Prachi R. Patel

Rational development and characterization of humanized anti-EGFR variant III chimeric antigen receptor T cells for glioblastoma.

A chimeric antigen receptor redirects T cells to treat glioblastoma. CAR T cells drive glioblastoma therapy Immunotherapy with chimeric antigen receptor (CAR) T cells can successfully treat B cell malignancies, but expansion into solid tumors has been limited by the lack of availability of tumor-specific antigens. Now, Johnson et al. target CAR T cells to a variant III mutation of the epidermal growth factor receptor (EGFRvIII), which is thought to be enriched in glioblastoma stem cells. They found that a low-affinity single-chain variable fragment was specific for EGFRvIII over wild-type EGFR and that CAR T cells transduced with this fragment were able to target antigen-expressing cells in vitro and in vivo in multiple mouse xenograft models of human glioblastoma. These cells are currently being moved into the clinic in a phase 1 clinical trial. Chimeric antigen receptors (CARs) are synthetic molecules designed to redirect T cells to specific antigens. CAR-modified T cells can mediate long-term durable remissions in B cell malignancies, but expanding this platform to solid tumors requires the discovery of surface targets with limited expression in normal tissues. The variant III mutation of the epidermal growth factor receptor (EGFRvIII) results from an in-frame deletion of a portion of the extracellular domain, creating a neoepitope. We chose a vector backbone encoding a second-generation CAR based on efficacy of a murine scFv–based CAR in a xenograft model of glioblastoma. Next, we generated a panel of humanized scFvs and tested their specificity and function as soluble proteins and in the form of CAR-transduced T cells; a low-affinity scFv was selected on the basis of its specificity for EGFRvIII over wild-type EGFR. The lead candidate scFv was tested in vitro for its ability to direct CAR-transduced T cells to specifically lyse, proliferate, and secrete cytokines in response to antigen-bearing targets. We further evaluated the specificity of the lead CAR candidate in vitro against EGFR-expressing keratinocytes and in vivo in a model of mice grafted with normal human skin. EGFRvIII-directed CAR T cells were also able to control tumor growth in xenogeneic subcutaneous and orthotopic models of human EGFRvIII+ glioblastoma. On the basis of these results, we have designed a phase 1 clinical study of CAR T cells transduced with humanized scFv directed to EGFRvIII in patients with either residual or recurrent glioblastoma (NCT02209376).

Ibrutinib enhances chimeric antigen receptor T-cell engraftment and efficacy in leukemia

Anti-CD19 chimeric antigen receptor (CAR) T-cell therapy is highly promising but requires robust T-cell expansion and engraftment. A T-cell defect in chronic lymphocytic leukemia (CLL) due to disease and/or therapy impairs ex vivo expansion and response to CAR T cells. To evaluate the effect of ibrutinib treatment on the T-cell compartment in CLL as it relates to CAR T-cell generation, we examined the phenotype and function of T cells in a cohort of CLL patients during their course of treatment with ibrutinib. We found that ≥5 cycles of ibrutinib therapy improved the expansion of CD19-directed CAR T cells (CTL019), in association with decreased expression of the immunosuppressive molecule programmed cell death 1 on T cells and of CD200 on B-CLL cells. In support of these findings, we observed that 3 CLL patients who had been treated with ibrutinib for ≥1 year at the time of T-cell collection had improved ex vivo and in vivo CTL019 expansion, which correlated positively together and with clinical response. Lastly, we show that ibrutinib exposure does not impair CAR T-cell function in vitro but does improve CAR T-cell engraftment, tumor clearance, and survival in human xenograft models of resistant acute lymphocytic leukemia and CLL when administered concurrently. Our collective findings indicate that ibrutinib enhances CAR T-cell function and suggest that clinical trials with combination therapy are warranted. Our studies demonstrate that improved T-cell function may also contribute to the efficacy of ibrutinib in CLL. These trials were registered at www.clinicaltrials.gov as #NCT01747486, #NCT01105247, and #NCT01217749.

Acute exposure to ZnO nanoparticles induces autophagic immune cell death

Abstract The increasing risk of incidental exposure to nanomaterials has led to mounting concerns regarding nanotoxicity. Zinc oxide nanoparticles (ZnO NPs) are produced in large quantities and have come under scrutiny due to their capacity to cause cytotoxicity in vitro and potential to cause harm in vivo. Recent evidence has indicated that ZnO NPs promote autophagy in cells; however, the signaling pathways and the role of ion release inducing toxicity remain unclear. In this study, we report that ZnO NPs are immunotoxic to primary and immortalized immune cells. Importantly, such immunotoxicity is observed in mice in vivo, since death of splenocytes is seen after intranasal exposure to ZnO NPs. We determined that ZnO NPs release free Zn2+ that can be taken up by immune cells, resulting in cell death. Inhibiting free Zn2+ ions in solution with EDTA or their uptake with CaCl2 abrogates ZnO NP-induced cell death. ZnO NP-mediated immune cell death was associated with increased levels of intracellular reactive oxygen species (ROS). ZnO NP death was not due to apoptosis, necroptosis or pyroptosis. Exposure of immune cells to ZnO NPs resulted in autophagic death and increased levels of LC3A, an essential component of autophagic vacuoles. Accordingly, ZnO NP-mediated upregulation of LC3A and induction of immune cell death were inhibited by blocking autophagy and ROS production. We conclude that release of Zn2+ from ZnO NPs triggers the production of excessive intracellular ROS, resulting in autophagic death of immune cells. Our findings suggest that exposure to ZnO NPs has the potential to impact host immunity.

Enhancing CAR T cell persistence through ICOS and 4-1BB costimulation

Successful tumor eradication by chimeric antigen receptor-expressing (CAR-expressing) T lymphocytes depends on CAR T cell persistence and effector function. We hypothesized that CD4+ and CD8+ T cells may exhibit distinct persistence and effector phenotypes, depending on the identity of specific intracellular signaling domains (ICDs) used to generate the CAR. First, we demonstrate that the ICOS ICD dramatically enhanced the in vivo persistence of CAR-expressing CD4+ T cells that, in turn, increased the persistence of CD8+ T cells expressing either CD28- or 4-1BB-based CARs. These data indicate that persistence of CD8+ T cells was highly dependent on a helper effect provided by the ICD used to redirect CD4+ T cells. Second, we discovered that combining ICOS and 4-1BB ICDs in a third-generation CAR displayed superior antitumor effects and increased persistence in vivo. Interestingly, we found that the membrane-proximal ICD displayed a dominant effect over the distal domain in third-generation CARs. The optimal antitumor and persistence benefits observed in third-generation ICOSBBz CAR T cells required the ICOS ICD to be positioned proximal to the cell membrane and linked to the ICOS transmembrane domain. Thus, CARs with ICOS and 4-1BB ICD demonstrate increased efficacy in solid tumor models over our current 4-1BB-based CAR and are promising therapeutics for clinical testing.

Induction of resistance to chimeric antigen receptor T cell therapy by transduction of a single leukemic B cell.

We report a patient relapsing 9 months after CD19-targeted CAR T cell (CTL019) infusion with CD19– leukemia that aberrantly expressed the anti-CD19 CAR. The CAR gene was unintentionally introduced into a single leukemic B cell during T cell manufacturing, and its product bound in cis to the CD19 epitope on the surface of leukemic cells, masking it from recognition by and conferring resistance to CTL019.A CAR gene unintentionally introduced in a contaminating leukemia cell during the manufacturing of CAR T cells caused a patient to relapse after therapy.

Enhancing T cell persistence of CAR-redirected T cells in solid tumors

T cell persistence is likely to promote long-term anti-tumor effects after adoptive T cell transfer. We have recently shown that incorporation of the ICOS intracellular domain into chimeric antigen receptors (CARs) significantly increased Th17 cell persistence in vivo, compared to CARs with CD28 or 4-1BB intracellular domains [1]. Here, we hypothesized that CD4+ and CD8+ T cells require distinct cytokine and costimulation signals for optimal persistence. To test this hypothesis, we compared the in vivo antitumor effects and persistence of combined CD4+ T cells (bulk or Th17-polarized) and CD8+ T cells redirected with CARs containing CD28, 4-1BB or ICOS-based costimulatory domains. Using multiple mouse tumor models, we demonstrate that the ICOS intracellular domain significantly enhanced the in vivo persistence of CAR-expressing CD4+ T cells, and that both persistence and tumor infiltration were further enhanced by culturing these cells under Th17-polarizing conditions. Importantly, Th17-polarized CD4+ T cells expressing an ICOS-based CAR significantly increased the circulatory persistence of bulk CD8+ T cells expressing either CD28- or 4-1BB-based CARs. We further demonstrate that the antitumor effect of CAR-expressing CD8+ T cells was enhanced when co-injected with ICOS-redirected Th17 cells. Collectively, our data suggest that combining Th17 CD4+ T cells redirected with an ICOS-based CAR with CD8+ CAR-T cells will enhance their persistence and antitumor efficacy.

Pre-clinical validation of a humanized anti-EGFR variant III chimeric antigen receptor and phase I trial of CART-EGFRvIII in glioblastoma

Chimeric antigen receptors are synthetic molecules designed to re-direct T cells to specific surface antigens; CAR-modified T cells can mediate long-term durable remissions in B cell malignancies, but expanding this platform to solid tumors requires the discovery of novel surface targets with limited expression. The variant III mutation of the epidermal growth factor receptor (EGFR variant III) is the most common variant of the EGF receptor observed in human tumors, and results from an in-frame deletion of a portion of the extracellular domain. In glioblastoma, the EGFRvIII mutation is oncogenic, portends a poor prognosis, and is thought to be enriched in glioblastoma stem cells. However, because the neoepitope of EGFR variant III is based on a small peptide sequence, an antibody or single-chain variable fragment (scFv) directed to this epitope must be rigorously tested to confirm lack of cross-reactivity to the ubiquitously expressed normal EGFR. Having selected a candidate murine scFv directed to EGFRvIII and a vector backbone encoding a second generation CAR, we generated a panel of humanized scFvs and tested their specificity and function as soluble proteins and in the form of CAR-transduced T cells. The lead candidate scFv was tested in vitro for its ability to direct CAR-transduced T cells to kill antigen-bearing targets effectively, and proliferate and secrete cytokines specifically in response to antigen. We further evaluated the specificity of the lead candidate CAR by comparing it to a cetuximab-based CAR which does not discriminate between EGFR and EGFR variant III; the two CARs, along with negative controls, were tested in vitro against primary cells derived from a panel of normal tissues, and in vivo in immunodeficient mice grafted with normal human skin, which naturally expresses EGFR. CAR-T cells were also able to control tumor growth in xenogeneic subcutaneous and orthotopic models of human EGFR variant III+ glioblastoma. We have designed a Phase I clinical study of CAR T cells transduced with humanized scFv directed to EGFR variant III in patients with glioblastoma.

Reducing Ex Vivo Culture Improves the Antileukemic Activity of Chimeric Antigen Receptor (CAR) T Cells

The efficacy of CAR T-cell therapy depends on the engraftment and persistence of T cells following adoptive transfer. Limiting ex vivo culture time of CD19-specific CAR T cells during manufacturing yielded improved persistence and effector function in vivo. The success of chimeric antigen receptor (CAR)–mediated immunotherapy in acute lymphoblastic leukemia (ALL) highlights the potential of T-cell therapies with directed cytotoxicity against specific tumor antigens. The efficacy of CAR T-cell therapy depends on the engraftment and persistence of T cells following adoptive transfer. Most protocols for T-cell engineering routinely expand T cells ex vivo for 9 to 14 days. Because the potential for engraftment and persistence is related to the state of T-cell differentiation, we hypothesized that reducing the duration of ex vivo culture would limit differentiation and enhance the efficacy of CAR T-cell therapy. We demonstrated that T cells with a CAR-targeting CD19 (CART19) exhibited less differentiation and enhanced effector function in vitro when harvested from cultures at earlier (day 3 or 5) compared with later (day 9) timepoints. We then compared the therapeutic potential of early versus late harvested CART19 in a murine xenograft model of ALL and showed that the antileukemic activity inversely correlated with ex vivo culture time: day 3 harvested cells showed robust tumor control despite using a 6-fold lower dose of CART19, whereas day 9 cells failed to control leukemia at limited cell doses. We also demonstrated the feasibility of an abbreviated culture in a large-scale current good manufacturing practice–compliant process. Limiting the interval between T-cell isolation and CAR treatment is critical for patients with rapidly progressing disease. Generating CAR T cells in less time also improves potency, which is central to the effectiveness of these therapies. Cancer Immunol Res; 6(9); 1100–9. ©2018 AACR.

719. Combination of ICOS and 4-1BB in a Third Generation CAR Exhibits Enhanced T Cell Persistence and Increased Antitumor Effect

T cell persistence is likely to promote long-term antitumor effects after adoptive T cell transfer; however, T cells expressing chimeric antigen receptors (CARs) have not persisted well in patients with solid tumors in trials reported to date. Here, we hypothesized that CD4+ and CD8+ T cells may need distinct costimulation signals to persist. To test this hypothesis, we compared the in vivo antitumor effects and persistence of combined CD4+ and CD8+ T cells redirected with CARs containing different costimulatory domains (CD28, 4-1BB or ICOS). Using multiple mouse tumor models, we demonstrate that the ICOS intracellular domain enhanced the in vivo persistence of CAR-expressing CD4+ T cells. Persistence of CD8+ T cells was highly dependent on the intracellular domain used to redirect CD4+ T cells; specifically, CD4+ T cells expressing an ICOS-based CAR significantly increased the persistence of CD8+ T cells expressing either CD28- or 4-1BB-based CARs. We further demonstrate that the combination of ICOS and 4-1BB in a third generation CAR (ICOSBBz) is more effective at eliminating large pancreatic tumors than either 4-1BB or ICOS alone. T cells redirected with ICOSBBz showed a less differentiated phenotype with superior proliferation and in vivo persistence. These results indicate that ICOSBBz-based CARs are promising therapeutics for clinical testing.