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Table ST1: Glossary of terms used in the paper Figure S1. Genetic modification and expansion of CAR+ T cells. Figure S2. In vitro expansion of CAR+ T cells. Figure S3. High-throughput cytotoxicity assay for monitoring T-cell target cell interactions in nanowell grids. Figure S4. CD19 expression on NALM-6 tumor cells or CD19+EL4 target cells as determined by immunofluoresecent staining. Figure S5. Composite micrographs illustrating representative examples of the interactions between single CAR+ T cells (E) and one or more NALM-6 tumor (T) cells. Figure S6. Increased probability of killing by individual CAR+ T cells at higher target cell densities. Figure S7. Donut plots summarizing the outcomes of the interaction between individual CAR8 cells and 1-3 CD19+-NALM-6 tumor cells. Figure S8. Timelapse Imaging Microscopy In Nanowell Grids (TIMING). Figure S9. At an E:T ratio of 1:1, identification of subgroups of killer CAR8 cells based on their motility and contact behavior with tumor cells. Figure S10. At an E:T of 1:1, the total duration of conjugation prior to NALM-6 tumor cell killing is no different for the CAR8 cells in the different subgroups. Figure S11. At an E:T ratio of 1:1, CAR8 cells in the S1 subgroup, demonstrate: (A) drop in motility, and (B) increased circularization upon conjugation to NALM-6 tumor cell. Figure S12. At an E:T ratio of 1:1, CAR8 cells in the different subgroups demonstrate different frequencies and kinetics of AICD subsequent to the interactions with NALM-6 cells. Figure S13. At an E:T ratio of 1:2-5, multi-killer CAR8 cells demonstrate no significant differences in their duration of conjugation prior to killing multiple NALM-6 tumor cells. Figure S14. Despite the increased crowding because of higher tumor cell density, multi-killer CAR8 cells displayed greater motility when conjugated to tumor cell in comparison to single-killer CAR8 cells that encountered only a single tumor cell. Figure S15. At an E:T ratio of 1:1, identification of subgroups of killer CAR4 cells based on their motility and contact behavior with tumor cells. Figure S16. At an E:T of 1:1, the total duration of conjugation prior to NALM-6 tumor cell killing is significantly longer for CAR4 cells in S2 subgroup in comparison to subgroups S1 and S3. Figure S17. At an E:T of 1:1, CAR4 cells in S2 subgroup induce apoptosis in tumor cells with delayed kinetics in comparison to CAR8 cells in the S2 subgroup. Figure S18. At an E:T ratio of 1:2-5, multi-killer CAR4 cells demonstrate increased circularization upon contact with one or more NALM-6 tumor cells. Figure S19. The ability of individual CAR4 cells to simultaneously conjugate to multiple NALM-6 tumor cells increases as the number of tumor cells within the nanowell increases. Figure S20. Comparison of the killing efficiency of individual single killer CAR+ T cells (E:T 1:1) with multi-killer CAR+ T cells (E:T 1:2-5) that killed multiple NALM-6 tumor cells.

T cells genetically modified to express a CD19-specific chimeric antigen receptor (CAR) for the investigational treatment of B-cell malignancies comprise a heterogeneous population, and their ability to persist and participate in serial killing of tumor cells is a predictor of therapeutic success. We implemented Timelapse Imaging Microscopy in Nanowell Grids (TIMING) to provide direct evidence that CD4+CAR+ T cells (CAR4 cells) can engage in multikilling via simultaneous conjugation to multiple tumor cells. Comparisons of the CAR4 cells and CD8+CAR+ T cells (CAR8 cells) demonstrate that, although CAR4 cells can participate in killing and multikilling, they do so at slower rates, likely due to the lower granzyme B content. Significantly, in both sets of T cells, a minor subpopulation of individual T cells identified by their high motility demonstrated efficient killing of single tumor cells. A comparison of the multikiller and single-killer CAR+ T cells revealed that the propensity and kinetics of T-cell apoptosis were modulated by the number of functional conjugations. T cells underwent rapid apoptosis, and at higher frequencies, when conjugated to single tumor cells in isolation, and this effect was more pronounced on CAR8 cells. Our results suggest that the ability of CAR+ T cells to participate in multikilling should be evaluated in the context of their ability to resist activation-induced cell death. We anticipate that TIMING may be used to rapidly determine the potency of T-cell populations and may facilitate the design and manufacture of next-generation CAR+ T cells with improved efficacy. Cancer Immunol Res; 3(5); 473–82. ©2015 AACR.See related commentary by June, p. 470

Movie M4. Representative example of a CAR8 cell that kills multiple tumor cells.

Movie M1. Representative example of a CAR8 cell from the S1 subgroup.

Movie M2. Representative example of a CAR8 cell from the S2 subgroup.

Movie M3. Representative example of a CAR8 cell from the S3 subgroup.

Movie M5. Representative example of a CAR4 cell that kills multiple tumor cells.

PDF file, 111K, Supplementary Table S1. Samples from 14 patients that received autologous TILs were studied. Supplementary Table S2.TCR Vα primers (the first 50 nt) and probes (the last 50 nt)used for DTEA analysis. Supplementary Table S3. TCR Vβ primers (the first 50 nt) and probes (the last 50 nt)used for DTEA analysis. Supplementary Table S4. House keeping genes used in DTEA analysis. Supplementary Table S5. DTEA can detect the low-frequency TCRVβ genes that cannot be readily identified by classical cloning.

Purpose: T-cell receptor (TCR) variable Vα and Vβ gene diversity is a surrogate biomarker for the therapeutic potential of adoptive immunotherapy and cellular immunity. Therefore, creating a straightforward, rapid, sensitive, and reliable method to view the global changes of both TCRVα and Vβ transcripts in heterogeneous populations of T cells is appealing.Experimental Design: We designed a “direct TCR expression assay” (DTEA) using a panel of customized bar-coded probes that simultaneously detects and quantifies 45 Vα and 46 Vβ transcripts in a nonenzymatic digital multiplexed assay from a small number of cells (104 cells) or as little as 100 ng of total RNA.Results: We evaluated DTEA on total RNA samples of tumor-infiltrating lymphocytes and peripheral blood obtained from patients with melanoma after adoptive T-cell therapy. DTEA detected a similar spectrum of the dominant patterns of TCRVβ gene usage as sequencing cloned TCRVβ CDR3 regions. However, DTEA was rapid, achieved a level of sensitivity to identify rare T-cell populations, and simultaneously tracked the full array of Vα and Vβ transcripts.Conclusions: DTEA can rapidly and sensitively track changes in TCRVα and Vβ gene usages in T-cell pools following immune interventions, such as adoptive T-cell transfer, and may also be used to assess impact of vaccination or reconstitution of T-cell compartment after hematopoietic stem cell transplantation. Clin Cancer Res; 18(17); 4733–42. ©2012 AACR.

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 Vδ1negVδ2neg 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>Vδ1negVδ2neg>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. Clin Cancer Res; 20(22); 5708–19. ©2014 AACR.

PDF file - 1405KB, Supplementary Materials and Methods. Supplementary Figures - (1) Tumor cell line culture conditions and media formulations (2) Co-culture conditions for gamma/delta T cells and designer aAPC (3) Intracellular cytokine production, Luminex, and neutralizing antibody cytolysis assays (4) Lentivirus packaging and transduction of CAOV3 cells Contents of the Supplemental Data include figures: (1) An example of the gating strategy for gamma/delta T-cell analyses (2) Schematic of DNA plasmid pLVU3G-effLuc-T2A-mKateS158A used to co-express enhanced firefly luciferase (effLuc) and mKate (3) Expression of activation markers CD38 and CD95 on propagated gamma/delta T cells (4) aAPC developed for co-culture with gamma/delta T cells to determine the impact of introduced co-stimulatory molecules (5) Expansion of UCB-derived gamma/delta T cells on aAPC with IL-2 and IL-21 (6) Surface expression of TCRdelta1 and TCRdelta2 chains on gamma/delta T cells derived from PBMC prior to propagation (7) Abundance of Vdelta and Vgamma mRNA species in gamma/delta T cells prior to ex vivo numeric expansion (8) Surface expression of TCRdelta1 and TCRdelta2 chains on gamma/delta T cells derived from UCB and propagated on aAPC with IL-2 and IL-21 (9) mRNA expression of shared Valpha/Vdelta alleles in gamma/delta T cells separated and then propagated on aAPC, IL-2, and IL-21 (10) Abundance of mRNA species coding for Vgamma chains in gamma/delta T-cell subsets (11) In vitro lysis of tumor cell line panel by polyclonal gamma/delta T cells (12) Specific lysis of hematological and solid tumor cells by Vdelta T-cell subsets (13) Immunophenotypes associated with PBMC-derived Vdelta T-cell subsets expanded on aAPC/IL-2/IL-21. Supplemental Table 1 - details the antibodies used in this study, where they were purchased, and their hybridoma clone.

PDF file, 264K, Supplementary Fig. S1. TCR Va and Vb gene expression in total RNA from TILs patient 303 and patient 232. Supplementary Fig S2. Global measurement of TCR Va (A-C) and TCR Vb (D-F) gene expression in patient 231 including in vitro rapid expanded TIL (A and D) and PBMC (B-C and E-F) collected after TIL infusion. Supplementary Fig S3. Global measurement of TCR Va (A-D) and TCR Vb (E-H) gene expression in patient 228 including in vitro rapid expanded TIL (A and E) and PBMC (B-D) and (F-H) collected after TIL infusion. Supplementary Fig S4. Global measurement of TCR Va (A-D) and TCR Vb (E-H) gene expression in patient 106 including in vitro rapid expanded TIL (A and E) and PBMC (B-D) and (F-H) collected after TIL infusion. Supplementary Fig S5. Global measurement of TCR Va (A-D) and TCR Vb (E-H) gene expression in patient 172 including in vitro rapid expanded TIL (A and E) and PBMC (B-D) and (F-H) collected after TIL infusion. Supplementary Fig S6. Global measurement of TCR Va (A-D) and TCR Vb (E-H) gene expression in patient 152 including in vitro rapid expanded TIL (A and E) and PBMC (B-D) and (F-H) collected after TIL infusion. Supplementary Fig S7. Global measurement of TCR Va (A-D) and TCR Vb (E-H) gene expression in patient 188 including in vitro rapid expanded TIL (A and E) and PBMC (B-D) and (F-H) collected after TIL infusion.

Supplementary Figure 1 from Reprogramming CD19-Specific T Cells with IL-21 Signaling Can Improve Adoptive Immunotherapy of B-Lineage Malignancies

Supplementary Materials and Methods

Activation and functional response of Cetux-CAR+ and Nimo-CAR+ T cells in response to cell lines with varying endogenous EGFR expression

Antibodies used in study

Engraftment of intracranial tumor and CAR+ T-cell phenotype prior to administration of T cells

Production of TNF-a by Cetux-CAR and Nimo-CAR+ T cells in response to different densities of EGFR

Ratio of CD4 and CD8 on Cetux-CAR+ and Nimo-CAR+ T cells

Surface expression of CAR during co-culture with tEGFR+ EL4 or CAR-L+ EL4

Schematic of plasmids and AaPC

Patients with acute myeloid leukaemia (AML) have a five-year survival rate of 28·7%. Natural killer (NK)-cell have anti-leukaemic activity. Here, we report on a series of 13 patients with high-risk R/R AML, treated with repeated infusions of double-bright (CD56bright /CD16bright ) expanded NK cells at an academic centre in Brazil. NK cells from HLA-haploidentical donors were expanded using K562 feeder cells, modified to express membrane-bound interleukin-21. Patients received FLAG, after which cryopreserved NK cells were thawed and infused thrice weekly for six infusions in three dose cohorts (106 -107 cells/kg/infusion). Primary objectives were safety and feasibility. Secondary endpoints included overall response (OR) and complete response (CR) rates at 28-30 days after the first infusion. Patients received a median of five prior lines of therapy, seven with intermediate or adverse cytogenetics, three with concurrent central nervous system (CNS) leukaemia, and one with concurrent CNS mycetoma. No dose-limiting toxicities, infusion-related fever, or cytokine release syndrome were observed. An OR of 78·6% and CR of 50·0% were observed, including responses in three patients with CNS disease and clearance of a CNS mycetoma. Multiple infusions of expanded, cryopreserved NK cells were safely administered after intensive chemotherapy in high-risk patients with R/R AML and demonstrated encouraging outcomes.

Patients with acute myeloid leukaemia (AML) have a five-year survival rate of 28·7%. Natural killer (NK)-cell have anti-leukaemic activity. Here, we report on a series of 13 patients with high-risk R/R AML, treated with repeated infusions of double-bright (CD56

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. Clin Cancer Res; 21(14); 3241–51. ©2015 AACR.

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 αβT-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.

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 Vδ1negVδ2neg 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>Vδ1negVδ2neg>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. Clin Cancer Res; 20(22); 5708–19. ©2014 AACR.

Purpose: Adoptive cell therapy with autologous tumor-infiltrating lymphocytes (TIL) is a therapy for metastatic melanoma with response rates of up to 50%. However, the generation of the TIL transfer product is challenging, requiring pooled allogeneic normal donor peripheral blood mononuclear cells (PBMC) used in vitro as “feeders” to support a rapid-expansion protocol. Here, we optimized a platform to propagate TIL to a clinical scale using K562 cells genetically modified to express costimulatory molecules such as CD86, CD137-ligand, and membrane-bound IL-15 to function as artificial antigen-presenting cells (aAPC) as an alternative to using PBMC feeders. Experimental Design: We used aAPC or g-irradiated PBMC feeders to propagate TIL and measured rates of expansion. The activation and differentiation state was evaluated by flow cytometry and differential gene expression analyses. Clonal diversity was assessed on the basis of the pattern of T-cell receptor usage. T-cell effector function was measured by evaluation of cytotoxic granule content and killing of target cells. Results: The aAPC propagated TIL at numbers equivalent to that found with PBMC feeders, whereas increasing the frequency of CD8 + T-cell expansion with a comparable effector-memory phenotype. mRNA profiling revealed an upregulation of genes in the Wnt and stem-cell pathways with the aAPC. The aAPC platform did not skew clonal diversity, and CD8 + T cells showed comparable antitumor function as those expanded with PBMC feeders. Conclusions: TIL can be rapidly expanded with aAPC to clinical scale generating T cells with similar phenotypic and effector profiles as with PBMC feeders. These data support the clinical application of aAPC to manufacture TIL for the treatment of melanoma.

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 β-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.

The advent of efficient approaches to the genetic modification of T cells has provided investigators with clinically appealing approaches to improve the potency of tumor-specific clinical grade T cells. For example, gene therapy has been successfully used to enforce expression of chimeric antigen receptors (CAR) that provide T cells with ability to directly recognize tumor-associated antigens without the need for presentation by human leukocyte antigen. Gene transfer of CARs can be undertaken using viral-based and non-viral approaches. We have advanced DNA vectors derived from the Sleeping Beauty (SB) system to avoid the expense and manufacturing difficulty associated with transducing T cells with recombinant viral vectors. After electroporation, the transposon/transposase system improves the efficiency of integration of plasmids used to express CAR and other transgenes in T cells. The SB system combined with artificial antigen-presenting cells (aAPC) can selectively propagate and thus retrieve CAR+ T cells suitable for human application. This review describes the translation of the SB system and aAPC for use in clinical trials and highlights how a nimble and cost-effective approach to developing genetically modified T cells can be used to implement clinical trials infusing next-generation T cells with improved therapeutic potential.

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 in10(9) CAR(+)γδ T cells from10(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.

The Sleeping Beauty (SB) transposon/transposase DNA plasmid system is used to genetically modify cells for long-term transgene expression. We adapted the SB system for human application and generated T cells expressing a chimeric antigen receptor (CAR) specific for CD19. Electrotransfer of CD19-specific SB DNA plasmids in peripheral blood mononuclear cells and propagation on CD19 artificial antigen presenting cells was used to numerically expand CD3 T cells expressing CAR. By day 28 of coculture, >90% of expanded CD3 T cells expressed CAR. CAR T cells specifically killed CD19 target cells and consisted of subsets expressing biomarkers consistent with central memory, effector memory, and effector phenotypes. CAR T cells contracted numerically in the absence of the CD19 antigen, did not express SB11 transposase, and maintained a polyclonal TCR Vα and TCR Vβ repertoire. Quantitative fluorescence in situ hybridization revealed that CAR T cells preserved the telomere length. Quantitative polymerase chain reaction and fluorescence in situ hybridization showed CAR transposon integrated on average once per T-cell genome. CAR T cells in peripheral blood can be detected by quantitative polymerase chain reaction at a sensitivity of 0.01%. These findings lay the groundwork as the basis of our first-in-human clinical trials of the nonviral SB system for the investigational treatment of CD19 B-cell malignancies (currently under 3 INDs: 14193, 14577, and 14739).

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.

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.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 CD19After 4 weeks, 90 % of cultured cells exhibited specific killing of CD19This 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.

Potential for on-target, but off-tissue toxicity limits therapeutic application of genetically modified T cells constitutively expressing chimeric antigen receptors (CARs) from tumor-associated antigens expressed in normal tissue, such as epidermal growth factor receptor (EGFR). Curtailing expression of CAR through modification of T cells by in vitro-transcribed mRNA species is one strategy to mitigate such toxicity. We evaluated expression of an EGFR-specific CAR coded from introduced mRNA in human T cells numerically expanded ex vivo to clinically significant numbers through coculture with activating and propagating cells (AaPC) derived from K562 preloaded with anti-CD3 antibody. The density of AaPC could be adjusted to affect phenotype of T cells such that reduced ratio of AaPC resulted in higher proportion of CD8 and central memory T cells that were more conducive to electrotransfer of mRNA than T cells expanded with high ratios of AaPC. RNA-modified CAR T cells produced less cytokine, but demonstrated similar cytolytic capacity as DNA-modified CAR T cells in response to EGFR-expressing glioblastoma cells. Expression of CAR by mRNA transfer was transient and accelerated by stimulation with cytokine and antigen. Loss of CAR abrogated T-cell function in response to tumor and normal cells expressing EGFR. We describe a clinically applicable method to propagate and modify T cells to transiently express EGFR-specific CAR to target EGFR-expressing tumor cells that may be used to limit on-target, off-tissue toxicity to normal tissue.

Adoptive transfer of primary T cells genetically modified to have desired specificity can exert an anti-tumor response in some patients. To improve our understanding of their therapeutic potential we have developed a clinically-appealing approach to reveal their in vivo biodistribution using nanoparticles that serve as a radiotracer for imaging by positron emission tomography (PET). T cells electroporated with DNA plasmids from the Sleeping Beauty transposon/transposase system to co-express chimeric antigen receptor (CAR) specific for CD19 and Firefly luciferase (ffLuc) were propagated on CD19+ K562-derived artificial antigen presenting cells. The approach to generating our clinical-grade CAR+ T cells was adapted to electro-transfer of gold nanoparticles (GNP) functionalized with 64Cu2+ using the macrocyclic chelator (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, DOTA) and polyethyleneglycol (GNP-64Cu/PEG2000). MicroPET/CT was used to visualize CAR+EGFPffLucHyTK+GNP-64Cu/PEG2000+ T cells and correlated with bioluminescence imaging and assessment of radioactivity. These data demonstrate that GNPs conjugated with 64Cu2+ can be prepared as radiotracer for PET and used to image T cells using an approach that has translational implications.

Clinical-grade T cells are genetically modified ex vivo to express a chimeric antigen receptor (CAR) to redirect specificity to a tumor associated antigen (TAA) thereby conferring antitumor activity in vivo. T cells expressing a CD19-specific CAR recognize B-cell malignancies in multiple recipients independent of major histocompatibility complex (MHC) because the specificity domains are cloned from the variable chains of a CD19 monoclonal antibody. We now report a major step toward eliminating the need to generate patient-specific T cells by generating universal allogeneic TAA-specific T cells from one donor that might be administered to multiple recipients. This was achieved by genetically editing CD19-specific CAR+ T cells to eliminate expression of the endogenous αβ T-cell receptor (TCR) to prevent a graft-versus-host response without compromising CAR-dependent effector functions. Genetically modified T cells were generated using the Sleeping Beauty system to stably introduce the CD19-specific CAR with subsequent permanent deletion of α or β TCR chains with designer zinc finger nucleases. We show that these engineered T cells display the expected property of having redirected specificity for CD19 without responding to TCR stimulation. CAR+TCRneg T cells of this type may potentially have efficacy as an off-the-shelf therapy for investigational treatment of B-lineage malignancies.

Improving the therapeutic efficacy of T cells expressing a chimeric antigen receptor (CAR) represents an important goal in efforts to control B-cell malignancies. Recently an intrinsic strategy has been developed to modify the CAR itself to improve T-cell signaling. Here we report a second extrinsic approach based on altering the culture milieu to numerically expand CAR+ T cells with a desired phenotype, for the addition of interleukin (IL)-21 to tissue culture improves CAR-dependent T-cell effector functions. We used electrotransfer of Sleeping Beauty system to introduce a CAR transposon and selectively propagate CAR+ T cells on CD19+ artificial antigen-presenting cells (aAPC). When IL-21 was present, there was preferential numeric expansion of CD19-specific T cells which lysed and produced IFN-γ in response to CD19. Populations of these numerically expanded CAR+ T cells displayed an early memory surface phenotype characterized as CD62L+CD28+ and a transcriptional profile of naïve T cells. In contrast, T cells propagated with only exogenous IL-2 tended to result in an overgrowth of CD19-specific CD4+ T cells. Furthermore, adoptive transfer of CAR+ T cells cultured with IL-21 exhibited improved control of CD19+ B-cell malignancy in mice. To provide coordinated signaling to propagate CAR+ T cells, we developed a novel mutein of IL-21 bound to the cell surface of aAPC that replaced the need for soluble IL-21. Our findings show that IL-21 can provide an extrinsic reprogramming signal to generate desired CAR+ T cells for effective immunotherapy. Cancer Res; 71(10); 3516–27. ©2011 AACR.

In the last decade, virus specific CTL manufacture has become a more widespread and reproducible technology. A number of clinical grade techniques have been described. Here we outline the approach used at Baylor College of Medicine (BCM) since 2004 to treat over 35 patients with EBV-positive lymphoma using T cells specific for the latent membrane proteins (LMP1 and LMP2) of EBV in phase I/II clinical trials. Clinical outcomes have been described, but briefly objective tumor responses were achieved in 11/16 patients and complete remissions in 8. [1], [2]Here we outline the CTL manufacturing process. We have made the detailed SOPs required available with appropriate URL links throughout the manuscript. These should facilitate the creation of protocols suitable for regulatory approval and provide the basis for GMP manufacture of LMP1- and LMP2-specific T cells.

Sleeping Beauty (SB3) transposon and transposase constitute a DNA plasmid system used for therapeutic human cell genetic engineering. Here we report a comparison of SB100X, a newly developed hyperactive SB transposase, to a previous generation SB11 transposase to achieve stable expression of a CD19-specific chimeric antigen receptor (CAR3) in primary human T cells. The electro-transfer of SB100X expressed from a DNA plasmid or as an introduced mRNA species had superior transposase activity in T cells based on the measurement of excision circles released after transposition and emergence of CAR expression on T cells selectively propagated upon CD19+ artificial antigen-presenting cells. Given that T cells modified with SB100X and SB11 integrate on average one copy of the CAR transposon in each T-cell genome, the improved transposition mediated by SB100X apparently leads to an augmented founder effect of electroporated T cells with durable integration of CAR. In aggregate, SB100X improves SB transposition in primary human T cells and can be titrated with an SB transposon plasmid to improve the generation of CD19-specific CAR+ T cells.

Nonviral integrating vectors can be used for expression of therapeutic genes. piggyBac (PB), a transposon/transposase system, has been used to efficiently generate induced pluripotent stems cells from somatic cells, without genetic alteration. In this paper, we apply PB transposition to express a chimeric antigen receptor (CAR) in primary human T cells. We demonstrate that T cells electroporated to introduce the PB transposon and transposase stably express CD19-specific CAR and when cultured on CD19(+) artificial antigen-presenting cells, numerically expand in a CAR-dependent manner, display a phenotype associated with both memory and effector T cell populations, and exhibit CD19-dependent killing of tumor targets. Integration of the PB transposon expressing CAR was not associated with genotoxicity, based on chromosome analysis. PB transposition for generating human T cells with redirected specificity to a desired target such as CD19 is a new genetic approach with therapeutic implications.

Mismatch of human leukocyte antigens (HLA) adversely impacts the outcome of patients after allogeneic hematopoietic stem-cell transplantation (alloHSCT). This translates into the clinical requirement to timely identify suitable HLA-matched donors which in turn curtails the chances of recipients, especially those from a racial minority, to successfully undergo alloHSCT. We thus sought to broaden the existing pool of registered unrelated donors based on analysis that eliminating the expression of the HLA-A increases the chance for finding a donor matched at HLA-B, -C and -DRB1 regardless of a patient’s race. Elimination of HLA-A expression in HSC was achieved using artificial zinc finger nucleases designed to target HLA-A alleles. Significantly, these engineered HSCs maintain their ability to engraft and reconstitute hematopoiesis in immunocompromised mice. This introduced loss of HLA-A expression decreases the need to recruit large number of donors to match with potential recipients and has particular importance for patients whose HLA repertoire is under-represented in the current donor pool. Furthermore, the genetic engineering of stem cells provides a translational approach to HLA-match a limited number of third-party donors with a wide number of recipients.

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection occurring after allogeneic hematopoietic stem-cell transplantation. Viral-specific T cells from allogeneic donors, including third party donors, can be propagated ex vivo in compliance with current good manufacturing practice (cGMP), employing repeated rounds of antigen-driven stimulation to selectively propagate desired T cells. The identification and isolation of antigen-specific T cells can also be undertaken based upon the cytokine capture system of T cells that have been activated to secrete gamma-interferon (IFN-γ). However, widespread human application of the cytokine capture system (CCS) to help restore immunity has been limited as the production process is time-consuming and requires a skilled operator. The development of a second-generation cell enrichment device such as CliniMACS Prodigy now enables investigators to generate viral-specific T cells using an automated, less labor-intensive system. This device separates magnetically labeled cells from unlabeled cells using magnetic activated cell sorting technology to generate clinical-grade products, is engineered as a closed system and can be accessed and operated on the benchtop. We demonstrate the operation of this new automated cell enrichment device to manufacture CMV pp65-specific T cells obtained from a steady-state apheresis product obtained from a CMV seropositive donor. These isolated T cells can then be directly infused into a patient under institutional and federal regulatory supervision. All the bio-processing steps including removal of red blood cells, stimulation of T cells, separation of antigen-specific T cells, purification, and washing are fully automated. Devices such as this raise the possibility that T cells for human application can be manufactured outside of dedicated good manufacturing practice (GMP) facilities and instead be produced in blood banking facilities where staff can supervise automated protocols to produce multiple products.