependent and non-MHC-dependent cytotoxic activity against a variety of tumor cells. Thus, such cell compositions can also be advantageously used to treat therapy-resistant tumors (e.g., resistant to immunotherapy or checkpoint molecule inhibitors) or tumors that exhibit an immune evasion phenotype associated with impaired MHCI antigen presentation, e.g., due to reduced expression of MHCI molecules, loss-of-function mutations, or other defects in the MHCI pathway characteristic of tumors.

In other embodiments, the cell composition has undergone a cryopreservation and subsequent thawing protocol prior to contact with the tumor cells. As shown herein, cell compositions according to the principles of the invention have demonstrated highly potent cytotoxicity after cryopreservation and thawing, and this activity was manifested even near the time of thawing (70-80% of the cytotoxic activity of the corresponding non-cryopreserved cells was retained within 24 hours of thawing) without the need for further expansion or manipulation steps. Thus, in other embodiments of the methods of the invention, the thawing protocol is performed within 1-3 days, e.g., within 36 hours, 24 hours, 12 hours, or 6 hours, of contacting the cell composition with the tumor cells. Each possibility represents a separate embodiment of the invention. In other embodiments, the cell composition can be autologous, histocompatible allogeneic, or non-histocompatible allogeneic to the subject, each possibility representing a separate embodiment of the invention.

In a further aspect, the invention provides a method for producing a cell composition for ACT, comprising the steps of:
a. providing a PBMC sample;
b. expanding the PBMCs for at least 9 days in the constant presence of an effective amount of IL-2 and a CD3 activator, and in the absence of other cytokines or antibodies, whereby the number of viable cells in the culture is increased by at least 20 fold by replenishing the effective amount of IL-2 and the CD3 activator every 2-4 days, thereby obtaining a cell composition comprising at least 5 x ¹⁰⁶ to 10 x ¹⁰⁸ viable cells;
and c. recovering the resulting cell composition.

In other embodiments, the expansion is carried out in the constant presence of IL-2 and a CD3 activator as the only exogenously added cell stimulatory factors. In a particular embodiment, step b comprises expanding the cells in the presence of 500-1500 IU/mL IL-2 and 10-60 ng/mL anti-CD3 antibody, which are replenished every 2-4 days for 10-16 days. In other embodiments, the cells are not subjected to further enrichment, stimulation or expansion steps. In other embodiments, the expansion is carried out to increase the number of viable cells by at least 30-fold. In other embodiments, the expansion is carried out to produce a cell composition comprising 5×10 ⁶ to 10×10 ⁸ viable mononuclear cells, of which 70-80% are CD3 ⁺ CD8 ⁺ cells expressing NKG2D and granzyme B. In other embodiments, the expansion is carried out to produce a cell composition capable of specifically eradicating hematopoietic tumor cells when incubated in vitro with hematopoietic tumor cells at a ratio of 0.25:1 for 24 hours. In other embodiments, expansion is performed to produce the cell compositions of the invention as disclosed herein.

In other embodiments, step b further comprises genetically modifying the cells. In other embodiments, the method comprises genetically modifying the cells to express a CAR and/or at least one cytokine, chemokine, or receptor thereof. In an exemplary embodiment, the method comprises modifying the cells to express (i) a CD19-specific CAR or a CD123-specific CAR, (ii) a modified CXCR4 receptor comprising a mutation or truncation of the C-terminal tail domain, and (iii) at least one cytokine selected from the group consisting of IL-2, IL-15, and IL-21. In other embodiments, the method further comprises cryopreserving the cell composition collected in step c.

In other embodiments, a cell composition prepared by the methods disclosed herein is provided.

In another aspect, an ACT cell composition is provided comprising ^5x106 to ^10x108 viable in vitro expanded PBMCs, 70-80% of which are CD3 ⁺ CD8 ⁺ cells expressing NKG2D and granzyme B, at least 70% of which are ^CD56- cells, and up to 5% of which are ^CD3- cells, and which exhibit significant cytotoxic activity against non-histocompatible tumor cells in vitro and in vivo.

In one embodiment, 7-12% of the live cells are CD3 ⁺ CD4 ⁺ cells, 20-28% are CD3 ⁺ CD56 ⁺ cells, and 10-14% are CD3 ⁺ CD8 ⁺ CD56 ⁺ cells. In another embodiment, the composition is characterized by a ratio of CD3 ⁺ CD8 ⁺ cells to CD3 ⁺ CD4 ⁺ cells of 7 to 8. In another (additional or alternative) embodiment, the composition comprises less than 3% CD3 ^- CD56 ⁺ cells.

According to certain embodiments, at least 40% of the CD3 ⁺ CD8 ⁺ cells are characterized by surface expression of at least one marker selected from the group consisting of perforin, FasL, CXCR4 and CXCR2. In other embodiments, at least 40% of the CD3 ⁺ CD8 ⁺ cells are characterized by surface expression of multiple markers selected from the group consisting of perforin, FasL, CXCR4 and CXCR2. In some embodiments, about 40% of the CD3 ⁺ CD8 ⁺ cells express FasL, about 50% of the CD3 ⁺ CD8 ⁺ cells express perforin, about 90% of the CD3 ⁺ CD8 ⁺ cells express CXCR2, and about 100% of the CD3 ⁺ CD8 ⁺ cells express CXCR4, granzyme B and NKG2D.

In other embodiments, at least 30%, typically at least about 40%, of the CD3 ⁺ CD8 ⁺ cells are characterized by surface expression of at least one marker selected from the group consisting of CXCR3, CXCR6, and DNAM-1. In one embodiment, at least 90% of the CD3 ⁺ CD8 ⁺ cells are characterized by surface expression of CXCR3. In another embodiment, at least 90%, typically substantially all of the CD3 ⁺ CD8 ⁺ cells are characterized by surface expression of DNAM-1. In another embodiment, at least 30% of the CD3 ⁺ CD8 ⁺ cells are characterized by surface expression of CXCR6. In another embodiment, about 90% of the CD3 ⁺ CD8 ⁺ cells are characterized by surface expression of CXCR3, substantially all of the CD3 ⁺ CD8 ⁺ cells are characterized by surface expression of DNAM-1, and about 30% of the CD3 ⁺ CD8 ⁺ cells are characterized by surface expression of CXCR6. Thus, an ACT cell composition according to an embodiment of the invention comprises a CD3 ^+CD8+ cell population defined as NKG2D ⁺ , Granzyme ^{B +} , Perforin ⁺ , FasL ⁺ , CXCR4 ⁺ , CXCR2 ⁺ , CXCR3 ⁺ , CXCR6 ⁺ and DNAM-1 ⁺ .

In other embodiments, up to 55% of the CD3 ⁺ CD8 ⁺ cells, typically up to about 50%, are characterized by surface expression of one or more immune checkpoint molecules selected from the group consisting of CTLA-4, PD-1, TIGIT, Lag-3, and Tim-3. In other embodiments, up to about 50% of the CD3 ⁺ CD8 ⁺ cells are characterized by surface expression of multiple immune checkpoint molecules selected from the group consisting of CTLA-4, PD-1, TIGIT, Lag-3, and Tim-3. In other embodiments, the CD3 ⁺ CD8 ⁺ cells are characterized by a substantial lack of surface expression of CTLA-4. In other embodiments, about 50-51% of the CD3 ⁺ CD8 ⁺ cells are characterized by surface expression of Lag-3. In other embodiments, about 50-54% of the CD3 ⁺ CD8 ⁺ cells are characterized by surface expression of Tim-3. In another embodiment, about 15% of the CD3 ⁺ CD8 ⁺ cells are characterized by surface expression of PD-1. In another embodiment, about 20-22% of the CD3 ⁺ CD8 ⁺ cells are characterized by surface expression of TIGIT. Thus, ACT cell compositions according to embodiments of the invention comprise a CD3 + CD8 ⁺ cell population defined as CTLA-4 ⁻ , PD-1 ⁻ , TIGIT ⁻ , Lag-3 ⁺ and Tim-3 ⁺ . ^In another embodiment, ACT cell compositions according to embodiments of the invention comprise a CD3 ⁺ CD8 ⁺ cell population defined as CTLA-4 ⁻ , PD-1 ⁻ , and TIGIT ⁻ . Each possibility represents a separate embodiment of the present invention.

In some embodiments, the cell composition exhibits MHC-dependent (or MHC-restricted) cytotoxic activity against hematopoietic tumor cells. In other embodiments, the cell composition exhibits non-MHC-dependent (or non-MHC-restricted) cytotoxic activity against hematopoietic tumor cells and solid tumor cells. In other embodiments, the cytotoxic activity is mediated by granzyme B and/or perforin. In other embodiments, the cytotoxic activity is not substantially mediated by FAS-FasL interactions. In other embodiments, the cell composition has both MHC-dependent and non-MHC-dependent cytotoxic activity.

In another embodiment, the composition is capable of specifically eradicating hematopoietic tumor cells when incubated in vitro for 24 hours at a ratio of 0.25:1. In another embodiment, the composition is capable of specifically eradicating hematopoietic tumor cells, eradicating 80-90% of the tumor cells when incubated in vitro for 24 hours at a ratio of 1:1 composition cells to tumor cells. In another embodiment, the tumor cells express at least one NKG2D ligand. In another embodiment, the tumor cells express multiple NKG2D ligands selected from the group consisting of MICA, MICB, HLAE, ULBP1, ULBP256, and ULBP3.

In other embodiments, the composition is capable of specifically eradicating non-histocompatible tumor cells in vivo without substantially inducing GVHD. In other embodiments, the composition is capable of inhibiting or preventing tumor development in a non-histocompatible subject in vivo without substantially inducing GVHD.

In other embodiments, the composition retains at least 70%, typically at least 80%, of its antitumor cytotoxic activity after cryopreservation and subsequent thawing protocol.

In other embodiments, the composition is prepared by the methods disclosed herein. According to some embodiments, the cell composition is prepared by a method comprising expanding PBMCs in the constant presence of an effective amount of IL-2 and a CD3 activator for at least 9 days, and replenishing the effective amount of IL-2 and a CD3 activator every 2-4 days. In other embodiments, the cell composition is incubated in the absence of other cytokines or antibodies.

In other embodiments, the cell composition is genetically modified. In other embodiments, the cell composition is genetically modified to express a CAR. In other embodiments, the CAR is directed to an antigen selected from the group consisting of BCMA, CD47, PDL-1, mesothelin, EpCAM, CD34, CD44, PSCA, MUC16, CD276, CD123, CD19, CD20, and EFFRvIII. In certain embodiments, the cells of the composition express a CD19-specific CAR or a CD123-specific CAR. In other embodiments, the cell composition is genetically modified to express at least one cytokine, chemokine, or receptor thereof. In other embodiments, the cell composition is genetically modified to express a modified chemokine receptor. In other embodiments, the modified chemokine receptor is a CXCR4 receptor comprising a mutation or truncation of the C-terminal tail domain. In other embodiments, the cell composition exogenously expresses IL-2, IL-15, and/or IL-21. In certain embodiments, the cell composition is modified to express (i) a CD19-specific CAR or a CD123-specific CAR, (ii) a modified CXCR4 receptor comprising a mutation or truncation of the C-terminal tail domain, and (iii) at least one cytokine selected from the group consisting of IL-2, IL-15, and IL-21.

In other embodiments, the method further comprises cryopreserving the cellular composition obtained in step c. In other embodiments, the method further comprises subjecting the cryopreserved cellular composition to a thawing protocol (e.g., prior to administration to a subject in need thereof). In other embodiments, the method further comprises cryopreservation and subsequent thawing protocols after step c, wherein the cellular composition retains at least 70%, typically at least 80%, of the antitumor cytotoxic activity properties of the cellular composition prior to cryopreservation.

In another aspect, a cell composition is provided comprising leukocytes genetically modified to express (i) a CD19-specific CAR or a CD123-specific CAR, (ii) a modified CXCR4 receptor comprising a mutation or truncation of the C-terminal tail domain, and (iii) at least one cytokine selected from the group consisting of IL-2, IL-15, and IL-21. In another embodiment, the cell composition is characterized in that 70-80% of the cells are CD3 ⁺ CD8 ⁺ cells expressing NKG2D and granzyme B, at least 70% are ^CD56- cells, and up to 5% are ^CD3- cells. According to another embodiment, the cell composition is prepared by a method comprising expanding PBMCs in the constant presence of an effective amount of IL-2 and a CD3 activator for at least 9 days, and replenishing the effective amount of IL-2 and the CD3 activator every 2-4 days.

In some embodiments, the cell compositions of the present invention disclosed herein are for use in treating a tumor in a subject in need of such treatment. In one embodiment, the tumor is a hematopoietic tumor. In another embodiment, the tumor is a solid tumor. In some embodiments, the tumor is selected from the group consisting of leukemia, multiple myeloma, prostate tumor, or mesothelioma. In another embodiment, the tumor is characterized by expression of at least one NKG2D ligand. In another embodiment, the tumor cells express multiple NKG2D ligands selected from the group consisting of MICA, MICB, HLAE, ULBP1, ULBP256, and ULBP3. In another embodiment, the tumor cells express MICA, MICB, HLAE, ULBP1, ULBP256, and ULBP3.

In other embodiments, the tumor is characterized by expression of at least one chemokine selected from the group consisting of a CXCR6 ligand, a CXCR4 ligand, a CXCR3 ligand, and a CXCR2 ligand. In other embodiments, the tumor is characterized by expression of at least one chemokine selected from the group consisting of a CXCR6 ligand, a CXCR4 ligand, and a CXCR3 ligand. In other embodiments, the tumor is characterized by expression of a CXCR6 ligand. In certain embodiments, the tumor is characterized by expression of the CXCR6 ligand, CXCL16.

In other embodiments, the tumor is characterized by expression of at least one inhibitory immune checkpoint molecule and/or its ligand. In some embodiments, the tumor may express at least one ligand of CTLA-4, PD-1, TIGIT, Lag-3, Tim-3, and combinations thereof, each possibility representing a separate embodiment of the invention. In other embodiments, the tumor is characterized by expression of at least one ligand of an inhibitory immune checkpoint molecule selected from the group consisting of CTLA-4, PD-1, and TIGIT. In other embodiments, the tumor is characterized by expression of ligands of multiple inhibitory immune checkpoint molecules selected from the group consisting of CTLA-4, PD-1, and TIGIT. In other embodiments, the tumor is characterized by expression of at least one CTLA-4 ligand (e.g., CD80 and/or CD86).

In other embodiments, the tumor is resistant to treatment with at least one checkpoint molecule inhibitor. In some embodiments, the tumor may be resistant to treatment with an inhibitor of at least one checkpoint molecule selected from the group consisting of CTLA-4, PD-1, TIGIT, Lag-3, Tim-3, and combinations thereof, each possibility representing a separate embodiment of the invention. In other embodiments, the tumor is resistant to treatment with an inhibitor of at least one checkpoint molecule selected from the group consisting of CTLA-4, PD-1, and TIGIT. In other embodiments, the tumor is resistant to treatment with an inhibitor of CTLA-4, PD-1, and TIGIT.

For example, the cell compositions of the invention are shown herein to lack substantial expression of CTLA-4 and may therefore be particularly advantageous for treating tumors that are resistant to treatment with a CTLA-4 blocking antibody (e.g., ipilimumab and/or tremelimumab) as the sole therapeutic agent. Thus, in other embodiments, the subject has not received a treatment regimen with a checkpoint molecule inhibitor.

In other examples, the cell compositions of the present invention can be used to treat tumors expressing Tim-3 and/or Lag-3, e.g., in combination therapy with a Tim-3 inhibitor and/or a Lag-3 inhibitor, respectively. Thus, in other embodiments, the subject is undergoing a treatment regimen with at least one checkpoint molecule inhibitor, e.g., against PD-1, TIGIT, Lag-3, Tim-3, or a combination thereof, each possibility representing a separate embodiment of the present invention. In other embodiments, the above-described use further comprises administration of at least one checkpoint molecule inhibitor, e.g., against PD-1, TIGIT, Lag-3, Tim-3, or a combination thereof, each possibility representing a separate embodiment of the present invention. In other embodiments, the use further comprises administration of at least one checkpoint molecule inhibitor, e.g., against Lag-3, Tim-3, or a combination thereof. In some embodiments, the use of the cell compositions of the present invention can reduce or minimize the dose of existing anti-cancer therapies, e.g., immune checkpoint inhibitors.

In another aspect, there is provided a method of treating a tumor in a subject in need thereof, the method comprising contacting tumor cells with a therapeutically effective amount of a cell composition of the invention disclosed herein.

In one embodiment, the cell composition is autologous, histocompatible allogeneic, or non-histocompatible allogeneic to the subject. In certain embodiments, the cell composition is non-histocompatible allogeneic to the subject. In other embodiments, the contacting occurs in vivo. In other embodiments, the contacting occurs ex vivo. In other embodiments, the tumor is a hematopoietic tumor. In other embodiments, the tumor is a solid tumor. In other embodiments, the tumor is characterized by expression of at least one NKG2D ligand. In other embodiments, the tumor cells express a plurality of NKG2D ligands selected from the group consisting of MICA, MICB, HLAE, ULBP1, ULBP256, and ULBP3. In other embodiments, the tumor cells express MICA, MICB, HLAE, ULBP1, ULBP256, and ULBP3.

The following examples are presented to more fully illustrate certain embodiments of the present invention. However, these examples should not be construed as limiting the broad scope of the invention.

Generation and Characterization of Superactivated Killer (SAK) Cells Peripheral blood mononuclear cells (PBMCs) were isolated from human blood by Ficoll-Hypaque density centrifugation (Sigma). PBMCs were resuspended at a concentration of ^1x106 cells/mL in RPMI medium (complete medium + 1x MEM NEAA and 0.1 mM 2-mercaptoethanol) and stimulated with 1000 IU/mL recombinant human IL-2 (rhIL-2, R&D Systems) and 30 ng/mL anti-human CD3 antibody (OKT-3, eBioscience) on day 0. Cultures were maintained by adding fresh medium + rhIL-2 and anti-CD3 on days 4, 6, and 8. Cells were expanded for at least 11 days and harvested at various time points for further analysis.

To assess the proliferation rate, the harvested cells were stained with trypan blue and the total number of viable cells was counted on days 0, 4, 6, 8 and 11. The results are shown in Figure 1A and represent the mean ± standard deviation from nine separate experiments. As shown in Figure 1A, the cells proliferated continuously, reaching a proliferation rate of 30.55-fold after 11 days of culture.

Immunophenotyping by flow cytometry was then performed as follows: Cells were harvested at various time points, washed, stained with various monoclonal antibodies for 30 min at 4°C, and analyzed by flow cytometry. The following antibodies were used: FITC anti-CD4, APC anti-CD8, Percp anti-CD3, and PE anti-CD56 (all from Biolegend). The results are shown in Figures 1B-1D, where each figure shows the mean ± standard deviation from nine separate experiments.

As shown in Figures 1B-1D, the percentages of CD3 ⁺ CD8 ⁺ and CD3 ⁺ CD56 ⁺ cells in the total expanded cell population gradually increased over time, whereas the percentages of CD3 ⁺ CD4 ⁺ cells decreased over time. The means and percentages of the various subpopulations at day 11 are summarized in Table 1 (mean ± SD of 9 independent experiments). The subpopulations at day 11 are further characterized in Table 2 (% of each subpopulation from total composition, mean ± SD of 5 independent experiments). CD8 ⁺ CD56 ⁺ /CD8 ⁺ represents the percentile of CD8 ⁺ CD56 ⁺ within the total CD8 ⁺ population.

Comprehensive analysis of samples from 25 individual donors revealed that the composition was characterized by an average of 74.6±1.6% CD3 ⁺ CD8 ⁺ cells and 12±1% CD3 ⁺ CD4 ⁺ cells. Furthermore, the ratio of CD3 ⁺ CD8 ⁺ cells to CD3 ⁺ CD4 ⁺ cells was at least 6:1, and typically about 7-8:1. The resulting cells, referred to herein as superactivated killer (SAK) cells, were subjected to further analysis as detailed in the Examples below.

As a comparison, other known protocols for preparing PBMC-derived effector cells for ACT (i.e., cytokine-induced killer cells (CIK)) were also tested. For preparation of CIK, PBMC were resuspended in RPMI medium (complete medium + 1x MEM NEAA and 0.1 mM 2-mercaptoethanol) at a concentration of ^1x106 cells/mL and stimulated with 1000 IU/mL recombinant human IFN-γ (Peprotec) on day 0. On day 1, 50 ng/mL anti-human CD3 antibody (OKT-3, eBioscience) and 500 IU/mL rhIL-2 (R and D system) were added. Cultures were maintained with fresh medium + 500 IU/mL rhIL-2 on days 4, 6, and 8. Cells were allowed to grow for at least 11 days. CIK cells were harvested on day 11 for further assays. SAK cells were also grown in parallel as detailed above. Characterization of the CIK population on day 11 is shown in Table 3.

As can be seen from Tables 1-3, SAK cells were phenotypically distinct from CIK cells on day 11 of expansion, with the SAK cell composition having a significantly higher percentage of CD8 ⁺ CD56 ⁺ cells among total CD8 ⁺ cells than the CIK cell composition and a significantly lower percentage of CD3 ⁺ CD4 ⁺ cells than the CIK cell composition.

Moreover, CIK cells intended for ACT are further distinguished from SAK cells described herein by the relative proportions of additional subpopulations, including but not limited to, the incidence of CD3 ⁻ cells and CD56 ⁺ cells, which is higher in CIK cells. CIK cell compositions are typically administered to cancer patients after 21 days of expansion in a manner essentially corresponding to the CIK protocol described above. Such compositions are typically characterized by an average content of at least 30% CD56 ⁺ cells, more typically up to about 50%-80% CD56 ⁺ cells, and an average content of at least 5%, more typically up to about 10%-70% CD3 ⁻ cells. For example, various exemplary CIK compositions reported thus far have been characterized as comprising, at day 21, 40-80% CD3 ⁺ CD56 ⁺ cells, 20-60% CD3 ⁺ CD56- cells, and 1-10% CD3 ^- CD56 ⁺ cells; 35% CD3 ⁺ CD56 ⁺ cells, 65% CD3 ⁺ CD56 ^- cells, and 2.23% CD3 ^- CD56 ⁺ cells (mean values, 90% of total CD3 ⁺ cells); or 31.6% CD3 ⁺ CD56 ⁺ cells, 41.5% CD3 ⁺ CD56 ^- cells, and 11.7% CD3 ^- CD56 ⁺ cells (median values).

Antitumor cytotoxic activity of SAK cells To evaluate the effect of effector cells (SAK) on various tumor cells (also referred to herein as target cells), cytotoxicity assays were performed using a carboxyfluorescein diacetate succinimidyl ester (CFSE)-based assay. SAK cells were harvested on day 11 of culture. Target cells were labeled with CFSE (eBioscience) according to the manufacturer's instructions and seeded at a concentration of 2 × ¹⁰⁴ cells/well in 96-well plates. Effector cells were added to target cells at effector-to-target (E:T) ratios of 0.25:1, 0.5:1, 1:1, 2:1, 5:1, 10:1, and 30:1. After 24 h, CFSE ⁺ PI ^- labeled target cells were analyzed by flow cytometry. The percentile of viable cells was calculated as (number of CFSE ⁺ PI ⁻ cells with effector cells/number of CFSE ⁺ PI ⁻ cells without effector cells)×100.

The results obtained using THP-1 leukemia cells and RPMI8266 multiple myeloma cells are shown in Figures 2A-2B, respectively, and show the mean ± standard deviation of triplicates at each ratio. As shown in Figures 2A-2B, SAK cells exhibited potent cytotoxic activity against both types of hematopoietic tumor cell lines, THP-1 and RPMI8266, even at an E:T ratio as low as 0.25:1. When an E:T ratio of 2:1 was used, SAK cells showed a remarkable killing efficiency of about 90%, demonstrating the efficacy of SAK cells as an antitumor cell therapy.

The experiment was repeated with various solid tumor cell lines, including PC3 prostate cancer cells and H28 mesothelioma cancer cells, and compared with hematopoietic MV4-11 leukemia cells. The results are shown in Figures 3A-3C, which show the mean ± standard deviation of triplicates at each ratio. As shown in Figures 3A-3C, SAK cells showed significant efficacy against hematopoietic MV4-11 cells and both solid tumor cell lines. Increasing the E:T ratio increased the efficacy of SAK cells against all cancer cell lines, especially MV4-11 leukemia cells. For example, an E:T ratio of 2:1 was required to achieve 90% efficiency against MV4-11 cells, while an E:T ratio of 30:1 was required to achieve 90% efficiency against PC3 prostate cancer cells and H28 mesothelioma cancer cells.

The SAK cell compositions were then separated into CD3 ⁺ CD56 ⁺ and CD3 ⁺ CD56 ^- cells using flow cytometry and the cytotoxic activity of each population was compared to that of the original SAK cell composition. For this purpose, each population was incubated with MV4-11 cells at various E:T ratios (0.25:1, 0.5:1, 1:1 and 2:1) as detailed above. Of note, no significant differences were found between the cytotoxic potential of the tested populations and that of the intact SAK composition at any of the E:T ratios tested. Moreover, similar results were obtained with SAK cell compositions harvested on days 10 and 12 of expansion.

Thus, SAK cells exhibited potent antitumor cytotoxic activity against various tumor cells in an MHCII-independent manner. Furthermore, the cytotoxic activity appears to be CD56-independent.

SAK cells exhibit significantly improved antitumor cytotoxic activity compared to other PBMC-derived effector cells Following the discovery that SAK cells exert superior antitumor activity, the cytotoxic activity of SAK cells was compared with two other PBMC-derived effector cells, namely, cytokine-induced killer cells (CIK) and an activated lymphocyte composition (IL-2-proliferated T cells).

To prepare CIK, PBMCs were resuspended in RPMI medium (complete medium + 1x MEM NEAA and 0.1 mM 2-mercaptoethanol) at a concentration of ^1x106 cells/mL and stimulated with 1000 IU/mL recombinant human IFN-γ (Peprotec) on day 0. 50 ng/mL anti-human CD3 antibody (OKT-3, eBioscience) and 500 IU/mL rhIL-2 (R and D system) were added on day 1. Cultures were maintained by adding fresh medium + 500 IU/mL rhIL-2 on days 4, 6 and 8. Cells were expanded for at least 11 days. CIK cells were harvested on day 11 for further assays. On day 0, PBMCs were incubated with 50 ng/mL anti-human CD3 and 300 IU/mL rhIL-2, and IL-2-expanded T cell cultures were obtained after further addition of fresh medium containing 300 IU/mL rhIL-2.

CFSE-based cytotoxicity assays were performed essentially as described in Example 2 using SAK, CIK or IL-2 expanded T cells as effector cells and MV4-11 leukemia cells as target cells, with untreated MV4-11 cells set as control. The results are shown in Figures 4A-4B, showing the mean ± standard deviation of triplicates at each ratio.

Unexpectedly, as shown in Figures 4A-4B, SAK cells were found to be significantly more effective at eradicating tumor cells than control effector cells. When compared to CIK cells (Figure 4A), a lower E:T ratio was required for SAK cells to exhibit significant cytotoxic activity, and SAK cells exhibited superior cytotoxic activity at each E:T ratio tested. For example, at an E:T ratio of 5:1, SAK cells were 90% efficient, whereas CIK cells were only 40% efficient at the same ratio (Figure 4A). SAK cells were found to be even more effective compared to IL-2-expanded T cells. For example, even at an E:T ratio of 0.25:1, SAK cells were 60% efficient, whereas IL-2-expanded T cells were completely ineffective at the same ratio. Also, even at an E:T ratio of 2:1, SAK cells exhibited over 90% killing activity, whereas IL-2-expanded T cells were only 25% active (Figure 4B).

Therefore, it was unexpectedly found that SAK cells exert highly efficient antitumor cytotoxic activity compared to previously known PBMC-derived effector cells.

Immunophenotypic characterization of SAK and tumor cells NKG2D expression on CD8 ⁺ SAK cells was assessed as follows: SAK cells prepared as described in Example 1 were harvested on day 11 of growth, washed, stained with FITC-labeled anti-NKG2D and APC-labeled anti-CD8 monoclonal antibodies (Biolegend) for 30 minutes at 4°C, and analyzed by flow cytometry. Antibody staining on CD8 ⁺ cells was detected and compared to isotype antibody control staining.

The results are shown in Figure 5. As shown in Figure 5, CD8 ⁺ SAK cells highly express NKG2D. Remarkably, 100% of CD8 ⁺ cells were found to express NKG2D, with an 8.4-fold increase in mean fluorescence intensity compared to the isotype control.

Because SAK cells showed expression of the NKG2D receptor, we tested the expression of various NKG2D ligands in various tumor cell types using flow cytometry. The results are summarized in Table 4 below.

As shown in Table 4, MV411 leukemia cells highly express various NKG2D ligands on their surface, such as ULBP3, HLAE, ULBP1, ULBP256, and MICA and/or MICB. In contrast, H28 mesothelioma cells express these markers at significantly lower, i.e., non-significant levels (e.g., ULBP256) or at undetectable levels (e.g., MICA and MICB).

Therefore, SAK cells are characterized by high expression of NKG2D and have been found to be particularly effective against target tumor cells characterized by high expression of NKG2D ligands, such as MV4-11 leukemia cells.

Cytotoxic activity of SAK is mediated primarily by secretion of granzyme B and perforin, not FasL-Fas interactions To examine the mechanism of killing of SAK cells, expression levels of FASL, granzyme B, and perforin in CD8 ⁺ SAK cells were examined essentially as described in Example 4. Briefly, SAK cells were harvested on day 11 of growth, washed, stained with PE anti-FasL APC anti-perforin antibody or PE anti-granzyme monoclonal antibody (all from Biolegend) for 30 min at 4°C, and analyzed by flow cytometry. Antibody staining on CD8 ⁺ cells was detected and compared to isotype antibody control staining.

The results shown in Figures 6A-6C demonstrate high expression of FasL, Granzyme B and Perforin by the CD8 ⁺ cytotoxic cell population. Expression levels, expressed as mean fluorescence intensity (MFI), in CD8 ⁺ cells stained with marker-specific antibodies compared to isotype control antibody expression levels are summarized below in Table 5. As shown in Table 5, 40% of the CD8 ⁺ cells are FasL positive, 50% are Perforin positive and 100% express Granzyme B.

Next, target-induced perforin secretion was examined as follows: 5 × 10 ³ SAK cells (day 11) were added to MV4-11 cells at a ratio of 0.25:1 (E:T) for 24 h. After 24 h, the concentration of perforin in the culture supernatant was examined using an enzyme-linked immunosorbent assay (ELISA) kit (Abcam). As shown in Figure 7, the level of perforin secretion was increased after incubation with target MV4-11 tumor cells.

To further characterize the killing mechanism, CFSE-based cytotoxicity assays were performed in the presence of various inhibitors, essentially as described in Example 2. For this purpose, leukemic MV4-11 cells were cultured with SAK cells with or without the tested inhibitors, and untreated MV4-11 cells were set as controls. The following inhibitors were used: 3,4-dichloroisocoumarin (DCI: granzyme B inhibitor, Sigma), concanamycin A (CMA: perforin inhibitor, Sigma) and anti-FasL blocking antibody (Biolegend). 50 μM DCI, 1000 mM CMA or 5 μg/mL anti-FasL were added to SAK cells 2 hours prior to incubation with target cells. The cytotoxic activity of SAK against leukemic MV4-11 target cells was expressed as the percentage of viable cells calculated by (number of CFSE ⁺ PI ⁻ cells in the presence of effector cells/number of CFSE ⁺ PI ⁻ cells in the absence of effector cells) × 100.

The results are shown in Figures 8A-8C, which show the mean ± standard deviation of triplicates for each ratio. As shown in the figures, granzyme B inhibitors and perforin inhibitors significantly reduced the cytotoxic activity of SAK cells (Figures 8A and 8C, respectively), whereas incubation with anti-FasL antibody had little or no effect on the reduction in the percentage of viable cells (Figure 8B).

Thus, without wishing to be bound by any particular theory or mechanism of action, SAK cells appear to exhibit antitumor cytotoxic activity that is primarily dependent on granzyme B and perforin secretion rather than FasL-Fas interactions.

Characterization of SAK migratory capacity To further characterize the SAK cell population, analysis of chemokine receptor expression was performed by flow cytometry as follows: Cells were harvested at various time points, washed, stained with APC anti-CXCR4 (12G5 clone), APC anti-CXCR3, PE anti-CXCR6 or FITC anti-CXCR2 monoclonal antibodies, as well as anti-CD8 antibodies labeled with FITC or APC (all from Biolegend), respectively, for 30 min at 4° C., and analyzed by flow cytometry.

The results shown in Figure 9A show the expression of the chemokine receptors CXCR2, CXCR3 (upper panels, left and right, respectively), CXCR4 and CXCR6 (lower panels, left and right, respectively) on SAK cells at day 11 of expansion. Notably, it was found that virtually all CD3 ⁺ CD8 ⁺ cells were characterized by surface expression of CXCR4, while the remaining chemokine receptors were found on average in approximately 90% (CXCR2 and CXCR3) or approximately 30% (CXCR6) of CD3 ⁺ CD8 ⁺ cells.

Migration assays were performed to test the function of chemokine receptors on SAK cells. For this purpose, migration of SAK cells in response to the chemokines CXCL12, IP-10 or rhIL-8 (PeproTech EC) was assessed using 5 μm pore size Transwells (Costar). SAK cells were resuspended in RPMI medium containing 1% fetal calf serum (FCS). Cells (2× ¹⁰⁵ cells/well) were added to the upper chamber in a total volume of 100 μL, and 600 μL of RPMI supplemented with 100 ng/mL CXCL12 or IL-8, or 500 ng/mL IP-10, was added to the lower chamber. The amount of cells migrating to the lower compartment within 4 h was determined by FACS and expressed as a percentage of cells entering the upper chamber.

The results shown in Figure 9B indicate that SAK cells migrate toward the chemokines IL-8, SDF-1, and IP-10. Notably, at the concentrations tested, 3% of the cells migrated toward IL-8, whereas approximately 40% of the cells migrated toward SDF-1 and approximately 30% migrated toward IP-10. Thus, CXCR4- and CXCR3-dependent migration appears to be more prominent than CXCR2-dependent migration in the SAK population. Expression of CXCR3 and CXCR6 in SAK cells also indicates that SAK cells may migrate toward the CXCR3 ligands CXCL9 and CXCL11, and the CXCR6 ligand CXCL16.

In summary, the results demonstrate the ability of SAK cells to be recruited to tumor tissues, particularly tumors expressing CXCR4, CXCR6 and CXCR6 ligands. Of further interest is the finding that certain chemokine receptors, such as CXCR6, are suggested to be particularly important for the survival and proliferation of cytotoxic T cells in the tumor microenvironment, thereby inhibiting T cell exhaustion. Thus, without wishing to be bound by any particular theory or mechanism of action, expression of CXCR6 on SAK cells may indicate phenotypic activation and improved likelihood of survival and proliferation in the tumor microenvironment.

To generate the xenograft model, NOD SCID gamma (NSG) mice (immunodeficient mice lacking mature T, B and NK cells) were irradiated with 300 cGy. 24 hours later, RAJI cells (a human B lymphoblastoid line) were injected intravenously (iv) via the dorsal tail vein in a final volume of ²⁰⁰ μL (1 × 10 cells/mouse).

Mice were then injected intravenously (iv) with ^20x106 SAK cells and intraperitoneally (ip) with 10μg IL-2 on days 5, 6 and 7. Control mice were injected intravenously (iv) with PBS and intraperitoneally (ip) with 10μg IL-2. Mice were sacrificed on day 12 and bone marrow (BM) and spleen were harvested for analysis. Cells were isolated from these organs and stained with anti-human CD20 antibody (which specifically recognizes engrafted tumor cells) and the percentage of engrafted cells in BM and spleen was assessed by FACS. The results are shown in Table 6 below. % inhibition was calculated as 100-(CD20 in SAK mice/CD20 in control mice x 100).

As shown in Table 6, adoptive transfer of SAK cells significantly inhibited engraftment of Raji cells in the bone marrow and spleen, with particularly remarkable efficacy resulting in approximately 78% inhibition of tumor development in the spleen.

The experiment was repeated as detailed above, but blood drawn from sacrificed NSG mice was stained with anti-human CD19 antibody along with BM and spleen cells. The results are shown in Table 7 below, where % inhibition was calculated as 100-(CD19 in SAK mice/CD19 in control mice x 100). Visualized in Figures 10A-10C, the percentage of CD19 ⁺ Raji cells in NSG mice versus control NSG mice following transfer of SAK cells into the spleen (Figure 10A), BM (Figure 10B), and blood (Figure 10C) are shown.

As shown in Table 7, adoptive transfer of SAK cells significantly inhibited engraftment of Raji cells in bone marrow, blood, and spleen, and was highly effective in inhibiting tumor development in the spleen by approximately 97%. These findings indicate that only SAK cells can effectively inhibit tumor growth and engraftment.

Genetically modified SAK
The improved genetically modified SAK cells were prepared as described below.

For the production of nucleic acid constructs, the retroviral MSGV-1D3-28Z All ITAM intact expression vector (#107226, Addgene) was used with the following modifications. The retroviral vector backbone, pMSGV1, is a derivative of the murine stem cell virus (MSCV)-based splice GAG vector (pMSGV) that uses the MSCV long terminal repeat sequences. The "1D3-28Z All ITAM intact" insert was removed from the plasmid using restriction enzymes NcoI and SalI. The various cDNA inserts, as detailed below, were amplified by PCR and ligated into plasmids with NcoI and SalI restriction sites. The resulting constructs A-D (detailed in Table 8 below) were confirmed by DNA sequencing.

The nucleic acid sequence encoding the anti-human CD123CAR (contained in construct A) is as follows:
ATGGCCCTGCCTGTGACAGCCCTGCTGCTGCCTCTGGCTCTGCTGCTGCTGCTGCATGCCGCTAGACCCGATATTGTCCTCACTCAATCGCCGGACTCACTGGCGGTGTCCCTCGGAGAGAGGGCGA CGATCAATTGCCGGGCTTCCGAATCCGTCGATAACTACGGAAACACCTTTATGCACTGGTACCAACAGAAGCCAGGACAGCCACCAAAGCTGTTGATCTACCGCGCTTCAAACCTTGAGTC GGGTGTGCCGGACCG CTTCAGCGGCAGCGGTTCCAGAACCGACTTTACCTCACCATCAGCTCGCTGCAGGCCGAAGATGTCGCCGTCTATTACTGCCAACAGAGCAACGAAGATCCGCCTACTTTCGGACAGGGG ACTAAAACTGGAAATCAAAGGGCGGAGGAGGCTCGGGTGGAGGAGGATCGGGAGGAGGCGGGTCCGGTGGTGGCGGATCGCAATCCAGCTGGTGCAGTCCGGCGCAGAAGTGAAGAAGCCG GAGCGTCCGTGAAAG TGAGCTGCAAGGCCTCAGGGTACATCTTCACCAATTACGGCATGAATTGGGTGCGGCAGGCACCCGGACAGCGCCTGGAGTGGATGGGCTGGATCAACACTTACACCGGGGAAAGCACGTA CTCGGCCGACTTCAAAGGACGGGTGACCATTACCCTGGATAACCTCGGCCTCAACCGCTTACATGGAGCTCTCACTCACTTAGATCCGAGGACACTGCCGTCTACTGTGCAAGGAGCGGA GGCTACGACCCTATG GACTATTGGGGACAAGGCACTACTGTGACTGTGTCGTCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCC GGCCAGCGGCGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTTA CTGCTAA (SEQ ID NO: 1).

The IRES nucleic acid sequence (contained in constructs A and B) is as follows:
GAATTCCCGGGTCGACCTGCAGAAGCTTAAAACAGCTCTGGGGTTGTACCCCACCAGAGGCCCACGTGGCGGCTAGTACTCCGGTATTGCGGTACCCTTGTACGCCTGTTTTATATACTCCC TTCCCGTAACTTAGACGCACAAAAACCCAAGTTCAATAGAAGGGGGTA CAAACCAGTACCACCACGAACAAGCACTTCTGTTTCCCCGGTGATGTCGTATAGACTGCTTGCGTGGTTGAAAGCGACGGATCCGTTATCCGCTTATGTACTTCGAGAAGCCCAGTACCAC CTCGGAATCTTCGATGCGTTGCGCTCAGCACTCAACCCCAGAGGTGT AGCTTAGGCTGATGAGTCTGGACATCCCTCACCGGTGACGGTGGTCCAGGCTGCGTTGGCGGCCTACCTATGGCTAACGCCATGGGACGCTAGTTGTGAACAAGGTGTGAAGAGCCCTATTG AGCTACATAAGAATCCTCCGGCCCCTGAATGCGGCTAATCCCAAACC TCGGAGCAGGTGGTCACAAACCAGTGATTGGCCTGTCGTAACGCGCAAGTCCGTGGCGGAACCGACTACTTTGGGTGTCCGTGTTTCCTTTTATTTTATTGTGGCTGCTTATGGTGACAAT CACAGATTGTTATCATAAAGCGAATTGGATTGCGGCCGC (SEQ ID NO: 2).

The nucleic acid sequence of human IL-2 (accession number BC066257, contained in constructs A and B) is as follows:
ATGTACAGGATGCAAACTCCTGTCTTGCATTGCACTAAGTCTTGCACTTGTCACAAACAGTGCACCTACTTCAAGTTCTACAAAGAAAACACAGCTACAACTGGAGCATTTACTGCTGGATT TACAGATGATTTTGAATGGAATTAATAATTACAAGAATCCCAAACTCACCAGGATGCTCACATTTAAGTTTTACATGCCCAAGAAGGCCACAGAACTGAAACATCTTCAGTGTC TAGAAGAAGAACTCAAACCTCTGGAGGAAGTGCTAAATTTAGCTCAAAGCAAAAACTTTTCACTTAAGACCCAGGGACTTAATCAGCAATATCGTAATAGTTCTGGAACTAAAGGGATC TGAAACAACATTCATGTGTGAATATGCTGATGAGACAGCAACCATTGTAGAATTTCTGAACAGATGGATTACCTTTTGTCAAGCATCATCTCAACACTGACTTGA (SEQ ID NO: 3).

The nucleic acid sequence of human IL-15 (accession number BC100961.1, contained in construct C) is as follows:
ATGAGAATTTCGAAACCACATTTGAGAAGTATTTCCATCCAGTGCTACTTGTGTTTACTTCTAAACAGTCATTTTCTAACTGAAGCTGGCATTCATGTCTTCATTTTGGGCTGTTTCAGTG CAGGGCTTCCTAAAACAGAAGCCAACTGGGTGAATGTAATAAGTGATTTGAAAAAAAATTGAAGATCTTATTCAATCTATTGCATATTGATGCTACTTTATACGGAAAGTGATGTTCACCC CAGTT GCAAAGTAACAGCAAGTGCTTTCTCTTGGAGTTACAAGTTATTTCACTTGAGTCCGGAGATGCAAGTATTCATGATACAGTAGAAATCTGATCATCCTAGCAAACAAGTTTGTC TTCTAATGGGAATGTAACAGAATCTGGATGCAAAGAATGTGAGGAACTGGAGGGAAAAATATTAAAGAATTTTTGCAGAGTTTGTACATATTGTCCAAATGTTCATCAACACTTCT (SEQ ID NO. 4).

The nucleic acid sequence of the 2A element (contained in construct C) is as follows:
GAGGGCAGGGGAAGTCTAACTAACATGCGGGGACGTGGAGGAAAATCCCGGCCCA (SEQ ID NO:5).

The nucleic acid sequence of GFP (contained in constructs B and C) is as follows:
ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACG GCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCCACCCT CGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTT AAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGA ACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCAT CAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCAC TACCAGCAGAACACCCCCATCGGCGACGGCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCCAACGAGAAGCGCGATCCACATGGTCCTGCTGG AGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA (SEQ ID NO: 6).

The wild-type CXCR4 chemokine receptor has the following amino acid sequence:
MSIPLPLLQIYTSDNYTEEMGSGDYDSMKEPCFREENANFNKIFLPTIYSIIFLTGIVGNGLVILVMGYQKKLRSMTDKYRLHLSVADLLFVITLPFWAVDAVANWYFGNFLCKAVHVHVIYT VNLYSSVLILAFISLDRYLAIVHATNSQRPRKLLAEKVVYVGVWIPALLLTIPDFIFANVS The nucleic acid sequence encoding the modified C' truncated CXCR4 (WHIM R334x mutation in which the 19 C-terminal amino acids of SEQ ID NO:7 have been removed, contained in NM_001008540.2, construct D) has the following nucleic acid sequence:
ATGTCCATTCCTTTGCCTCTTTTGCAGATATACACTTCAGATAACTACACCGAGGAAATGGGCTCAGGGGACTATGACTCCATGAAGGAACCCTGTTTCCGTGAAGAAAATGCTAATTTCA ATAAAATCTTCCTGCCCACCATCTACTCCATCATCTTCTTAACTGGCATTGTGGGCAATGGATTGGTCATCCTGGTCATGGGTTACCAGAAGAAACTGAGAAGCATGACGGACAAGTACAG GCTGCACCTGTCA GTGGCCGACCTCCTCTTTGTCATCACGCTTCCCTTCTGGGCAGTTGATGCCGTGGCAAACTGGTACTTTGGGAACTTCCTATGCAAGGCAGTCCATGTCATCTACACAGTCAACCTCTACA GCAGTGTCCTCATCCTGGCCTTCATCAGTCTGGACCGCTACCTGGCCATCGTCCACGCCACCAACAGTCAGAGGCCAAGGAAGCTGTTGGCTGAAAAGGTGGTCTATGTTGGCGTCTGGAT CCCTGCCCTCCTG CTGACTATTCCCGACTTCATCTTTGCCAACGTCAGTGAGGCAGATGACAGATATATCTGTGACCGCTTCTACCCCAATGACTTGTGGGTGGTTGTGTTCCAGTTTCAGCACATCATGGTTG GCCTTATCCTGCCTGGTATTGTCATCCTGTCCTGCTATTGCATTATCCATCCAAGCTGTCACACTCCAAGGGCCACCAGAAGCGCAAGGCCCTCAAGACCACAGTCATCCTCCATCCTGGC TTTCTTCGCCTGT TGGCTGCCTTACTACATTGGGATCAGCATCGACTCCTTCATCCTCCTGGAAATCATCAAGCAAGGGTGTGAGTTTGAGAACACTGTGCACAAGTGATTTCCATCACCGAGGCCCTAGCTTT CTTCCACTGTTGTCTGAACCCCATCCTCTATGCTTTCCTTGGAGCCAAATTTAAAACCTCTGCCCAGCACGCACTCACCTCTGTGAGCAGAGGGTCCAGCCTCAAAGATCCTCTCCAAAGGA AAGTAA (SEQ ID NO: 8).

To prepare retroviral particles for transduction, eco-phoenix cells were seeded at a density of ^5x105 cells in 6-well plates and allowed to reach 60-70% confluence on the day of infection. Eco-phoenix cells were transfected with 2μg/well of retroviral plasmid MSGV (containing the tested construct) for 5 hours at 37°C, 5% _CO2 using Lipofectamine transfection reagent according to the manufacturer's protocol. After 48 hours, the supernatant containing retroviral particles was harvested and filtered through a 0.45μm filter (EMD Millipore, Billerica, MA, USA) to remove cell debris. The supernatant was used to transduce PG13 cells to produce a PG13-based packaging cell line and produce the modified MSGV virus.

PG13 transduction was mediated by RetroNectin® (Takara Shuzo). Non-tissue culture 24-well plates were coated with RetroNectin® (diluted 1:40 in PBS). Plates were coated overnight at 4°C, then blocked with 2% BSA for 30 min at room temperature (RT) and washed once with PBS before use. Virus-containing supernatants from transfected eco-phoenix cells were added to RetroNectin®-coated plates and centrifuged for 2 h. In the next step, PG13 cells were added to the wells and incubated with the virus for 24 h. After 3 days, positive PG13 cells (detected by FACS) were selected and expanded.

The obtained viral particles were used to transduce SAK cells during the expansion method, which were prepared essentially as described in Example 1. Specifically, freshly isolated PBMCs were stimulated for 48 hours in the presence of 30 ng/mL OKT3 and 1000 IU/mL IL-2. After stimulation, the cells were transduced with the retroviral vector (produced by PG13 cells as described above) by transferring them to culture dishes precoated with the retroviral vector as described above. For this purpose, non-tissue culture 24-well plates were coated with RetroNectin®, and the virus-containing supernatant (from PG13) was added to the RetroNectin®-coated plates and centrifuged for 2 hours. In the next step, the stimulated cells were added to the wells and incubated with the viral particles for 24 hours. The cells were then further expanded with 30 ng anti-CD3 and 1000 IU/mL IL-2 as described in Example 1. Positive (construct-containing) cells were detected and quantified by FACS using APC anti-CXCR4 antibody, CD123-Fc and PE anti-Fc antibodies, or GFP analysis, confirming expression of the corresponding exogenous gene.

Similarly, constructs A and D are cloned into a plasmid backbone along with the 2A sequence to produce a nucleic acid construct capable of constitutively expressing a CD123-specific CAR, a modified CXCR4 receptor, and IL-2 (anti-CD123-IRES-IL-2-2A-CXCR4whim). The combined construct is transduced into SAK cells as described above. Similarly, construct CXCR4whim-IRES-IL-2 is produced and transduced into freshly prepared SAK cells.

Cells transduced with the various constructs will be assayed for antitumor activity in vitro and in vivo essentially as described in Examples 2 and 7, respectively.

Cytotoxic Activity of Genetically Modified SAK Cells Expressing CD19-Specific CAR Genetically modified SAK cells expressing CD19 CAR (modified to recognize CD19-expressing B-cell malignancies) were prepared essentially as described in Example 8 with the following modifications.

The vector was engineered to express the scFv targeting domain (specific for CD19), the CD8-derived hinge region, the CD8-derived transmembrane region, the 4-1BB/CD137 costimulatory domain, and the CD3 ζ chain intracellular signaling domain. The retroviral MSGV-1D3-28Z All ITAM intact expression vector carrying the CD19CAR sequence was used to generate the nucleic acid construct. The "1D3-28Z All ITAM intact" insert was removed from the plasmid using restriction enzymes NcoI and SalI. The CD19CAR cDNA was amplified by PCR and ligated into a plasmid carrying NcoI and SalI restriction sites. The resulting construct was verified by DNA sequencing.

The nucleic acid sequence encoding CD19CAR (sense strand) is as follows:
gccaccatggctctgcccgtgaccgcactcctcctgccactggctctgctgcttcacgccgctcgcccacaagtccagcttcaagaatcagggcctggtctggtgaagccatctgagactc tgtccctcacttgcaccgtgagcggagtgtccctcccagactacggagtgagctggattagaca gcctcccggaaagggactggagtggatcggagtgatttggggtagcgaaaccacttactataactctcttccctgaagtcacgggtcaccatttcaaaggataactcaaagaatcaagtgagc ctcaagctctcatcagtcaccgccgctgacaccgccgtgtattactgtgccaagcattactacta tggaggtcctacgccatggactactgggggccaggaactctggtcactgtgtcatctggtggaggaggtagcggaggagcgggagcggtggaggtggctccggaggtggcggaagcgaa atcgtgatgacccagagccctgcaacctgtccctttctcccgggaacggggctaccctttcttg tcggcatcacaagatatctcaaaatacctcaattggtatcaacagaagccgggacaggcccctaggcttcttatccacacctctcgcctgcatagcgggattcccgcacgctttagc gggtctggaagcgggaccgactacactctgaccatctcatctctccagcccgaggacttcgccgt ctacttctgccagcaggtaacaccctgccgtacaccttcggccaggcaccaagcttgagatcaaaaccactactcccgctccaaggccacccacccctgccccgaccatcgcctctcag ccgctttccctgcgtccggaggcatgtagacccgcagctggtgggggccgtgcatacccggggtct tgacttcgcctgcgatatctacttgggcccctctggctggtacttgcgggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatcttt aagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgccggttccc agaggaggaggaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggag tacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaa tccccaagagggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccaggactc agcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg (SEQ ID NO: 9).

Preparation of retroviral particles for transduction was performed as described in Example 8. Detection and quantification of positive (construct-containing) cells was performed by FACS analysis using CD19-Fc and PE anti-Fc antibodies, confirming expression of the CD19CAR gene.

The cytotoxic activity of CD19CAR SAK cells against CD19 positive cells (Toledo) and CD19 negative cells (U937) was measured in comparison with control SAK cells. CFSE-based cytotoxicity assays were performed essentially as described in Example 2 using SAK and CD19CAR SAK cells as effector cells and Toledo and U937 cells as target cells. The results for CD19 ⁺ (Toledo) and CD19 ^- (U937) cells are shown in Figures 11A and 11B, respectively, and represent the mean ± standard deviation of triplicates at each ratio.

As shown in FIG. 11A, CD19CAR SAK cells were found to be significantly more effective at eradicating Toledo cells (CD19 positive cells) than control SAK cells.

CD19CAR SAK cells showed excellent cytotoxic activity at each E:T ratio tested. For example, at an E:T ratio of 1:1, CD19CAR SAK cells showed 93% efficiency, whereas control SAK cells at the same ratio showed only 54% efficiency. In contrast, the cytotoxic activity of CD19CAR SAK and control SAK against U937 cells was identical, meaning that the enhanced activity of CD19CAR cells is specific to CD19-expressing tumor cells.

These results indicate that CAR SAK can be an effective alternative to conventional CAR T cells, particularly for solid tumors for which conventional CAR T cells have poor therapeutic effects.

In vivo alloreactivity To characterize the potential of SAK cells to induce graft-versus-host (GVH) responses and graft-versus-host disease (GVHD), NSG mice were injected with SAK cells or PBMCs and monitored for the development of GVHD for one month. For this purpose, NSG mice were first irradiated with 300 cGy. 24 hours later, SAK cells or PBMCs (purified from human blood) were injected intravenously (iv) via the dorsal tail vein in a final volume of 200 μL (5× ¹⁰⁶ cells per mouse). Acute GVHD clinical scores were calculated three times a week. Each of the following four parameters (posture, activity, fur texture, and skin integrity) was scored as 0 (if absent) or 2 (if present). Mouse weights were also measured three times a week. The results are shown in Figures 12A-12C.

Approximately 15 days after injection, mice receiving PBMCs began to show clinical signs of GVHD (Figure 12C). The mice were not groomed and looked dirty. They also began to lose weight. All mice treated with PBMCs developed severe acute GVHD and died within 17 days (Figure 12A). In contrast, mice treated with SAK cells showed minimal signs of GVHD and all survived. These mice experienced milder weight loss compared to the PBMC group (Figure 12B).

These results demonstrate the suitability of SAK cells for allogeneic clinical use, including treatment of histoincompatible subjects, and suggest that SAK cells may be a useful improvement to existing adoptive cell transfer treatment protocols.

Evaluation of Cytotoxicity of Cryopreserved SAK Cells For SAK cells to be used as a commercial therapeutic product, their production and freezing methods must be advantageously evaluated prior to clinical use. To evaluate the cytotoxic activity of thawed SAK cells, the cells were harvested 24 hours after freezing and co-cultured with MV411 and THP-1 leukemia target cells at various ratios.

For this purpose, fresh SAK cells (described in Example 1) at day 11 of growth were frozen in freezing medium (containing 90% fetal calf serum (FCS) + 10% dimethyl sulfoxide (DMSO)) and transferred to liquid nitrogen for cryopreservation. The resulting cryopreserved SAK cells were used in cytotoxicity assays 24 hours after thawing and compared to the cytotoxic activity of fresh SAK cells at day 11 (which had not been subjected to freezing and subsequent thawing). The results are shown in Figures 13A-13B.

As shown in Figures 13A and 13B, thawed SAK had reduced cytotoxic activity compared to fresh SAK, indicating that freeze-thaw cycles may affect the viability and/or activity of SAK cells to some extent. However, cryopreserved SAK cells showed significant and potent cytotoxic activity against MV411 (Figure 13B) and THP-1 (Figure 13A) cells even at low E:T ratios, retaining at least about 70-80% of the cytotoxic activity of the corresponding fresh SAK cells.

The results therefore indicate that SAK cells may be used not only immediately after preparation, but also after cryopreservation. Furthermore, it is demonstrated herein that the cell composition exhibits very potent cytotoxicity immediately after thawing, without the need for additional growth or manipulation steps. Thus, cell compositions containing SAK cells can be manufactured and distributed as stable products with long shelf lives, which is facilitated by cryopreservation. This is in contrast to certain cell therapies based on other immune cell populations, such as NK cells, which have been reported to be highly sensitive to cryopreservation, a property suggested to be associated with numerous failures in the clinical treatment of solid tumors (Mark et al., Nat Commun 11, 5224 (2020)).

Expression of immune checkpoint molecules in SAK cells The presence of immune checkpoint molecules in CD8 ⁺ SAK cells was evaluated as follows. SAK cells prepared as described in Example 1 were harvested on day 11 of growth, washed, and stained with either PE-labeled anti-Tim-3 antibody ( FIG. 14 , upper panel, left), APC-labeled anti-CTLA-4 antibody ( FIG. 14 , upper panel, right), APC-labeled anti-PD-1 antibody ( FIG. 14 , center panel, left), APC-labeled anti-TIGIT antibody ( FIG. 14 , center panel, right), or PE-labeled anti-Lag-3 antibody ( FIG. 14 , lower panel, left) for 30 minutes at 4° C., and analyzed by flow cytometry. APC- or PE-labeled anti-CD8 monoclonal antibody (Biolegend) and isotype control antibody were also used. In addition to the inhibitory checkpoint molecules Tim-3, CTLA-4, PD-1, TIGIT and Lag-3, the presence of the activating receptor DNAM-1 (FIG. 14 lower panel, right) was also assessed on SAK cells.

Notably, as shown in Figure 14, virtually all CD8 ⁺ cells were found to express the activating receptor DNAM-1. In contrast, inhibitory immune checkpoint molecules were either undetectable or detected at low levels. Notably, SAK cells did not express any CTLA-4 and expressed low levels of PD-1 and TIGIT, indicating a low likelihood of inhibition by tumors expressing the corresponding ligands.

Quantification of the percentage of cells expressing various surface markers in samples from different donors revealed that 99%-100% of CD8 ⁺ cells expressed DNAM-1, approximately 0% of cells expressed CTLA-4, 12%-15% of cells expressed PD-1, and 20%-22% of cells expressed TIGIT. Surface expression of Lag-3 and Tim-3 was found in approximately 51%-63% and 54%-97% of CD8 ⁺ cells, respectively.

Next, expression of checkpoint molecules on SAK cells was compared to expression of checkpoint molecules on CIK cells. Compositions were prepared from the same donors essentially as described in Example 1, and cells were harvested on day 11 and subjected to phenotypic analysis by flow cytometry as described above. The results are shown in Table 9 below, which shows the mean fluorescence intensity (MFI) measured upon staining for each indicated marker in each cell type, as well as the relative expression in SAK cells compared to CIK cells (fold increase, MFI of SAK cells/MFI of CIK cells, denoted as "SAK/CIK").

As shown in Table 9, cells prepared by the various growth protocols exhibited various phenotypes with respect to unique expression profiles of surface markers. In particular, SAK cells exhibit significantly higher levels of the tested markers than the corresponding CIK cells harvested on day 11. Thus, the results demonstrate that SAK cells can be readily identified and distinguished from other cell compositions by readily available methods. Additionally, the presence of surface markers known to be elevated during lymphocyte activation at higher levels in SAK cells than in CIK cells on day 11 further suggests that SAK cells adopt a "hyperactivated" phenotype during the improved growth method.

It is known that NK and T cell functions are regulated and maintained by the balance between inhibitory receptors (e.g., PD-1, TIGIT, Lag-3, Tim-3 and/or CTLA-4) and activating receptors (e.g., DNAM-1). Furthermore, blockade of inhibitory immune checkpoint receptors may be important in effectively reversing T cell exhaustion and restoring the antitumor capabilities of T cells. Thus, there is growing interest that blockade of inhibitory immune checkpoint molecules may also affect SAK cell activity.

Of note, it has been demonstrated herein that SAK cells have significantly enhanced cytotoxicity against tumor cells compared to CIK cells after 11 days of growth (e.g., Example 3). Without wishing to be bound by a particular theory or mechanism of action, the "hyperactivated" phenotype of SAK cells, characterized by a unique balance of various receptors and effector molecules disclosed herein, may be associated with favorable functional capabilities against tumor cells.

In summary, SAK cells produced by the advantageous protocols disclosed herein were found to exhibit a favorable profile of cell surface receptors, such as immune checkpoint molecules and chemokine receptors. In particular, SAK cells were found to be characterized by expression of DNAM-1 and CXCR6, which are known to positively affect immune effector survival, proliferation and/or activity. Without wishing to be bound by a particular theory or mechanism of action, these results are consistent with the ability to produce ACT compositions characterized by an activated phenotype that can minimize tumor evasion.

Involvement of Major Histocompatibility Complex Class I (MHCI) The killing mechanism of SAK cells against MV4-11 leukemia cells (a human cell line of mixed B-myelomonocytic leukemia, also referred to herein as MV411) was examined using an inhibitor of MHC class I. Cytotoxicity assays were performed essentially as described in Example 2, except that MV411 cells were incubated with 1 μg/mL anti-MHCI antibody (W6/32 clone, Biolegend) 1 hour before the addition of SAK cells .

Thus, the results show that SAK cells exhibit both MHCI-dependent and non-MHCI-dependent cytotoxic activity against leukemia cells. Notably, both activities did not require compatibility of HLA alleles, as the effector SAK cells were not histocompatible. Thus, compositions comprising SAK cells according to the present invention can also be advantageously used to treat immune-evasive or therapy-resistant tumors characterized by downregulated MHC-I expression and/or activity.

In summary, the results described herein demonstrate the suitability of SAK cells for the treatment of tumors of various origins in both histocompatible and non-histocompatible subjects. As demonstrated herein, SAK cells exhibited significant antitumor activity against hematopoietic and solid tumors, retained antitumor efficacy even in the absence of HLA compatibility, and further demonstrated improved safety as evidenced by a significant reduction in the risk of inducing graft-versus-host disease (GVHD). Without wishing to be bound by a particular theory or mechanism of action, the cytotoxic activity of SAK cells against various tumor cells may involve NKG2D and/or T cell receptor (TCR)-mediated activity, as shown herein.

The above description of the specific embodiments fully reveals the general nature of the present invention, so that others, applying current knowledge, can easily modify and/or adapt such specific embodiments to various applications without undue experimentation and without departing from the general concept. Such adaptations and modifications should therefore be understood and are intended to be within the meaning and scope of equivalents of the disclosed embodiments. It should be understood that the expressions or terms used herein are for the purpose of description and not for the purpose of limitation. The means, materials and steps for carrying out the various disclosed functions may take various alternative forms without departing from the invention.

SEQ ID NO: 1: Anti-human CD123 CAR
SEQ ID NO:2: IRES nucleic acid sequence SEQ ID NO:5: 2A element SEQ ID NO:6: GFP
SEQ ID NO: 9: CD19 CAR

Claims (15)

A cell composition for adoptive cell transfer (ACT) comprising in vitro expanded peripheral blood mononuclear cells (PBMCs), 70-85% of which are CD3 ⁺ CD8 ⁺ cells expressing NKG2D and granzyme B, at least 70% of which are CD56 ^- cells, and up to 5% of which are CD3 ^- cells, and which exhibit significant cytotoxic activity against tumor cells in vitro and in vivo. 2. The cell composition of claim 1, wherein 7-12% of the cells are CD3 ⁺ CD4 ⁺ cells, 20-28% are CD3 ⁺ CD56 ⁺ cells, and 10-14% are CD3 ⁺ CD8 ⁺ CD56 ⁺ cells. The cell composition of claim 2, wherein the amount of CD3 ⁺ CD8 ⁺ cells in the composition is 6 to 8 times the amount of CD3 ⁺ CD4 ⁺ cells in the composition. The cell composition of any one of claims 1 to 3, comprising less than 3% CD3 ^- CD56 ⁺ cells. The cell composition of any one of claims 1 to 4, wherein at least 40% of the CD3 ⁺ CD8 ⁺ cells are characterized by surface expression of at least one marker selected from the group consisting of perforin, Fas ligand (FasL), CXCR4 and CXCR2. The cell composition of any one of claims 1 to 5, wherein the CD3 ⁺ CD8 ⁺ cells are further characterized as DNAM-1 ⁺ , CTLA-4 ^- cells. The cell composition of any one of claims 1 to 6, wherein at least 90% of the CD3 ⁺ CD8 ⁺ cells are characterized by surface expression of CXCR3 and at least 30% of the CD3 ⁺ CD8 ⁺ cells are characterized by surface expression of CXCR6. The cell composition of any one of claims 1 to 7 , which exhibits non-MHC-restricted cytotoxic activity mediated by granzyme B and/or perforin against the tumor cells. The cell composition of any one of claims 1 to 8, wherein the cells are genetically modified to express at least one of a chimeric antigen receptor (CAR), a cytokine, a chemokine, a cytokine or chemokine receptor, or any combination thereof. 1. A method for producing a cell composition for adoptive cell transfer (ACT), comprising:
a. providing a peripheral blood mononuclear cell (PBMC) sample;
b. Propagating the PBMCs in the constant presence of an effective amount of IL-2 and a CD3 activator, and in the absence of other cytokines or antibodies, for at least 9 days, thereby increasing the number of viable cells in the culture by at least 20 fold and obtaining a cell composition comprising at least 5 x ¹⁰ to 10 x ¹⁰ viable cells. The method according to claim 10 , wherein the IL-2 and the CD3 activator are the only exogenously added cell stimulating factors, and proliferation is carried out under conditions in which they are constantly present. 12. The method of claim 10 or 11, wherein step b comprises expanding the cells in the presence of 500-1500 IU/mL IL-2 and 10-60 ng/mL anti-CD3 antibody, which are replenished every 48-96 hours for 10-16 days. The cell composition of any one of claims 1 to 9 , for use in treating a tumor in a subject in need thereof, the tumor being any one selected from the group consisting of leukemia, multiple myeloma, prostate tumor, or mesothelioma . The cell composition of claim 13 , wherein the tumor is a hematopoietic tumor or a solid tumor. The cell composition of claim 13 or 14, wherein the tumor is resistant to treatment with at least one CTLA-4 specific blocking antibody and/or has not undergone a therapeutic regimen with a checkpoint molecule inhibitor .