Tethered IL-15 augments antitumor activity and promotes a stem-cell memory subset in tumor-specific T cells.

Adoptive immunotherapy retargeting T cells to CD19 via a chimeric antigen receptor (CAR) is an investigational treatment capable of inducing complete tumor regression of B-cell malignancies when there is sustained survival of infused cells. T-memory stem cells (TSCM) retain superior potential for long-lived persistence, but challenges exist in manufacturing this T-cell subset because they are rare among circulating lymphocytes. We report a clinically relevant approach to generating CAR+ T cells with preserved TSCM potential using the Sleeping Beauty platform. Because IL-15 is fundamental to T-cell memory, we incorporated its costimulatory properties by coexpressing CAR with a membrane-bound chimeric IL-15 (mbIL15). The mbIL15-CAR T cells signaled through signal transducer and activator of transcription 5 to yield improved T-cell persistence independent of CAR signaling, without apparent autonomous growth or transformation, and achieved potent rejection of CD19+ leukemia. Long-lived T cells were CD45ROnegCCR7+CD95+, phenotypically most similar to TSCM, and possessed a memory-like transcriptional profile. Overall, these results demonstrate that CAR+ T cells can develop long-term persistence with a memory stem-cell phenotype sustained by signaling through mbIL15. This observation warrants evaluation in clinical trials.

Bioengineering T cells to target carbohydrate to treat opportunistic fungal infection.

Clinical-grade T cells are genetically modified ex vivo to express chimeric antigen receptors (CARs) to redirect their specificity to target tumor-associated antigens in vivo. We now have developed this molecular strategy to render cytotoxic T cells specific for fungi. We adapted the pattern-recognition receptor Dectin-1 to activate T cells via chimeric CD28 and CD3-ζ (designated "D-CAR") upon binding with carbohydrate in the cell wall of Aspergillus germlings. T cells genetically modified with the Sleeping Beauty system to express D-CAR stably were propagated selectively on artificial activating and propagating cells using an approach similar to that approved by the Food and Drug Administration for manufacturing CD19-specific CAR(+) T cells for clinical trials. The D-CAR(+) T cells exhibited specificity for ß-glucan which led to damage and inhibition of hyphal growth of Aspergillus in vitro and in vivo. Treatment of D-CAR(+) T cells with steroids did not compromise antifungal activity significantly. These data support the targeting of carbohydrate antigens by CAR(+) T cells and provide a clinically appealing strategy to enhance immunity for opportunistic fungal infections using T-cell gene therapy.

Bispecific T-cells expressing polyclonal repertoire of endogenous γδ T-cell receptors and introduced CD19-specific chimeric antigen receptor.

Even though other γδ T-cell subsets exhibit antitumor activity, adoptive transfer of γδ Tcells is currently limited to one subset (expressing Vγ9Vδ2 T-cell receptor (TCR)) due to dependence on aminobisphosphonates as the only clinically appealing reagent for propagating γδ T cells. Therefore, we developed an approach to propagate polyclonal γδ T cells and rendered them bispecific through expression of a CD19-specific chimeric antigen receptor (CAR). Peripheral blood mononuclear cells (PBMC) were electroporated with Sleeping Beauty (SB) transposon and transposase to enforce expression of CAR in multiple γδ T-cell subsets. CAR(+)γδ T cells were expanded on CD19(+) artificial antigen-presenting cells (aAPC), which resulted in >10(9) CAR(+)γδ T cells from <10(6) total cells. Digital multiplex assay detected TCR mRNA coding for Vδ1, Vδ2, and Vδ3 with Vγ2, Vγ7, Vγ8, Vγ9, and Vγ10 alleles. Polyclonal CAR(+)γδ T cells were functional when TCRγδ and CAR were stimulated and displayed enhanced killing of CD19(+) tumor cell lines compared with CAR(neg)γδ T cells. CD19(+) leukemia xenografts in mice were reduced with CAR(+)γδ T cells compared with control mice. Since CAR, SB, and aAPC have been adapted for human application, clinical trials can now focus on the therapeutic potential of polyclonal γδ T cells.

Sleeping beauty generated CD19 CAR T-Cell therapy for advanced B-Cell hematological malignancies.

Chimeric antigen receptor (CAR) T-cell therapy has emerged recently as a standard of care treatment for patients with relapsed or refractory acute lymphoblastic leukemia (ALL) and several subtypes of B-cell non-Hodgkin lymphoma (NHL). However, its use remains limited to highly specialized centers, given the complexity of its administration and its associated toxicities. We previously reported our experience in using a novel Sleeping Beauty (SB) CD19-specific CAR T-cell therapy in the peri-transplant setting, where it exhibited an excellent safety profile with encouraging survival outcomes. We have since modified the SB CD19 CAR construct to improve its efficacy and shorten its manufacturing time. We report here the phase 1 clinical trial safety results. Fourteen heavily treated patients with relapsed/refractory ALL and NHL were infused. Overall, no serious adverse events were directly attributed to the study treatment. Three patients developed grades 1-2 cytokine release syndrome and none of the study patients experienced neurotoxicity. All dose levels were well tolerated and no dose-limiting toxicities were reported. For efficacy, 3 of 8 (38%) patients with ALL achieved CR/CRi (complete remission with incomplete count recovery) and 1 (13%) patient had sustained molecular disease positivity. Of the 4 patients with DLBCL, 2 (50%) achieved CR. The SB-based CAR constructs allow manufacturing of targeted CAR T-cell therapies that are safe, cost-effective and with encouraging antitumor activity.

A high throughput microelectroporation device to introduce a chimeric antigen receptor to redirect the specificity of human T cells.

It has been demonstrated that a chimeric antigen receptor (CAR) can directly recognize the CD19 molecule expressed on the cell surface of B-cell malignancies independent of major histocompatibility complex (MHC). Although T-cell therapy of tumors using CD19-specific CAR is promising, this approach relies on using expression vectors that stably integrate the CAR into T-cell chromosomes. To circumvent the potential genotoxicity that may occur from expressing integrating transgenes, we have expressed the CD19-specific CAR transgene from mRNA using a high throughput microelectroporation device. This research was accomplished using a microelectroporator to achieve efficient and high throughput non-viral gene transfer of in vitro transcribed CAR mRNA into human T cells that had been numerically expanded ex vivo. Electro-transfer of mRNA avoids the potential genotoxicity associated with vector and transgene integration and the high throughput capacity overcomes the expected transient CAR expression, as repeated rounds of electroporation can replace T cells that have lost transgene expression. We fabricated and tested a high throughput microelectroporator that can electroporate a stream of 2 x 10(8) primary T cells within 10 min. After electroporation, up to 80% of the passaged T cells expressed the CD19-specific CAR. Video time-lapse microscopy (VTLM) demonstrated the redirected effector function of the genetically manipulated T cells to specifically lyse CD19+ tumor cells. Our biomedical microdevice, in which T cells are transiently and safely modified to be tumor-specific and then can be re-infused, offers a method for redirecting T-cell specificity, that has implications for the development of adoptive immunotherapy.

Very rapid cloning, expression and identifying specificity of T-cell receptors for T-cell engineering.

Neoantigens can be predicted and in some cases identified using the data obtained from the whole exome sequencing and transcriptome sequencing of tumor cells. These sequencing data can be coupled with single-cell RNA sequencing for the direct interrogation of the transcriptome, surfaceome, and pairing of αß T-cell receptors (TCRαß) from hundreds of single T cells. Using these 2 large datasets, we established a platform for identifying antigens recognized by TCRαßs obtained from single T cells. Our approach is based on the rapid expression of cloned TCRαß genes as Sleeping Beauty transposons and the determination of the introduced TCRαßs' antigen specificity and avidity using a reporter cell line. The platform enables the very rapid identification of tumor-reactive TCRs for the bioengineering of T cells with redirected specificity.

Combining adoptive cellular and immunocytokine therapies to improve treatment of B-lineage malignancy.

Currently, the lineage-specific cell-surface molecules CD19 and CD20 present on many B-cell malignancies are targets for both antibody- and cell-based therapies. Coupling these two treatment modalities is predicted to improve the antitumor effect, particularly for tumors resistant to single-agent biotherapies. This can be shown using an immunocytokine, composed of a CD20-specific monoclonal antibody fused to biologically active interleukin 2 (IL-2), combined with ex vivo expanded human umbilical cord blood-derived CD8(+) T cells, that have been genetically modified to be CD19 specific, for adoptive transfer after allogeneic hematopoietic stem-cell transplantation. We show that a benefit of targeted delivery of recombinant IL-2 by the immunocytokine to the CD19(+)CD20(+) tumor microenvironment is improved in vivo persistence of the CD19-specific T cells, and this results in an augmented cell-mediated antitumor effect. Phase I trials are under way using anti-CD20-IL-2 immunocytokine and CD19-specific T cells as monotherapies, and our results warrant clinical trials using combination of these two immunotherapies.

CD28 costimulation provided through a CD19-specific chimeric antigen receptor enhances in vivo persistence and antitumor efficacy of adoptively transferred T cells.

Chimeric antigen receptors (CAR) combine an antigen-binding domain with a CD3-zeta signaling motif to redirect T-cell specificity to clinically important targets. First-generation CAR, such as the CD19-specific CAR (designated CD19R), may fail to fully engage genetically modified T cells because activation is initiated by antigen-dependent signaling through chimeric CD3-zeta, independent of costimulation through accessory molecules. We show that enforced expression of the full-length costimulatory molecule CD28 in CD8(+)CD19R(+)CD28(-) T cells can restore fully competent antigen-dependent T-cell activation upon binding CD19(+) targets expressing CD80/CD86. Thus, to provide costimulation to T cells through a CD19-specific CAR, independent of binding to CD80/CD86, we developed a second-generation CAR (designated CD19RCD28), which includes a modified chimeric CD28 signaling domain fused to chimeric CD3-zeta. CD19R(+) and CD19RCD28(+) CD8(+) T cells specifically lyse CD19(+) tumor cells. However, the CD19RCD28(+) CD8(+) T cells proliferate in absence of exogenous recombinant human interleukin-2, produce interleukin-2, propagate, and up-regulate antiapoptotic Bcl-X(L) after stimulation by CD19(+) tumor cells. For the first time, we show in vivo that adoptively transferred CD19RCD28(+) T cells show an improved persistence and antitumor effect compared with CD19R(+) T cells. These data imply that modifications to the CAR can result in improved therapeutic potential of CD19-specific T cells expressing this second-generation CAR.

Antitumor activity of CD56-chimeric antigen receptor T cells in neuroblastoma and SCLC models.

The CD56 antigen (NCAM-1) is highly expressed on several malignancies with neuronal or neuroendocrine differentiation, including small-cell lung cancer and neuroblastoma, tumor types for which new therapeutic options are needed. We hypothesized that CD56-specific chimeric antigen receptor (CAR) T cells could target and eliminate CD56-positive malignancies. Sleeping Beauty transposon-generated CD56R-CAR T cells exhibited αßT-cell receptors, released antitumor cytokines upon co-culture with CD56+ tumor targets, demonstrated a lack of fratricide, and expression of cytolytic function in the presence of CD56+ stimulation. The CD56R-CAR+ T cells are capable of killing CD56+ neuroblastoma, glioma, and SCLC tumor cells in in vitro co-cultures and when tested against CD56+ human xenograft neuroblastoma models and SCLC models, CD56R-CAR+ T cells were able to inhibit tumor growth in vivo. These results indicate that CD56-CARs merit further investigation as a potential treatment for CD56+ malignancies.

Redirecting Specificity of T cells Using the Sleeping Beauty System to Express Chimeric Antigen Receptors by Mix-and-Matching of VL and VH Domains Targeting CD123+ Tumors.

Adoptive immunotherapy infusing T cells with engineered specificity for CD19 expressed on B- cell malignancies is generating enthusiasm to extend this approach to other hematological malignancies, such as acute myelogenous leukemia (AML). CD123, or interleukin 3 receptor alpha, is overexpressed on most AML and some lymphoid malignancies, such as acute lymphocytic leukemia (ALL), and has been an effective target for T cells expressing chimeric antigen receptors (CARs). The prototypical CAR encodes a VH and VL from one monoclonal antibody (mAb), coupled to a transmembrane domain and one or more cytoplasmic signaling domains. Previous studies showed that treatment of an experimental AML model with CD123-specific CAR T cells was therapeutic, but at the cost of impaired myelopoiesis, highlighting the need for systems to define the antigen threshold for CAR recognition. Here, we show that CARs can be engineered using VH and VL chains derived from different CD123-specific mAbs to generate a panel of CAR+ T cells. While all CARs exhibited specificity to CD123, one VH and VL combination had reduced lysis of normal hematopoietic stem cells. This CAR's in vivo anti-tumor activity was similar whether signaling occurred via chimeric CD28 or CD137, prolonging survival in both AML and ALL models. Co-expression of inducible caspase 9 eliminated CAR+ T cells. These data help support the use of CD123-specific CARs for treatment of CD123+ hematologic malignancies.

Imaging of Sleeping Beauty-Modified CD19-Specific T Cells Expressing HSV1-Thymidine Kinase by Positron Emission Tomography.

PURPOSE: We have incorporated a positron emission tomography (PET) functionality in T cells expressing a CD19-specific chimeric antigen receptor (CAR) to non-invasively monitor the adoptively transferred cells. PROCEDURES: We engineered T cells to express CD19-specific CAR, firefly luciferase (ffLuc), and herpes simplex virus type-1 thymidine kinase (TK) using the non-viral-based Sleeping Beauty (SB) transposon/transposase system adapted for human application. Electroporated primary T cells were propagated on CD19+ artificial antigen-presenting cells. RESULTS: After 4 weeks, 90 % of cultured cells exhibited specific killing of CD19+ targets in vitro, could be ablated by ganciclovir, and were detected in vivo by bioluminescent imaging and PET following injection of 2'-deoxy-2'-[18F]fluoro-5-ethyl-1-ß-D-arabinofuranosyl-uracil ([18F]FEAU). CONCLUSION: This is the first report demonstrating the use of SB transposition to generate T cells which may be detected using PET laying the foundation for imaging the distribution and trafficking of T cells in patients treated for B cell malignancies.

Phase I trials using Sleeping Beauty to generate CD19-specific CAR T cells.

BACKGROUND: T cells expressing antigen-specific chimeric antigen receptors (CARs) improve outcomes for CD19-expressing B cell malignancies. We evaluated a human application of T cells that were genetically modified using the Sleeping Beauty (SB) transposon/transposase system to express a CD19-specific CAR. METHODS: T cells were genetically modified using DNA plasmids from the SB platform to stably express a second-generation CD19-specific CAR and selectively propagated ex vivo with activating and propagating cells (AaPCs) and cytokines. Twenty-six patients with advanced non-Hodgkin lymphoma and acute lymphoblastic leukemia safely underwent hematopoietic stem cell transplantation (HSCT) and infusion of CAR T cells as adjuvant therapy in the autologous (n = 7) or allogeneic settings (n = 19). RESULTS: SB-mediated genetic transposition and stimulation resulted in 2,200- to 2,500-fold ex vivo expansion of genetically modified T cells, with 84% CAR expression, and without integration hotspots. Following autologous HSCT, the 30-month progression-free and overall survivals were 83% and 100%, respectively. After allogeneic HSCT, the respective 12-month rates were 53% and 63%. No acute or late toxicities and no exacerbation of graft-versus-host disease were observed. Despite a low antigen burden and unsupportive recipient cytokine environment, CAR T cells persisted for an average of 201 days for autologous recipients and 51 days for allogeneic recipients. CONCLUSIONS: CD19-specific CAR T cells generated with SB and AaPC platforms were safe, and may provide additional cancer control as planned infusions after HSCT. These results support further clinical development of this nonviral gene therapy approach. TRIAL REGISTRATION: Autologous, NCT00968760; allogeneic, NCT01497184; long-term follow-up, NCT01492036. FUNDING: National Cancer Institute, private foundations, and institutional funds. Please see Acknowledgments for details.

A quantitative high-throughput chemotaxis assay using bioluminescent reporter cells.

Here we report on a novel biophotonic assay system for the detection and quantitation of chemotaxis, the directed movement of cells in response to chemokine concentration gradients. Our assay employs a firefly luciferase (ffLuc)-generated biophotonic signal to quantify cellular migration in 96-well microplate chemotaxis instruments. When compared to direct cell enumeration, the biophotonic reporter method is superior in accuracy, reproducibility, and sensitivity. As a proof-of-concept, we demonstrate the utility of this assay for quantifying the chemotactic response of ex vivo expanded ffLuc(+) primary human T-cells to recombinant human chemokines MCP-1, RANTES, and IP-10. The 96-well microplate format and in situ biophotonic detection of cells are amenable to high-throughput screening of peptides and small molecule libraries to identify agonists and antagonists of cellular chemotaxis, to analyze biological fluids for chemotactic activity, and to study chemotaxis in a variety of cell types.

Biophotonic cytotoxicity assay for high-throughput screening of cytolytic killing.

We have developed a highly sensitive biophotonic luciferase assay as an alternative to (51)Cr-release for assessment of cell-mediated cytotoxicity. The luciferin/ATP-dependent luminescent signal of target cells stably or transiently transfected with a firefly luciferase reporter gene (fLuc:Zeo) linearly correlates with viable target cell number. Upon incubation of fLuc:Zeo(+) target cells with CD8(+) CTLs, a rapid decrease in bioluminescence was detected that correlated with antigen-specific target cell lysis. The levels of specific lysis measured by (51)Cr-release assays correlated with the attenuation in biophotonic target cell signal, thus validating this approach as a sensitive and accurate method for the measurement of cytolysis. We show that this luminescent-based cytolytic assay (LCA) is amenable for high-throughput screening of effector cell cytolytic activity, allows for the rate of cytolysis to be measured in a single micro-plate, and permits the multiplexing of cytolytic killing with other lymphocyte functional assays such as cytokine release. Importantly, this method accurately measures the cytolytic killing of target cells that are either stably or transiently transfected with a fLuc reporter gene, and thus is ideal for monitoring cytolysis of both primary autologous and immortalized target cell lines. The versatility of the non-radioactive, high-throughput, biophotonic cytolytic assay should make this method an attractive alternative to chromium-release for quantifying effector cell cytolytic activity.

Genetic Engineering of T Cells to Target HERV-K, an Ancient Retrovirus on Melanoma.

PURPOSE: The human endogenous retrovirus (HERV-K) envelope (env) protein is a tumor-associated antigen (TAA) expressed on melanoma but not normal cells. This study was designed to engineer a chimeric antigen receptor (CAR) on T-cell surface, such that they target tumors in advanced stages of melanoma. EXPERIMENTAL DESIGN: Expression of HERV-K protein was analyzed in 220 melanoma samples (with various stages of disease) and 139 normal organ donor tissues using immunohistochemical (IHC) analysis. HERV-K env-specific CAR derived from mouse monoclonal antibody was introduced into T cells using the transposon-based Sleeping Beauty (SB) system. HERV-K env-specific CAR(+) T cells were expanded ex vivo on activating and propagating cells (AaPC) and characterized for CAR expression and specificity. This includes evaluating the HERV-K-specific CAR(+) T cells for their ability to kill A375-SM metastasized tumors in a mouse xenograft model. RESULTS: We detected HERV-K env protein on melanoma but not in normal tissues. After electroporation of T cells and selection on HERV-K(+) AaPC, more than 95% of genetically modified T cells expressed the CAR with an effector memory phenotype and lysed HERV-K env(+) tumor targets in an antigen-specific manner. Even though there is apparent shedding of this TAA from tumor cells that can be recognized by HERV-K env-specific CAR(+) T cells, we observed a significant antitumor effect. CONCLUSIONS: Adoptive cellular immunotherapy with HERV-K env-specific CAR(+) T cells represents a clinically appealing treatment strategy for advanced-stage melanoma and provides an approach for targeting this TAA on other solid tumors.

Sleeping Beauty Transposition of Chimeric Antigen Receptors Targeting Receptor Tyrosine Kinase-Like Orphan Receptor-1 (ROR1) into Diverse Memory T-Cell Populations.

T cells modified with chimeric antigen receptors (CARs) targeting CD19 demonstrated clinical activity against some B-cell malignancies. However, this is often accompanied by a loss of normal CD19+ B cells and humoral immunity. Receptor tyrosine kinase-like orphan receptor-1 (ROR1) is expressed on sub-populations of B-cell malignancies and solid tumors, but not by healthy B cells or normal post-partum tissues. Thus, adoptive transfer of T cells specific for ROR1 has potential to eliminate tumor cells and spare healthy tissues. To test this hypothesis, we developed CARs targeting ROR1 in order to generate T cells specific for malignant cells. Two Sleeping Beauty transposons were constructed with 2nd generation ROR1-specific CARs signaling through CD3ζ and either CD28 (designated ROR1RCD28) or CD137 (designated ROR1RCD137) and were introduced into T cells. We selected for T cells expressing CAR through co-culture with γ-irradiated activating and propagating cells (AaPC), which co-expressed ROR1 and co-stimulatory molecules. Numeric expansion over one month of co-culture on AaPC in presence of soluble interleukin (IL)-2 and IL-21 occurred and resulted in a diverse memory phenotype of CAR+ T cells as measured by non-enzymatic digital array (NanoString) and multi-panel flow cytometry. Such T cells produced interferon-γ and had specific cytotoxic activity against ROR1+ tumors. Moreover, such cells could eliminate ROR1+ tumor xenografts, especially T cells expressing ROR1RCD137. Clinical trials will investigate the ability of ROR1-specific CAR+ T cells to specifically eliminate tumor cells while maintaining normal B-cell repertoire.

Tuning Sensitivity of CAR to EGFR Density Limits Recognition of Normal Tissue While Maintaining Potent Antitumor Activity.

Many tumors overexpress tumor-associated antigens relative to normal tissue, such as EGFR. This limits targeting by human T cells modified to express chimeric antigen receptors (CAR) due to potential for deleterious recognition of normal cells. We sought to generate CAR(+) T cells capable of distinguishing malignant from normal cells based on the disparate density of EGFR expression by generating two CARs from monoclonal antibodies that differ in affinity. T cells with low-affinity nimotuzumab-CAR selectively targeted cells overexpressing EGFR, but exhibited diminished effector function as the density of EGFR decreased. In contrast, the activation of T cells bearing high-affinity cetuximab-CAR was not affected by the density of EGFR. In summary, we describe the generation of CARs able to tune T-cell activity to the level of EGFR expression in which a CAR with reduced affinity enabled T cells to distinguish malignant from nonmalignant cells.

Universal artificial antigen presenting cells to selectively propagate T cells expressing chimeric antigen receptor independent of specificity.

T cells genetically modified to stably express immunoreceptors are being assessed for therapeutic potential in clinical trials. T cells expressing a chimeric antigen receptor (CAR) are endowed with a new specificity to target tumor-associated antigen (TAA) independent of major histocompatibility complex. Our approach to nonviral gene transfer in T cells uses ex vivo numeric expansion of CAR T cells on irradiated artificial antigen presenting cells (aAPC) bearing the targeted TAA. The requirement for aAPC to express a desired TAA limits the human application of CARs with multiple specificities when selective expansion through coculture with feeder cells is sought. As an alternative to expressing individual TAAs on aAPC, we expressed 1 ligand that could activate CAR T cells for sustained proliferation independent of specificity. We expressed a CAR ligand (designated CARL) that binds the conserved IgG4 extracellular domain of CAR and demonstrated that CARL aAPC propagate CAR T cells of multiple specificities. CARL avoids technical issues and costs associated with deploying clinical-grade aAPC for each TAA targeted by a given CAR. Using CARL enables 1 aAPC to numerically expand all CAR T cells containing the IgG4 domain, and simplifies expansion, testing, and clinical translation of CAR T cells of any specificity.

Activating and propagating polyclonal gamma delta T cells with broad specificity for malignancies.

PURPOSE: To activate and propagate populations of γδ T cells expressing polyclonal repertoire of γ and δ T-cell receptor (TCR) chains for adoptive immunotherapy of cancer, which has yet to be achieved. EXPERIMENTAL DESIGN: Clinical-grade artificial antigen-presenting cells (aAPC) derived from K562 tumor cells were used as irradiated feeders to activate and expand human γδ T cells to clinical scale. These cells were tested for proliferation, TCR expression, memory phenotype, cytokine secretion, and tumor killing. RESULTS: γδ T-cell proliferation was dependent upon CD137L expression on aAPC and addition of exogenous IL2 and IL21. Propagated γδ T cells were polyclonal as they expressed TRDV1, TRDV2-2, TRDV3, TRDV5, TRDV7, and TRDV8 with TRGV2, TRGV3F, TRGV7, TRGV8, TRGV9*A1, TRGV10*A1, and TRGV11 TCR chains. IFNγ production by Vδ1, Vδ2, and Vδ1(neg)Vδ2(neg) subsets was inhibited by pan-TCRγδ antibody when added to cocultures of polyclonal γδ T cells and tumor cell lines. Polyclonal γδ T cells killed acute and chronic leukemia, colon, pancreatic, and ovarian cancer cell lines, but not healthy autologous or allogeneic normal B cells. Blocking antibodies demonstrated that polyclonal γδ T cells mediated tumor cell lysis through combination of DNAM1, NKG2D, and TCRγδ. The adoptive transfer of activated and propagated γδ T cells expressing polyclonal versus defined Vδ TCR chains imparted a hierarchy (polyclonal>Vδ1>Vδ1(neg)Vδ2(neg)>Vδ2) of survival of mice with ovarian cancer xenografts. CONCLUSIONS: Polyclonal γδ T cells can be activated and propagated with clinical-grade aAPCs and demonstrate broad antitumor activities, which will facilitate the implementation of γδ T-cell cancer immunotherapies in humans.

Clinical application of Sleeping Beauty and artificial antigen presenting cells to genetically modify T cells from peripheral and umbilical cord blood.

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer a biologic product with the potential for superior (i) recognition of tumor-associated antigens (TAAs), (ii) persistence after infusion, (iii) potential for migration to tumor sites, and (iv) ability to recycle effector functions within the tumor microenvironment. Most approaches to genetic manipulation of T cells engineered for human application have used retrovirus and lentivirus for the stable expression of CAR(1-3). This approach, although compliant with current good manufacturing practice (GMP), can be expensive as it relies on the manufacture and release of clinical-grade recombinant virus from a limited number of production facilities. The electro-transfer of nonviral plasmids is an appealing alternative to transduction since DNA species can be produced to clinical grade at approximately 1/10(th) the cost of recombinant GMP-grade virus. To improve the efficiency of integration we adapted Sleeping Beauty (SB) transposon and transposase for human application(4-8). Our SB system uses two DNA plasmids that consist of a transposon coding for a gene of interest (e.g. 2(nd) generation CD19-specific CAR transgene, designated CD19RCD28) and a transposase (e.g. SB11) which inserts the transgene into TA dinucleotide repeats(9-11). To generate clinically-sufficient numbers of genetically modified T cells we use K562-derived artificial antigen presenting cells (aAPC) (clone #4) modified to express a TAA (e.g. CD19) as well as the T cell costimulatory molecules CD86, CD137L, a membrane-bound version of interleukin (IL)-15 (peptide fused to modified IgG4 Fc region) and CD64 (Fc-γ receptor 1) for the loading of monoclonal antibodies (mAb)(12). In this report, we demonstrate the procedures that can be undertaken in compliance with cGMP to generate CD19-specific CAR(+) T cells suitable for human application. This was achieved by the synchronous electro-transfer of two DNA plasmids, a SB transposon (CD19RCD28) and a SB transposase (SB11) followed by retrieval of stable integrants by the every-7-day additions (stimulation cycle) of γ-irradiated aAPC (clone #4) in the presence of soluble recombinant human IL-2 and IL-21(13). Typically 4 cycles (28 days of continuous culture) are undertaken to generate clinically-appealing numbers of T cells that stably express the CAR. This methodology to manufacturing clinical-grade CD19-specific T cells can be applied to T cells derived from peripheral blood (PB) or umbilical cord blood (UCB). Furthermore, this approach can be harnessed to generate T cells to diverse tumor types by pairing the specificity of the introduced CAR with expression of the TAA, recognized by the CAR, on the aAPC.