David Torres

Characterization and Functional Analysis of scFv-based Chimeric Antigen Receptors to Redirect T Cells to IL13Rα2-positive Glioma.

Immunotherapy with T cells expressing chimeric antigen receptors (CARs) is an attractive approach to improve outcomes for patients with glioblastoma (GBM). IL13Rα2 is expressed at a high frequency in GBM but not in normal brain, making it a promising CAR T-cell therapy target. IL13Rα2-specific CARs generated up to date contain mutated forms of IL13 as an antigen-binding domain. While these CARs target IL13Rα2, they also recognize IL13Rα1, which is broadly expressed. To overcome this limitation, we constructed a panel of IL13Rα2-specific CARs that contain the IL13Rα2-specific single-chain variable fragment (scFv) 47 as an antigen binding domain, short or long spacer regions, a transmembrane domain, and endodomains derived from costimulatory molecules and CD3.ζ (IL13Rα2-CARs). IL13Rα2-CAR T cells recognized IL13Rα2-positive target cells in coculture and cytotoxicity assays with no cross-reactivity to IL13Rα1. However, only IL13Rα2-CAR T cells with a short spacer region produced IL2 in an antigen-dependent fashion. In vivo, T cells expressing IL13Rα2-CARs with short spacer regions and CD28.ζ, 41BB.ζ, and CD28.OX40.ζ endodomains had potent anti-glioma activity conferring a significant survival advantage in comparison to mice that received control T cells. Thus, IL13Rα2-CAR T cells hold the promise to improve current IL13Rα2-targeted immunotherapy approaches for GBM and other IL13Rα2-positive malignancies.

CD123-Engager T Cells as a Novel Immunotherapeutic for Acute Myeloid Leukemia

Immunotherapy with CD123-specific T-cell engager proteins or with T cells expressing CD123-specific chimeric antigen receptors is actively being pursued for acute myeloid leukemia. T cells secreting bispecific engager molecules (ENG-T cells) may present a promising alternative to these approaches. To evaluate therapeutic potential, we generated T cells to secrete CD123/CD3-bispecific engager molecules. CD123-ENG T cells recognized primary acute myeloid leukemia (AML) cells and cell lines in an antigen-dependent manner as judged by cytokine production and/or tumor killing, and redirected bystander T cells to AML cells. Infusion of CD123-ENG T cells resulted in regression of AML in xenograft models conferring a significant survival advantage of treated mice in comparison to mice that received control T cells. At high effector to target ratios, CD123-ENG T cells recognized normal hematopoietic stem and progenitor cells (HSPCs) with preferential recognition of HSPCs from cord blood compared to bone marrow. We therefore introduced the CD20 suicide gene that can be targeted in vivo with rituximab into CD123-ENG T cells. The expression of CD20 did not diminish the anti-AML activity of CD123-ENG T cells, but allowed for rituximab-mediated ENG-T cell elimination. Thus, ENG-T cells coexpressing CD20 suicide and CD123 engager molecules may present a promising immunotherapeutic approach for AML.

T cells expressing CD19-specific Engager Molecules for the Immunotherapy of CD19-positive Malignancies.

T cells expressing chimeric antigen receptors (CARs) or the infusion of bispecific T-cell engagers (BITEs) have shown antitumor activity in humans for CD19-positive malignancies. While BITEs redirect the large reservoir of resident T cells to tumors, CAR T cells rely on significant in vivo expansion to exert antitumor activity. We have shown that it is feasible to modify T cells to secrete solid tumor antigen-specific BITEs, enabling T cells to redirect resident T cells to tumor cells. To adapt this approach to CD19-positive malignancies we now generated T cells expressing secretable, CD19-specific BITEs (CD19-ENG T cells). CD19-ENG T cells recognized tumor cells in an antigen-dependent manner as judged by cytokine production and tumor killing, and redirected bystander T cells to tumor cells. Infusion of CD19-ENG T cells resulted in regression of leukemia or lymphoma in xenograft models and a survival advantage in comparison to control mice. Genetically modified T cells expressing engager molecules may present a promising addition to current CD19-targeted immunotherapies.

T Cell-Activating Mesenchymal Stem Cells as a Biotherapeutic for HCC

The outcome for advanced stage hepatocellular carcinoma (HCC) remains poor, highlighting the need for novel therapies. Genetically modified mesenchymal stem cells (MSCs) are actively being explored as cancer therapeutics due to their inherent ability to migrate to tumor sites. We reasoned that MSCs can be genetically modified to redirect T cells to Glypican-3 (GPC3)+ HCC, and genetically modified these with viral vectors encoding a GPC3/CD3 bispecific T cell engager (GPC3-ENG), a bispecifc T cell engager specific for an irrelevant antigen (EGFRvIII), and/or costimulatory molecules (CD80 and 41BBL). Coculture of GPC3+ cells, GPC3-ENG MSCs, and T cells resulted in T cell activation, as judged by interferon γ (IFNγ) production and killing of tumor cells by T cells. Modification of GPC3-ENG MSCs with CD80 and 41BBL was required for antigen-dependent interleukin-2 (IL-2) production by T cells and resulted in faster tumor cell killing by redirected T cells. In vivo, GPC3-ENG MSCs ± costimulatory molecules had antitumor activity in the HUH7 HCC xenograft model, resulting in a survival advantage. In conclusion, MSCs genetically modified to express GPC3-ENG ± costimulatory molecules redirect T cells to GPC3+ tumor cells and have potent antitumor activity. Thus, further preclinical exploration of our modified approach to GPC3-targeted immunotherapy for HCC is warranted.

282. A scFv-Based CAR To Redirect T Cells To IL13Ra2-Positive Glioma

BACKGROUND: Glioblastoma (GBM) is the most aggressive primary brain tumor in humans, and is virtually incurable with conventional therapies. Immunotherapy with T cells expressing chimeric antigen receptors (CARs) is an attractive approach to improve outcomes. IL13Ra2 is aberrantly expressed in GBM therefore it is a promising target for CAR T-cell immunotherapy. The antigen recognition domain of CARs normally consists of a single chain variable fragment (scFv), however current IL13Ra2-specific CARs use IL13 muteins as an antigen recognition domain. We have previously shown that IL13 mutein-based CARs also recognize IL13Ra1, raising significant safety concerns. To overcome this obstacle we have recently generated a high affinity IL13Ra2-specific scFv. The goal of this project is now to develop a scFv-based IL13Ra2-specific CAR (IL13Ra2-CAR) and evaluate the effector function of IL13Ra2-CAR T cells.METHODS: We constructed a panel of IL13Ra2-CARs that contain our new IL13Ra2-specific scFv (m47) as an ectodomain, a short hinge (SH) or long hinge (LH), a CD28 transmembrane domain, and endodomains that contain signaling domains derived from CD3ζ and co-stimulatory molecules (CD28.ζ, CD137.ζ, CD28.CD137.ζ, CD28.CD134.ζ). IL13Ra2-CAR T cells were generated by retroviral transduction, and we determined their effector function in vitro, in co-culture and cytotoxicity assays, and in vivo, in the U373 brain xenograft model.RESULTS: Expression of all CARs in T cells was similar as judged by Western blot analysis. However, cell surface expression varied depending on used hinge and endodomain. In cytotoxicity assays, T cells expressing the different IL13Ra2-CARs only killed target cells expressing IL13Ra2, but not IL13Ra1 thus confirming the specificity of receptor-CAR interaction. While all IL13Ra2-CAR T cells secreted significant levels of IFNγ in co-culture assays with the IL13Ra2+ glioma cell line U373, only short hinge CAR T cells secreted significant amounts of IL2. T cells expressing IL13Ra2-CARs with a deleted endodomain (IL13Ra2.Δ-CAR) secreted no cytokines confirming that cytokine production depends on the presence of a functional IL 13Ra2-CAR. In vivo, injection of IL13Ra2.SH.CD28.ζ-CAR T cells into U373-bearing mice resulted in regression of gliomas as judged by bioluminescence imaging. IL13Ra2.LH.CD28.ζ- or IL13Ra2.Δ-CAR T cells had no antitumor effects.CONCLUSION: For the first time we have generated a CAR that only recognizes IL13Ra2 and not IL13Ra1. Comparison of several IL13Ra2-CARs revealed so far that a CAR with a SH and a CD28.ζ endodomain results in optimal T-cell activation as judged by IL2 production and in vivo anti-glioma activity. Our results warrant further active exploration of IL13Ra2-specific scFv-based CAR T cells for the adoptive immunotherapy of high grade glioma.

Costimulation to enhance the antitumor activity of CD19 eng T cells

Meeting abstracts Immunotherapy with T cells or bispecific T cell engagers is one promising approach to improve outcomes for patients with CD19-positive hematological malignancies. We had previously shown that T cells expressing bispecific T cell engagers that recognize CD19 and CD3 (CD19-ENG T

746. Go-TCR: Inducible MyD88/CD40 (iMC) Enhances Proliferation and Survival of Tumor-Specific TCR-Modified T Cells, Increasing Anti-Tumor Efficacy

Introduction: Use of tumor antigen-specific T cell receptors (TCRs) to refocus T cell killing has shown tantalizing clinical efficacy; however, durable responses have been limited by poor T cell persistence and expansion in vivo. Also, MHC class I downregulation in tumors further reduces therapeutic efficacy. Therefore, we co-expressed in human T cells a small molecule dimerizer (rimiducid)-dependent “activation switch”, called inducible MyD88/CD40 (iMC), along with tumor-targeted TCRs to regulate T cell expansion and activation, while affecting upregulation of MHC class I on tumors.Methods: Human T cells were CD3/CD28-activated and transduced with αβTCR-encoding γ-retroviruses recognizing either the CT antigen, PRAME (HLA-A*0201/SLLQHLIGL), or the B-cell-specific transcriptional co-activator, Bob1/OBF-1 (HLA-B*0702/APAPTAVVL). Parallel “Go-TCR” vectors co-expressed iMC, comprising MyD88 and CD40 signaling domains along with rimiducid-binding FKBP12-V36. Proliferation, cytokine production and cytotoxicity of modified T cells was assessed using peptide-pulsed T2 cells (PRAME only) or against PRAME+/Bob1+, HLA-A2+ -B7+ U266 myeloma cells +/- 10 nM rimiducid. MHC class I induction was measured using transwell assays and flow cytometry. In vitro tumor killing was analyzed by T cell and tumor coculture assays at various effector to target ratios over a 7-day period. Finally, in vivo efficacy was determined using immune-deficient NSG mice engrafted i.v. with U266 cells and treated i.v. with 1×107 transduced T cells. iMC was activated in vivo by weekly i.p. rimiducid injections (1-5 mg/kg). Tumor size and T cell expansion was measured using in vivo BLI imaging and flow cytometry.Results: All vectors efficiently (~85%) transduced activated T cells and showed antigen-specific IFN-γ production and cytotoxicity against peptide-pulsed T2 cells and/or PRAME+Bob1+ U266 cells. However, both iMC signaling and TCR ligation of PRAME peptide-pulsed T2 Cells were required for IL-2 production. Coculture assays with U266 cells showed that tumor elimination, IL-2 secretion and robust (~ 50-fold) T cell proliferation (vs TCR signaling alone) was optimized with concurrent rimiducid-driven iMC activation in both “Go-PRAME” and “Go-Bob1” constructs. Further, iMC activation produced TCR-independent IFN-γ that increased (~100-fold) MHC class I expression on tumor cells. In NSG mice engrafted with U266 tumors, iMc-PRAME TCR-modified T cells persisted for at least 81 days post-injection and prevented tumor growth, unlike other T cell groups. Importantly, weekly rimiducid injection dramatically expanded iMC-PRAME TCR-expressing T cell numbers by ~1000-fold on day 81 post-injection vs T cells expressing only the PRAME TCR (p ~ 0.001).Summary: The novel rimiducid-regulated “Go” switch, iMC, greatly augments activation and expansion of TCR-engineered T cells while sensitizing tumors to T cells via cytokine-induced MHC class I upregulation. iMC-enhanced TCRs are prototypes of novel “Go-TCR” engineered T cell therapies that increase efficacy, safety and durability of adoptive T cell therapies.

Abstract B078: GoTCR: Inducible MyD88/CD40 (iMC) enhances proliferation and survival of tumor-specific TCR-modified T cells and improves antitumor efficacy in myeloma

Introduction: Use of T cells engineered to express antigen-specific T cell receptors (TCRs) has shown promise as a cancer immunotherapy treatment; however, durable responses have been limited by poor T cell persistence and expansion in vivo . Additionally, MHC class I downregulation on tumor cells further reduces therapeutic efficacy. Therefore, we co-expressed in human T cells a novel, small molecule dimerizer (rimiducid)-dependent T cell “activation switch”, called inducible MyD88/CD40 (iMC), along with tumor antigen-specific TCRs to regulate T cell activation and expansion, while upregulating MHC class I expression on tumor cells. Methods: Human T cells were activated with anti-CD3/CD28 and transduced with γ-retroviruses encoding TCR α and β chains recognizing either the cancer-testes antigen PRAME (HLA-A*201-restricted SLLQHLIGL) or the B cell-specific transcriptional co-activator, Bob1/OBF-1 (HLA-B*702-restricted APAPTAVVL). Parallel “GoTCR” vectors co-expressed the αβ TCR and iMC, comprising signaling domains from MyD88 and CD40 fused in frame with tandem rimiducid-binding FKBP12v36 domains. Proliferation, cytokine production and cytotoxicity of modified T cells was assessed using peptide-pulsed EGFPluc-expressing T2 cells (PRAME only) or PRAME + /Bob1 + , HLA-A2 + HLA-B7 + EGFPluc-expressing U266 myeloma cells ± rimiducid (10 nM). MHC class I upregulation on tumor cells was measured using transwell assays and flow cytometry. In vitro tumor killing and T cell proliferation were analyzed using T cell and tumor coculture assays by either measuring loss of luciferase activity overnight or by flow cytometry over a period of 4-7 days. Finally, in vivo efficacy was determined using immune-deficient NSG mice engrafted i.v. with U266 cells and treated i.v. with 5×10 6 -1×10 7 transduced T cells. iMC was activated in vivo by weekly or biweekly i.p. rimiducid injections (1-5 mg/kg). Tumor size and T cell expansion was measured using in vivo bioluminescence imaging and flow cytometry, respectively. Results: All vectors efficiently (∼85%) transduced activated T cells and showed antigen-specific IFN-γ production and cytolytic function against peptide-pulsed T2 cells and/or PRAME + Bob1 + U266 myeloma cells. However, both TCR ligation and rimiducid-dependent iMC costimulation were required for IL-2 production against PRAME peptide-pulsed T2 cells. Coculture assays against U266 cells showed that tumor elimination was optimized with concurrent rimiducid-driven iMC activation in both “GoPRAME” and “GoBob1” constructs, and this was accompanied by greatly increased IL-2 secretion and robust T cell proliferation (∼ 50-fold vs PRAME or Bob1-specific TCRs alone). Further, iMC activation produced IFN-γ independently of TCR ligation, which significantly increased MHC class I expression on tumor cells (∼ 7-fold) relative to PRAME TCR-transduced T cells. In NSG mice engrafted with PRAME + U266 myeloma tumors, GoPRAME TCR-modified T cells persisted for 81 days post-injection and prevented tumor growth, unlike any of the other T cell groups. Importantly, weekly rimiducid injection dramatically expanded iMC-PRAME TCR-expressing T cell numbers by ∼1000-fold on day 81 post-injection compared to T cells expressing only the PRAME TCR (p Summary: iMC is a novel “Go” switch that utilizes rimiducid, a small molecule dimerizer, to provide costimulation to PRAME and Bob1-specific TCR-engineered T cells while sensitizing tumors to TCR-mediated recognition via cytokine-induced MHC I upregulation. These iMC-enhanced TCRs are prototypes of novel “GoTCR” engineered T cell therapies that may increase efficacy, safety and durability of adoptive T cell therapies. Citation Format: Tsvetelina Pentcheva-Hoang, David Torres, Tania Rodriguez, Ana Korngold, An Lu, Jeannette Crisostomo, Annemarie Moseley, Lorenz Jahn, Mirjam H.M. Heemskerk, Kevin Slawin, David Spencer, Aaron Foster. GoTCR: Inducible MyD88/CD40 (iMC) enhances proliferation and survival of tumor-specific TCR-modified T cells and improves antitumor efficacy in myeloma [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B078.

Abstract LB-084: Go-TCR™: Inducible MyD88/CD40 (iMC) enhances proliferation and survival of tumor-specific TCR-modified T cells, increasing anti-tumor efficacy

Introduction: Use of tumor antigen-specific T cell receptors (TCRs) to refocus T cell killing has shown tantalizing clinical efficacy; however, durable responses have been limited by poor T cell persistence and expansion in vivo. Also, MHC class I downregulation in tumors further reduces therapeutic efficacy. Therefore, we co-expressed in human T cells a small molecule dimerizer (rimiducid)-dependent “activation switch”, called inducible MyD88/CD40 (iMC), along with tumor-targeted TCRs to regulate T cell expansion and activation, while affecting upregulation of MHC class I on tumors. Methods: Human T cells were CD3/CD28-activated and transduced with αβTCR-encoding γ-retroviruses recognizing either the CT antigen, PRAME (HLA-A*02:01/SLLQHLIGL), or the B-cell-specific transcriptional co-activator, Bob1/OBF-1 (HLA-B*07:02/APAPTAVVL). Parallel “Go-TCR” vectors co-expressed iMC, comprising MyD88 and CD40 signaling domains along with rimiducid-binding FKBP12-V36. Proliferation, cytokine production and cytotoxicity of modified T cells was assessed using peptide-pulsed T2 cells (PRAME only) or against PRAME+/Bob1+, HLA-A2+ -B7+ U266 myeloma cells +/- 10 nM rimiducid. MHC class I induction was measured using transwell assays and flow cytometry. In vitro tumor killing was analyzed by T cell and tumor coculture assays at various effector to target ratios over a 7-day period. Finally, in vivo efficacy was determined using immune-deficient NSG mice engrafted i.v. with U266 cells and treated i.v. with 1×107 transduced T cells. iMC was activated in vivo by weekly i.p. rimiducid injections (1-5 mg/kg). Tumor size and T cell expansion was measured using in vivo BLI imaging and flow cytometry. Results: All vectors efficiently (∼85%) transduced activated T cells and showed antigen-specific IFN-γ production and cytotoxicity against peptide-pulsed T2 cells and/or PRAME+Bob1+ U266 cells. However, both iMC signaling and TCR ligation of PRAME peptide-pulsed T2 Cells were required for IL-2 production. Coculture assays with U266 cells showed that tumor elimination, IL-2 secretion and robust (∼ 50-fold) T cell proliferation (vs TCR signaling alone) was optimized with concurrent rimiducid-driven iMC activation in both “Go-PRAME” and “Go-Bob1” constructs. Further, iMC activation produced TCR-independent IFN-γ that increased (∼100-fold) MHC class I expression on tumor cells. In NSG mice engrafted with U266 tumors, iMC-PRAME TCR-modified T cells persisted for at least 81 days post-injection and prevented tumor growth, unlike other T cell groups. Importantly, weekly rimiducid injection dramatically expanded iMC-PRAME TCR-expressing T cell numbers by ∼1000-fold on day 81 post-injection vs T cells expressing only the PRAME TCR (p Summary: The novel rimiducid-regulated “Go” switch, iMC, greatly augments activation and expansion of TCR-engineered T cells while sensitizing tumors to T cells via cytokine-induced MHC class I upregulation. iMC-enhanced TCRs are prototypes of novel “Go-TCR” engineered T cell therapies that increase efficacy, safety and durability of adoptive T cell therapies. Citation Format: David M. Spencer, Tsvetelina P. Hoang, Aaron Foster, Tania Rodriguez, David Torres, An Lu, Jeannette Crisostomo, Lorenz Jahn, Mirjam H.M. Heemskerk. Go-TCR™: Inducible MyD88/CD40 (iMC) enhances proliferation and survival of tumor-specific TCR-modified T cells, increasing anti-tumor efficacy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-084.

Charachterization and functional analysis of scFv-based CARs to redirect T cells to IL13Rα2-positive glioma

Meeting abstracts The goal of this project is to develop T cells that express chimeric antigen receptors (CARs) as an effective immunotherapy for glioblastoma (GBM), the most aggressive, primary brain tumor in humans, which outcome remains poor. IL13Rα2 is aberrantly expressed in GBM and therefore