Kirsten Switzer

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.

Reprogramming CD19-Specific T Cells with IL-21 Signaling Can Improve Adoptive Immunotherapy 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.

Evaluating risks of insertional mutagenesis by DNA transposons in gene therapy

Investigational therapy can be successfully undertaken using viral- and nonviral-mediated ex vivo gene transfer. Indeed, recent clinical trials have established the potential for genetically modified T cells to improve and restore health. Recently, the Sleeping Beauty (SB) transposon/transposase system has been applied in clinical trials to stably insert a chimeric antigen receptor (CAR) to redirect T-cell specificity. We discuss the context in which the SB system can be harnessed for gene therapy and describe the human application of SB-modified CAR(+) T cells. We have focused on theoretical issues relating to insertional mutagenesis in the context of human genomes that are naturally subjected to remobilization of transposons and the experimental evidence over the last decade of employing SB transposons for defining genes that induce cancer. These findings are put into the context of the use of SB transposons in the treatment of human disease.

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.

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 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.

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

Significance We describe an approach based on cytokine therapeutics to enhance the persistence and effectiveness of T-cell–based immunotherapies using chimeric antigen receptors (CARs). This strategy is effective without the use of high-dose exogenous cytokines that are typically associated with toxicities. Moreover, we report that the persistence of the least differentiated memory T cell, the T-memory stem cell, was promoted by signaling induced by a membrane-bound chimeric IL-15 cytokine-fusion molecule. These findings may contribute to improving the safety and therapeutic efficacy of CAR-based immunotherapies of patients with advanced cancer. 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.

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

Genetic editing of HLA expression in hematopoietic stem cells to broaden their human application.

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.

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 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.