Engineering Tolerance toward Allogeneic CAR-T Cells by Regulation of MHC Surface Expression with Human Herpes Virus-8 Proteins.

Allogeneic, off-the-shelf (OTS) chimeric antigen receptor (CAR) cell therapies have the potential to reduce manufacturing costs and variability while providing broader accessibility to cancer patients and those with other diseases. However, host-versus-graft reactivity can limit the durability and efficacy of OTS cell therapies requiring new strategies to evade adaptive and innate-immune responses. Human herpes virus-8 (HHV8) maintains infection, in part, by evading host T and natural killer (NK) cell attack. The viral K3 gene encodes a membrane-tethered E3 ubiquitin ligase that discretely targets major histocompatibility complex (MHC) class I components, whereas K5 encodes a similar E3 ligase with broader specificity, including MHC-II and the MHC-like MHC class I polypeptide-related sequence A (MIC-A)- and sequence B (MIC-B)-activating ligands of NK cells. We created γ-retroviruses encoding K3 and/or K5 transgenes that efficiently transduce primary human T cells. Expression of K3 or K5 resulted in dramatic downregulation of MHC-IA (human leukocyte antigen [HLA]-A, -B, and -C) and MHC class II (HLA-DR) cell-surface expression. K3 expression was sufficient for T cells to resist exogenously loaded peptide-MHC-specific cytotoxicity, as well as recognition in one-way allogeneic mixed lymphocyte reactions. Further, in immunodeficient mice engrafted with allogeneic T cells, K3-transduced T cells selectively expanded in vivo. Ectopic K5 expression in MHC class I-, MIC-A+/B+ K562 cells also reduced targeting by primary NK cells. Coexpression of K3 in prostate stem cell antigen (PSCA)-directed, inducible MyD88/CD40 (iMC)-enhanced CAR-T cells did not impact cytotoxicity, T cell growth, or cytokine production against HPAC pancreatic tumor target cells, whereas K5-expressing cells showed a modest reduction in interleukin (IL)-2 production without effect on cytotoxicity. Together, these results support application of these E3 ligases to advance development of OTS CAR-T cell products.

Streamlined production of genetically modified T cells with activation, transduction and expansion in closed-system G-Rex bioreactors.

BACKGROUND: Gas Permeable Rapid Expansion (G-Rex) bioreactors have been shown to efficiently expand immune cells intended for therapeutic use, but do not address the complexity of the viral transduction step required for many engineered T-cell products. Here we demonstrate a novel method for transduction of activated T cells with Vectofusin-1 reagent. Transduction is accomplished in suspension, in G-Rex bioreactors. The simplified transduction step is integrated into a streamlined process that uses a single bioreactor with limited operator intervention. METHODS: Peripheral blood mononuclear cells (PBMCs) from healthy donors were thawed, washed and activated with soluble anti-CD3 and anti-CD28 antibodies either in cell culture bags or in G-Rex bioreactors. Cells were cultured in TexMACS GMP medium with interleukin (IL)-7 and IL-15 and transduced with RetroNectin in bags or Vectorfusin-1 in the G-Rex. Total viable cell number, fold expansion, viability, transduction efficiency, phenotype and function were compared between the two processes. RESULTS: The simplified process uses a single vessel from activation through harvest and achieves 56% transduction with 29-fold expansion in 11 days. The cells generated in the simplified process do not differ from cells produced in the conventional bag-based process functionally or phenotypically. DISCUSSION: This study demonstrates that T cells can be transduced in suspension. Further, the conventional method of generating engineered T cells in bags for clinical use can be streamlined to a much simpler, less-expensive process without compromising the quality or function of the cell product.

Regulated Expansion and Survival of Chimeric Antigen Receptor-Modified T Cells Using Small Molecule-Dependent Inducible MyD88/CD40.

Anti-tumor efficacy of T cells engineered to express chimeric antigen receptors (CARs) is dependent on their specificity, survival, and in vivo expansion following adoptive transfer. Toll-like receptor (TLR) and CD40 signaling in T cells can improve persistence and drive proliferation of antigen-specific CD4+ and CD8+ T cells following pathogen challenge or in graft-versus-host disease (GvHD) settings, suggesting that these costimulatory pathways may be co-opted to improve CAR-T cell persistence and function. Here, we present a novel strategy to activate TLR and CD40 signaling in human T cells using inducible MyD88/CD40 (iMC), which can be triggered in vivo via the synthetic dimerizing ligand, rimiducid, to provide potent costimulation to CAR-modified T cells. Importantly, the concurrent activation of iMC (with rimiducid) and CAR (by antigen recognition) is required for interleukin (IL)-2 production and robust CAR-T cell expansion and may provide a user-controlled mechanism to amplify CAR-T cell levels in vivo and augment anti-tumor efficacy.

Systematically probing the bottom-up synthesis of AuPAMAM conjugates for enhanced transfection efficiency.

BACKGROUND: Gold nanoparticles (AuNPs) have shown great promise as scaffolds for gene therapy vectors due to their attractive physiochemical properties which include biocompatibility, ease of functionalization via the nearly covalent gold-sulfur dative bond, and surface plasmon optical properties. Previously, we synthesized stable AuNP-polyamidoamine (AuPAMAM) conjugates and showed their success in vitro as non-viral gene delivery vectors. RESULTS: In this study, we systematically perturbed each component of the AuPAMAM conjugates and analyzed the resulting effect on transfection efficiency. Due to the modular, bottom-up nature of the AuPAMAM synthesis, we were able to probe each step of the fabrication process. The relationship between each conjugation parameter and the function of the final vector were investigated. More than fourfold enhanced transfection efficiency was achieved by modifying the PAMAM concentration, PAMAM core chemistry, PAMAM terminus chemistry, and self-assembled monolayer composition of the AuPAMAM conjugates. CONCLUSIONS: This work suggest that AuPAMAM synthesis platform is a promising non-viral gene therapy approach and highlights the importance of inspecting the role of each individual constituent in all nanotechnology hybrid materials.

The miR-223/nuclear factor I-A axis regulates glial precursor proliferation and tumorigenesis in the CNS.

Contemporary views of tumorigenesis regard its inception as a convergence of genetic mutation and developmental context. Glioma is the most common and deadly malignancy in the CNS; therefore, understanding how regulators of glial development contribute to its formation remains a key question. Previously we identified nuclear factor I-A (NFIA) as a key regulator of developmental gliogenesis, while miR-223 has been shown to repress NFIA expression in other systems. Using this relationship as a starting point, we found that miR-223 can suppress glial precursor proliferation via repression of NFIA during chick spinal cord development. This relationship is conserved in glioma, as miR-223 and NFIA expression is negatively correlated in human glioma tumors, and the miR-223/NFIA axis suppresses tumorigenesis in a human glioma cell line. Subsequent analysis of NFIA function revealed that it directly represses p21 and is required for tumorigenesis in a mouse neural stem cell model of glioma. These studies represent the first characterization of miR-223/NFIA axis function in glioma and demonstrate that it is a conserved proliferative mechanism across CNS development and tumorigenesis.

An adaptable system for improving transposon-based gene expression in vivo via transient transgene repression.

Transposons permit permanent cellular genome engineering in vivo. However, transgene expression falls rapidly postdelivery due to a variety of mechanisms, including immune responses. We hypothesized that delaying initial transgene expression would improve long-term transgene expression by using an engineered piggyBac transposon system that can regulate expression. We found that a 2-part nonviral Tet-KRAB inducible expression system repressed expression of a luciferase reporter in vitro. However, we also observed nonspecific promoter-independent repression. Thus, to achieve temporary transgene repression after gene delivery in vivo, we utilized a nonintegrating version of the repressor plasmid while the gene of interest was delivered in an integrating piggyBac transposon vector. When we delivered the luciferase transposon and repressor to immunocompetent mice by hydrodynamic injection, initial luciferase expression was repressed by 2 orders of magnitude. When luciferase expression was followed long term in vivo, we found that expression was increased >200-fold compared to mice that received only the luciferase transposon and piggyBac transposase. We found that repression of early transgene expression could prevent the priming of luciferase-specific T cells in vivo. Therefore, transient transgene repression postgene delivery is an effective strategy for inhibiting the antitransgene immune response and improving long-term expression in vivo without using immunosuppression.

Hypoimmune anti-CD19 chimeric antigen receptor T cells provide lasting tumor control in fully immunocompetent allogeneic humanized mice.

Manufacturing autologous chimeric antigen receptor (CAR) T cell therapeutics is complex, and many patients experience treatment delays or cannot be treated at all. Although current allogeneic CAR products have the potential to overcome manufacturing bottlenecks, they are subject to immune rejection and failure to persist in the host, and thus do not provide the same level of efficacy as their autologous counterparts. Here, we aimed to develop universal allogeneic CAR T cells that evade the immune system and produce a durable response. We generated human hypoimmune (HIP) T cells with disrupted B2M, CIITA, and TRAC genes using CRISPR-Cas9 editing. In addition, CD47 and anti-CD19 CAR were expressed using lentiviral transduction. These allogeneic HIP CD19 CAR T cells were compared to allogeneic CD19 CAR T cells that only expressed the anti-CD19 CAR (allo CAR T). In vitro assays for cancer killing and exhaustion revealed no differences between allo CAR T and HIP CAR T cells, confirming that the HIP edits did not negatively affect T cell performance. Clearance of CD19+ tumors by HIP CAR T cells in immunodeficient NSG mice was comparable to that of allo CAR T cells. In fully immunocompetent humanized mice, HIP CAR T cells significantly outperformed allo CAR T cells, showed improved persistence and expansion, and provided lasting cancer clearance. Furthermore, CD47-targeting safety strategies reliably and specifically eliminated HIP CAR T cells. These findings suggest that universal allogeneic HIP CAR T cell-based therapeutics might overcome the limitations associated with poor persistence of allogeneic CAR T cells and exert durable anti-tumor responses.

IRAK-M removal counteracts dendritic cell vaccine deficits in migration and longevity.

To function optimally as vaccines, dendritic cells (DCs) must actively migrate to lymphoid organs and maintain a viable, mature state for sufficient time to effectively present their Ag to cognate T cells. Unfortunately, mature DCs rapidly lose viability and function after injection, and only a minority leaves the vaccine site and migrates to lymph nodes. We show that all of these functions can be enhanced in DCs by removal of IL-1R-associated kinase M (IRAK-M). We found that IRAK-M is induced in DCs by TLR ligation and that its absence from these cells leads to increased activation of the p38-MAPK and NF-κB pathways, which, in turn, improves DC migration to lymph nodes, increases their longevity, and augments their secretion of Th1-skewing cytokines and chemokines. These biological effects have immunological consequences. IRAK-M(-/-) DCs increase the proliferation and activation of Ag-specific T cells, and a single vaccination with Ag-pulsed, LPS-matured IRAK-M(-/-) DCs eliminates established tumors and prolongs the survival of EG7 or B16.f10 tumor-bearing mice, without discernible induction of autoimmune disease. Thus, manipulation of IRAK-M levels can increase the potency of DC vaccines by enhancing their Ag-presenting function, migration, and longevity.

T lymphocytes coexpressing CCR4 and a chimeric antigen receptor targeting CD30 have improved homing and antitumor activity in a Hodgkin tumor model.

For the adoptive transfer of tumor-directed T lymphocytes to prove effective, there will probably need to be a match between the chemokines the tumor produces and the chemokine receptors the effector T cells express. The Reed-Stemberg cells of Hodgkin lymphoma (HL) predominantly produce thymus- and activation-regulated chemokine/CC chemokine ligand 17 (TARC/CCL17) and macrophage-derived chemokine (MDC/CCL22), which preferentially attract type 2 T helper (Th2) cells and regulatory T cells (Tregs) that express the TARC/MDC-specific chemokine receptor CCR4, thus generating an immunosuppressed tumor environment. By contrast, effector CD8(+) T cells lack CCR4, are nonresponsive to these chemokines and are rarely detected at the tumor site. We now show that forced expression of CCR4 by effector T cells enhances their migration to HL cells. Furthermore, T lymphocytes expressing both CCR4 and a chimeric antigen receptor directed to the HL associated antigen CD30 sustain their cytotoxic function and cytokine secretion in vitro, and produce enhanced tumor control when infused intravenously in mice engrafted with human HL. This approach may be of value in patients affected by HL.

T cells as vehicles for cancer vaccination.

The success of cancer vaccines is dependent on the delivery of tumor-associated antigens (TAAs) within lymphoid tissue in the context of costimulatory molecules and immune stimulatory cytokines. Dendritic cells (DCs) are commonly utilized to elicit antitumor immune responses due to their attractive costimulatory molecule and cytokine expression profile. However, the efficacy of DC-based vaccines is limited by the poor viability and lymph-node migration of exogenously generated DCs in vivo. Alternatively, adoptively transferred T cells persist for long periods of time in vivo and readily migrate between the lymphoid and vascular compartments. In addition, T cells may be genetically modified to express both TAA and DC-activating molecules, suggesting that T cells may be ideal candidates to serve as cellular vehicles for antigen delivery to lymph node-resident DCs in vivo. This paper discusses the concept of using T cells to induce tumor-specific immunity for vaccination against cancer.

T cells expressing constitutively active Akt resist multiple tumor-associated inhibitory mechanisms.

Adoptive transfer of antigen-specific cytotoxic T lymphocytes has shown promise for the therapy of cancer. However, tumor-specific T cells are susceptible to diverse inhibitory signals from the tumor microenvironment. The Akt/protein kinase B plays a central role in T-cell proliferation, function, and survival and we hypothesized that expression of constitutively active Akt (caAkt) in T cells could provide resistance to many of these tumor-associated inhibitory mechanisms. caAkt expression in activated human T cells increased proliferation and cytokine production, a likely result of their sustained expression of nuclear factor-κB (NF-κB) and provided resistance to apoptosis by upregulating antiapoptotic molecules. caAkt expressing T cells (caAkt-T-cells) were also relatively resistant to suppression by and conversion into regulatory T cells (Tregs). These characteristics provided a survival advantage to T cells cocultured with tumor cells in vitro; CD3/28-stimulated T cells expressing a chimeric antigen receptor (CAR) specific for disialoganglioside (GD2) that redirected their activity to the immunosuppressive, GD2-expressing neuroblastoma cell line, LAN-1, resisted tumor-induced apoptosis when co-expressing transgenic caAkt. In conclusion, caAkt-transduced T cells showed resistance to several evasion strategies employed by tumors and may therefore enhance the antitumor activity of adoptively transferred T lymphocytes.

Genetic manipulation of tumor-specific cytotoxic T lymphocytes to restore responsiveness to IL-7.

Adoptive transfer of antigen-specific cytotoxic T lymphocytes (CTLs) can induce objective clinical responses in patients with malignant diseases. The option of providing a proliferative and survival advantage to adoptively transferred CTLs remains a challenge to improve their efficacy. Host lymphodepletion and administration of recombinant interleukin-2 (IL-2) are currently used to improve CTL survival and expansion after adoptive transfer, but these approaches are frequently associated with significant side effects and may increase proliferation of T regulatory cells. IL-7 is a crucial homeostatic cytokine that has been safely administered as a recombinant protein. However, while IL-7 induces robust expansion of naive and memory T lymphocytes, the lack of expression of the IL-7 receptor alpha chain (IL-7Ralpha) by CTLs precludes their response to this cytokine. We found that CTLs can be genetically modified to re-express IL-7Ralpha, and that this manipulation restores the response of these cells to IL-7 without apparent modification of their antigen specificity or dependency, and without changing their response to other common gamma (gammac) chain cytokines. This approach may allow selective expansion of CTLs without the unwanted effects associated with IL-2.

Inducible MyD88/CD40 synergizes with IL-15 to enhance antitumor efficacy of CAR-NK cells.

Natural killer (NK) cells expressing chimeric antigen receptors (CARs) are a promising anticancer immunotherapy, leveraging both innate NK cell antitumor activity and target-specific cytotoxicity. Inducible MyD88/CD40 (iMC) is a potent, rimiducid-regulated protein switch that has been deployed previously as a T-cell activator to enhance proliferation and persistence of CAR-modified T cells. In this study, iMC was extended to CAR-NK cells to enhance their growth and augment cytotoxicity against tumor cells. iMC-activated NK cells substantially increased cytokine and chemokine secretion and displayed higher levels of perforin and granzyme B degranulation. In addition, iMC activation could be coupled with ectopic interleukin-15 (IL-15) to further enhance NK cell proliferation. When coexpressed with a target-specific CAR (CD123 or BCMA), this IL-15/iMC system showed further augmented antitumor activity through enhanced CAR-NK cell expansion and cytolytic activity. To protect against potential toxicity from engineered NK cells, an orthogonal rapamycin-regulated Caspase-9 (iRC9) was included in a 4-gene, dual-switch platform. After infusion of dual-switch NK cells, pharmacologic iRC9 dimerization led to rapid elimination of a majority of expanded transduced NK cells. Thus, CAR-NK cells utilizing dual molecular switches provide an innovative and effective approach to cancer immunotherapy with controlled specificity, efficacy, and safety.

Selective depletion of a minor subpopulation of B-chronic lymphocytic leukemia cells is followed by a delayed but progressive loss of bulk tumor cells and disease regression.

Cancer precursor/progenitor cells may initiate and sustain the growth of tumors, but evidence for their existence in human disease is indirect, relying on their in vitro properties and animal models. More directly, specific elimination of these rare cells from cancer patients should produce a delayed but progressive disappearance of differentiated malignant progeny. Here, we describe selective eradication of a putative precursor population in a patient with B-cell chronic lymphocytic leukemia, followed 6 months later by a progressive loss of mature tumor cells without further treatment. This outcome supports the presence of a rare population of precursor/progenitor cells in human malignancies, and suggests benefit from their removal.

Constitutively active MyD88/CD40 costimulation enhances expansion and efficacy of chimeric antigen receptor T cells targeting hematological malignancies.

Successful adoptive chimeric antigen receptor (CAR) T-cell therapies against hematological malignancies require CAR-T expansion and durable persistence following infusion. Balancing increased CAR-T potency with safety, including severe cytokine-release syndrome (sCRS) and neurotoxicity, warrants inclusion of safety mechanisms to control in vivo CAR-T activity. Here, we describe a novel CAR-T cell platform that utilizes expression of the toll-like receptor (TLR) adaptor molecule, MyD88, and tumor-necrosis factor family member, CD40 (MC), tethered to the CAR molecule through an intentionally inefficient 2A linker system, providing a constitutive signal that drives CAR-T survival, proliferation, and antitumor activity against CD19+ and CD123+ hematological cancers. Robust activity of MC-enhanced CAR-T cells was associated with cachexia in animal models that corresponded with high levels of human cytokine production. However, toxicity could be successfully resolved by using the inducible caspase-9 (iC9) safety switch to reduce serum cytokines, by administration of a neutralizing antibody against TNF-α, or by selecting "low" cytokine-producing CD8+ T cells, without loss of antitumor activity. Interestingly, high basal activity was essential for in vivo CAR-T expansion. This study shows that co-opting novel signaling elements (i.e., MyD88 and CD40) and development of a unique CAR-T architecture can drive T-cell proliferation in vivo to enhance CAR-T therapies.

Two-Dimensional Regulation of CAR-T Cell Therapy with Orthogonal Switches.

Use of chimeric antigen receptors (CARs) as the basis of targeted adoptive T cell therapies has enabled dramatic efficacy against multiple hematopoietic malignancies, but potency against bulky and solid tumors has lagged, potentially due to insufficient CAR-T cell expansion and persistence. To improve CAR-T cell efficacy, we utilized a potent activation switch based on rimiducid-inducible MyD88 and CD40 (iMC)-signaling elements. To offset potential toxicity risks by this enhanced CAR, an orthogonally regulated, rapamycin-induced, caspase-9-based safety switch (iRC9) was developed to allow in vivo elimination of CAR-T cells. iMC costimulation induced by systemic rimiducid administration enhanced CAR-T cell proliferation, cytokine secretion, and antitumor efficacy in both in vitro assays and xenograft tumor models. Conversely, rapamycin-mediated iRC9 dimerization rapidly induced apoptosis in a dose-dependent fashion as an approach to mitigate therapy-related toxicity. This novel, regulatable dual-switch system may promote greater CAR-T cell expansion and prolonged persistence in a drug-dependent manner while providing a safety switch to mitigate toxicity concerns.

Adoptive T-cell immunotherapy of chronic lymphocytic leukaemia.

Immunotherapy for B-cell chronic lymphocytic leukaemia (B-CLL) and other haematological malignancies may consist of passive antibody, active immunization or adoptive T-cell transfer. This chapter will focus on T-lymphocyte immunotherapy; an approach supported by earlier observations that the beneficial effects of allogeneic stem cell transplantation depend, in part, on the graft-versus-leukaemia effects mediated by these cells. One promising strategy consists of the genetic manipulation of effector T lymphocytes to express tumour-specific T-cell receptors or chimeric antigen receptors directed against surface antigens on the B-CLL cells. This methodology is now being integrated with the concept that tumour recurrence may be due to the persistence of a reservoir of more primitive and chemoresistant tumour cells, dubbed 'cancer stem cells', with self-renewal capacity. Identification and characterization of these cancer stem cells in B-CLL is crucial for the development of new anti-tumour agents, and for the identification of target antigens for cellular immunotherapy. This chapter will describe how immunotherapy may be directed to a more primitive side population of B-CLL cells.

In vivo fluorescent optical imaging of cytotoxic T lymphocyte migration using IRDye800CW near-infrared dye.

We describe a method to measure in vivo migration of human T cells by using the near-infrared (NIR) dye IRDye800CW. Labeling of Epstein-Barr virus-specific T cells with IRDye800CW did not affect viability, proliferation, or T cell function. Following tail vein injection into mice bearing subcutaneous tumors, the NIR signal could be measured in vivo at the tumor site. Analysis of tumors revealed T cell infiltration and an increased NIR signal, confirming T cell migration. To test specific migration with IRDye800CW, tumors were modified to express CCL5 to measure site-specific migration. The NIR signal was increased at CCL5-secreting tumors compared with control tumors. Together, these data suggest that IRDye800CW may be used to study the trafficking of T cells in a small animal model and may have potential as a short-term reporter molecule for human immunotherapy studies.

Anti-Tumor Effects after Adoptive Transfer of IL-12 Transposon-Modified Murine Splenocytes in the OT-I-Melanoma Mouse Model.

Adoptive transfer of gene modified T cells provides possible immunotherapy for patients with cancers refractory to other treatments. We have previously used the non-viral piggyBac transposon system to gene modify human T cells for potential immunotherapy. However, these previous studies utilized adoptive transfer of modified human T cells to target cancer xenografts in highly immunodeficient (NOD-SCID) mice that do not recapitulate an intact immune system. Currently, only viral vectors have shown efficacy in permanently gene-modifying mouse T cells for immunotherapy applications. Therefore, we sought to determine if piggyBac could effectively gene modify mouse T cells to target cancer cells in a mouse cancer model. We first demonstrated that we could gene modify cells to express murine interleukin-12 (p35/p40 mIL-12), a transgene with proven efficacy in melanoma immunotherapy. The OT-I melanoma mouse model provides a well-established T cell mediated immune response to ovalbumin (OVA) positive B16 melanoma cells. B16/OVA melanoma cells were implanted in wild type C57Bl6 mice. Mouse splenocytes were isolated from C57Bl6 OT-I mice and were gene modified using piggyBac to express luciferase. Adoptive transfer of luciferase-modified OT-I splenocytes demonstrated homing to B16/OVA melanoma tumors in vivo. We next gene-modified OT-I cells to express mIL-12. Adoptive transfer of mIL-12-modified mouse OT-I splenocytes delayed B16/OVA melanoma tumor growth in vivo compared to control OT-I splenocytes and improved mouse survival. Our results demonstrate that the piggyBac transposon system can be used to gene modify splenocytes and mouse T cells for evaluating adoptive immunotherapy strategies in immunocompetent mouse tumor models that may more directly mimic immunotherapy applications in humans.

Ex-vivo uses and applications of cytokines for adoptive immunotherapy in cancer.

Harnessing the power of the immune system to eliminate infection and cancer is a long-standing goal in clinical immunology. The development of a robust immune response to foreign antigen relies, in part, on communication between cellular components of the immune system. The proteins involved in governing the magnitude and longevity of an immune response are collectively called cytokines. Cytokines act directly on immune cells to induce proliferation, differentiation and tolerance, and signaling errors can lead to autoimmune disease or immune deficiency. Identification of the molecules involved in these signaling processes has allowed investigators to manipulate immune cells for therapeutic effect, both in vivo and ex vivo. While in vivo immune modulation using cytokines has produced some exciting results, the toxicity involved with systemic administration has limited their broad use in the clinic. Therefore, research has been focused on the ex vivo manipulation of immune cells for adoptive transfer to treat cancer and infection. This review will focus on the ex vivo manipulation of immune cells with particular emphasis on stimulating cytotoxic T cell responses. Adoptive transfer of ex vivo generated cell types may then be used to treat malignant and viral disease.