Antigen Recognition Domain Research Articles

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.

419. Allogenic CAR T-Cells Targeting CD123 for Adoptive Immunotherapy of Acute Myeloid Leukemia (AML)

Chimeric antigen receptor (CAR)-redirected T-cells have given rise to long-term durable remissions and remarkable objective response rates in patients with refractory leukemia, raising hopes that a wider application of CAR technology may lead to a new paradigm in cancer treatment. A limitation of the current autologous approach is that CAR T-cells must be manufactured on a “per patient basis”. We have developed a standardized platform for manufacturing T-cells from third-party healthy donors to generate allogeneic “off-the-shelf” engineered CAR+ T-cell–based frozen products. This platform utilizes Transcription Activator-Like Effector Nuclease (TALEN) gene editing technology to inactivate the TCRα constant (TRAC) gene, eliminating the potential for T-cells bearing alloreactive TCR's to mediate Graft versus Host Disease (GvHD). We have previously demonstrated that editing of the TRAC gene can be achieved at high frequencies, obtaining up to 80% of TCRα negative cells. This allows us to efficiently produce TCR-deficient T-cells that have been shown to no longer mediate alloreactivity in a xeno-GvHD mouse model. In addition to CAR expression, our T-cells are engineered to co-express the RQR8 gene as a safety feature, with the aim of rendering them sensitive to the monoclonal antibody rituximab.In this work we present the adaptation of this allogeneic platform to the production of T cells targeting CD123, the transmembrane alpha chain of the interleukin-3 receptor, which is expressed in tumor cells from the majority of patients with Acute Myeloid Leukemia (AML). To identify an effective CAR targeting CD123, we have screened multiple antigen recognition domains in the context of several different CAR architectures to identify candidates displaying activity against cell lines expressing variable levels of the CD123 antigen. Furthermore, experiments in an AML mouse model using anti-CD123 CAR T cells demonstrate important anti-tumor activity in vivo. The ability to carry out large scale manufacturing of allogeneic, non alloreactive CD123 specific T cells from a single healthy donor will thus offer the possibility of an off-the-shelf treatment that would be immediately available for administration to a large number of AML patients.

3β-Hydroxysterol Δ24-Reductase on the Surface of Hepatitis C Virus-Related Hepatocellular Carcinoma Cells Can Be a Target for Molecular Targeting Therapy

In our previous study, we demonstrated that 3β-hydroxysterol Δ24-reductase (DHCR24) was overexpressed in hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC), and that its expression was induced by HCV. Using a monoclonal antibody against DHCR24 (2-152a MAb), we found that DHCR24 was specifically expressed on the surface of HCC cell lines. Based on these findings, we aimed to establish a novel targeting strategy using 2-152a MAb to treat HCV-related HCC. In the present study, we examined the antitumor activity of 2-152a MAb. In the presence of complement, HCC-derived HuH-7 cells were killed by treatment with 2-152a MAb, which was mediated by complement-dependent cytotoxicity (CDC). In addition, the antigen recognition domain of 2-152a MAb was responsible for the unique anti-HCV activity. These findings demonstrate the feasibility of using 2-152a MAb for antibody therapy against HCV-related HCC. In addition, surface DHCR24 on HCC cells exhibited a functional property, agonist-induced internalization. We showed that 2-152a MAb-mediated binding of a cytotoxic agent (a saponin-conjugated secondary antibody) to surface DHCR24 led to significant cytotoxicity. This suggests that surface DHCR24 on HCC cells can function as a carrier for internalization. Therefore, surface DHCR24 could be a valuable target for HCV-related HCC therapy, and 2-152a MAb appears to be useful for this targeted therapy.

ErbB-targeted CAR T-cell immunotherapy of cancer.

Chimeric antigen receptor (CAR) based immunotherapy has been under development for the last 25 years and is now a promising new treatment modality in the field of cancer immunotherapy. The approach involves genetically engineering T cells to target malignant cells through expression of a bespoke fusion receptor that couples an HLA-independent antigen recognition domain to one or more intracellular T-cell activating modules. Multiple clinical trials are now underway in several centers to investigate CAR T-cell immunotherapy of diverse hematologic and solid tumor types. The most successful results have been achieved in the treatment of patients with B-cell malignancies, in whom several complete and durable responses have been achieved. This review focuses on the preclinical and clinical development of CAR T-cell immunotherapy of solid cancers, targeted against members of the ErbB family.

T Cell Engineering.

T cells are a new and promising antigen-specific therapeutic option for the treatment of malignant diseases. To achieve antigen specificity against tumor antigens, T cells can be manipulated by gene transfer to express chimeric antigen receptors (CARs). CAR-expressing T cells are called redirected T cells. CARs are composed of an extracellular antibody-derived antigen recognition domain, a transmembrane domain and a cytoplasmatic signal domain. Therefore, redirected T cells combine the exchangeable specificity of an antibody with the cytotoxic machinery of a T cell. Early clinical trials with redirected T cells targeting cluster of differentiation (CD) 19 have shown impressive results in CD19-positive hematological cancers. However, for solid cancers only limited clinical experience exists and new and innovative concepts have to be developed to overcome tumor-mediated immune suppression. Herein, we describe the general design of a CAR, the function of the different domains and the different strategies to produce redirected T cells. Furthermore, we summarize and discuss the preclinical and clinical data indicating the tremendous potential of redirected T cells to become a mainstay of cancer immunotherapy.

Allogenic T-Cells Targeting CD123 for Adoptive Immunotherapy of Acute Myeloid Leukemia (AML)

Adoptive immunotherapy using autologous T-cells endowed with chimeric antigen receptors (CARs) has emerged as a promising new approach to treating cancer. However, a limitation of this approach is that CAR T-cells must be generated on a bespoke basis. To overcome this limitation, we have developed an allogeneic based platform for large scale production of “off-the-shelf” CAR T-cells from unrelated 3rd party donors. This platform utilizes Transcription Activator-Like Effector Nuclease (TALENTM) gene editing technology to inactivate the TCRα constant (TRAC) gene, eliminating the potential for T-cells bearing alloreactive TCR’s to mediate Graft versus Host Disease (GvHD). We have previously demonstrated that editing of the TRAC gene can be achieved at high frequencies, obtaining up to 80% of TCRαβ negative cells. This allows us to efficiently produce TCR-deficient T-cells that have been shown to no longer mediate alloreactivity in a xeno-GvHD mouse model. Furthermore, the capacity to perform efficient multiplex genome editing using TALENTM offers the possibility of simultaneously rendering cells resistant to standard chemotherapy or to tumor evasion mechanisms.In this work we present the adaptation of this allogeneic platform to the production of T cells targeting CD123, the transmembrane alpha chain of the interleukin-3 receptor, which is expressed in tumor cells from the majority of patients with Acute Myeloid Leukemia (AML). In a first step, we have screened human primary T-cells harboring CARs with different antigen recognition domains in the context of multiple CAR architectures in order to identify candidates displaying specific activity against cell lines expressing variable levels of the CD123 antigen. To provide proof of concept for the general applicability of the allogeneic approach we have manufactured a TCR-deficient CD123 CAR T-cell (UCART123) and demonstrated that this product maintains a potent anti-tumoral activity in vitro. Experiments in an orthotopic AML mouse model using UCART123 cells are currently ongoing, in order to establish the absence of alloreactivity and the anti-tumoral activity in vivo. The ability to carry out large scale manufacturing of allogeneic, non alloreactive CD123 specific T Cells from a single healthy donor could thus offer the possibility of an off-the-shelf treatment that would be immediately available for administration to a large number of AML patients. DisclosuresGaletto:Cellectis SA: Employment. Lebuhotel:Cellectis SA: Employment. Gouble:Cellectis SA: Employment. Schiffer-Mannioui:Cellectis SA: Employment. Smith:Cellectis SA: Employment.

CD38 Chimeric Antigen Receptor Engineered T Cells As Therapeutic Tools for Multiple Myeloma

Chimeric Antigen Receptors (CARs) are engineered constructs consisting of an antibody-derived antigen recognition domain linked to intracellular T cell signaling domains. Cytotoxic T cells transduced to express tumor-reactive CARs are highly promising tools for immunotherapy of cancer. The CD38 molecule, with its high and homogenous expression on Multiple Myeloma (MM) tumor cells, is considered a suitable target for antibody therapy of MM. Prompted by this, we evaluated the feasibility and efficacy of targeting MM cells with CD38-CAR-transduced T cells (CD38-CAR T cells). To this end, we generated three different retroviral CAR constructs based on human CD38 antibodies as antigen recognition domain, CD3zeta and 41BB (CD137) as signaling domains and transduced them into PBMCs of a healthy donor. After in vitro selection and expansion, all CD38-CAR T cells, either unsorted or CD4/CD8 sorted, effectively lysed MM cell lines in a dose-, and CD38 expression-dependent manner, with a better efficacy for the CD8+ fraction. CD38-CAR T cells also effectively eradicated primary MM cells in the bone marrow mononuclear cells derived from MM patients, indicating their clinical relevancy. Although CD38-CAR T cells also displayed cytotoxic activity against the CD38+ fraction of mature monocytes and NK cells and to a lesser extent CD38+ B and T cells, they did not affect the outgrowth of CD34+ cells into various myeloid lineages. In addition,CD38-CAR T cell activity was effectively controllable by transducing them with a caspase 9-based inducible suicide gene. More interestingly, we discovered that the CD38-CAR T cells were themselves devoid of CD38 surface expression, indicating that CD38 was not essential for T cell expansion and function. Finally, in a novel in vivo xenotransplant model (UM9 cell line), in which myeloma cells were grown in a humanized bone marrow microenvironment, i.v. as well as intra tumor administration of CD38-CAR T cells established significant anti-tumor effects, proving that CD38-CAR endowed cytotoxic T lymphocytes, even with no CD38 expression, can efficiently migrate, infiltrate and eliminate human MM tumors growing in their natural niche. These results demonstrate the feasibility and potency of CAR mediated targeting of CD38+ MM cells. Optimization of CD38-CAR and suicide-gene control of CD38 CAR T cellsmay provide next steps towards safe clinical implementation of CD38-CAR T cell immune therapy. DisclosuresDrent:Genmab BV: Guest employee (unpaid) Other. Lammerts van Bueren:Genmab : Employment. Parren:Genmab: Employment, Equity Ownership. van de Donk:Genmab BV: Research Funding; J&J: Research Funding; Celgene: Research Funding. Martens:Genmab BV: Research Funding; J&J: Research Funding; Celgene: Research Funding. Lokhorst:Celgene: Research Funding; J&J: Research Funding; Genmab: Research Funding. Mutis:Celgene: Research Funding; J&J: Research Funding; Genmab BV: Research Funding.

A Novel Chimeric Antigen Receptor Against Prostate Stem Cell Antigen Mediates Tumor Destruction in a Humanized Mouse Model of Pancreatic Cancer

Despite advances in the understanding of its molecular pathophysiology, pancreatic cancer remains largely incurable, highlighting the need for novel therapies. We developed a chimeric antigen receptor (CAR) specific for prostate stem cell antigen (PSCA), a glycoprotein that is overexpressed in pancreatic cancer starting at early stages of malignant transformation. To optimize the CAR design, we used antigen-recognition domains derived from mouse or human antibodies, and intracellular signaling domains containing one or two T cell costimulatory elements, in addition to CD3zeta. Comparing multiple constructs established that the CAR based on human monoclonal antibody Ha1-4.117 had the greatest reactivity in vitro. To further analyze this CAR, we developed a human pancreatic cancer xenograft model and adoptively transferred CAR-engineered T cells into animals with established tumors. CAR-engineered human lymphocytes induced significant antitumor activity, and unlike what has been described for other CARs, a second-generation CAR (containing CD28 cosignaling domain) induced a more potent antitumor effect than a third-generation CAR (containing CD28 and 41BB cosignaling domains). While our results provide evidence to support PSCA as a target antigen for CAR-based immunotherapy of pancreatic cancer, the expression of PSCA on selected normal tissues could be a source of limiting toxicity.

Abstract B8: Adoptive immunotherapy targeting tumor heterogeneity in high-grade glioma (HGG)

Abstract Background: Highly selective targeted T cell therapies using chimeric antigen receptors (CAR) are emerging as safe and effective modalities for the treatment of cancer. Downregulation and/or mutation of targeted antigens is a common tactic used by cancer cells to create antigen loss escape variants; culminating in relapse. Hence, targeting two or more antigens on tumor cells simultaneously could result in better therapeutic efficacy. However, developing two separate cellular products for clinical use as combination therapy is difficult, owing to regulatory hurdles and cost. In contrast, rendering an individual T cell bispecific could increase target cell selectivity, improve T cell activation and offset tumor escape due to antigen loss. This approach could particularly be applicable in heterogeneous tumors like HGG. We have characterized and quantified the heterogeneity of antigen expression in HGG tissues and primary cell lines using immunofluorescent staining as well as flow cytometry. While dual targeting was clearly superior to single targeting, targeting of three antigens (or more) did not predict a significantly superior advantage in our patient cohort (Hegde et al. Mol Therapy 2013). Objective: The intent of this project is to develop an effective adoptive cell therapy for HGG using T cells genetically modified to express a bispecific CAR that incorporates two scFv antigen-recognition domains, in tandem (TanCAR), simultaneously targeting two glioma-restricted antigens, HER2 and IL-13Rα2. Methods: A model for the bispecific TanCAR was constructed using ModWeb as described previously by us (Grada and Hegde et al., Mol Ther Nucleic Acids 2013). Extracellular-domain was assembled on Clone Manager®, then cloned into the Gateway® entry vector pDONR™221 and sequence-verified. This antigen recognition domain was then cloned inline with a signaling domain of the co-stimulatory molecule, CD28 and T cell receptor ζ-chain. We used a retroviral system to graft T cells with HER2 and IL-13Rα2-specific TanCAR and confirmed their surface expression using flow cytometry. Their functionality was tested against plate-bound HER2 and IL-13Rα2 proteins as well as against HGG cells in co-cultures and cytotoxicity assays. Cytokine release was studied using ELISA. HER2-specific, IL-13Rα2-specific CAR T cells and non transduced (NT) T cells from the same donor served as controls. Results: The extracellular domain of the TanCAR includes an IL-13Rα2 scFv followed by a Gly-Ser linker, a HER2 specific scFv (FRP5) and another Gly-Ser tandem repeat hinge. The intracytoplasmic domain consists of a CD28/CD3ζ signaling-moiety. Fifty to 80% of T cells expressed HER2/IL-13Rα2 TanCAR on the cell surface. In Cr51 cytotoxicity assays, TanCAR-grafted bispecific T cells showed improved killing of IL-13Rα2 and HER2 positive HGG cell lines as well as autologous HGG cells over the control T cells from the same donor. In co-cultures with autologous HGG cells, HER2/IL-13Rα2 TanCAR T cells proliferated and released immunostimulatory cytokines. Though HER2/IL-13Rα2 TanCAR T cells were effectively activated and secreted IFN-γ and IL-2 on recognizing HER2 and IL-13Rα2 proteins individually, cytokine secretion was significantly higher on simultaneous exposure to both target antigens. Conclusion: The pattern of heterogeneity in HGG favors near complete targeting of tumor subpopulations using bispecific T cell approach. Simultaneous targeting of two glioma-restricted antigens using TanCAR modified T cells could improve T cell activation and can potentially be used to offset antigen escape variants and reduce tumor recurrence. Citation Format: Meenakshi Hegde, Amanda Corder, Zakaria Grada, Tiara T. Byrd, Vita S. Brawley, Helen E. Heslop, Stephen Gottschalk, Nabil Ahmed. Adoptive immunotherapy targeting tumor heterogeneity in high-grade glioma (HGG). [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B8.

Abstract LB-199: A rationally designed bispecific chimeric antigen receptor molecule that simultaneously redirects T cells to target HER2 and GD2 in osteosarcoma

Abstract Tumor immunotherapy with chimeric antigen receptor (CAR)-modified T cells has shown substantial efficacy in early phase clinical trials. Targeting a single tumor antigen could however result in creation of antigen escape variants. One strategy to overcome this immune evasion mechanism is to simultaneously target two or more tumor restricted antigens (TAAs). We have created a novel bispecific CAR molecule that can effectively target HER2 and GD2 in osteosarcoma (OS), using in silico modeling. The extracellular domain of the bispecific CAR consists of two distinct antigen-recognition domains linked by a Gly-Ser linker; one for HER2 (scFv derived from monoclonal antibody FRP5) and the other for GD2 (scFv derived from monoclonal antibody 14g2a), joined in tandem (TanCAR). This extracellular-domain was assembled on Clone Manager®, then cloned into the Gateway® entry vector pDONR™221, sequence-verified and cloned inline with a signaling domain of the co-stimulatory molecule, CD28 and T-cell receptor zeta-chain. Healthy donor and patients’ T cells were transduced with HER2/GD2 TanCAR using a retroviral system. Surface expression of the TanCAR extracellular domain in its entirety was confirmed by flowcytometry using a HER2-scFv and another GD2-scFv specific method. We studied the varying expression pattern of HER2 and GD2 in 12 OS cell lines; e.g. LM7 was HER2/GD2 +/+, HOS HER2/GD2 +/- and IOR/OS9 was HER2/GD2 -/+. Burkitt's lymphoma cell line Raji, was used as a HER2/GD2 -/- control. TanCAR T cells released immunostimulatory cytokines (IL-2, IFN-γ) in co-cultures with tumor cells as detected by ELISA. In standard 51Cr cytotoxicity assays, TanCAR T cells distinctly recognized and killed HER2 and GD2 expressing OS cells as well as tumor cells expressing either HER2 or GD2 only. Importantly, bispecific TanCAR T cells attained full activation only upon encounter of both target antigens simultaneously yet they sustained their anti-tumor activity at low T-cell to tumor cell ratios and at modest or absent expression of either. CONCLUSION: The described bispecific single chimeric antigen receptor molecule (TanCAR) could widen the therapeutic application of a T-cell product by recognition of either target antigens (the Boolean OR), offsetting antigen escape. Importantly, if could improve selective full T cell activation (the Boolean AND) to maintain safety by minimizing off-target effects. Citation Format: Meenakshi G. Hegde, Amanda Corder, Tiara T. Byrd, Kristen Fousek, Helen E. Heslop, Stephen Gottschalk, Eric Yvon, Gianpietro Dotti, Nabil Ahmed. A rationally designed bispecific chimeric antigen receptor molecule that simultaneously redirects T cells to target HER2 and GD2 in osteosarcoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-199. doi:10.1158/1538-7445.AM2014-LB-199

Genetic modification of T cells with a novel bispecific chimeric antigen receptor to enhance the control of high-grade glioma (HGG).

10027 Background: Highly targeted immunotherapy with adoptively transferred chimeric antigen receptor (CAR) T cells has shown considerable efficacy against some refractory childhood malignancies. Downregulation/mutation of targeted antigen can however create antigen loss escape variants. Multispecific T-cell approach could therefore further improve their therapeutic efficacy. Methods: We created a novel bispecific CAR that incorporates two extracellular antigen-recognition domains; one for HER2 (from HER2 monoclonal antibody FRP5) and the other for IL-13Rα2 (a mutated IL-13 molecule), in tandem (TanCAR). TanCAR was rationally designed by in silico modeling. Intracellular domain consists of a CD28/zeta-signaling chain. TanCAR encoding DNA was then synthesized and force expressed on T cells using retroviral system. Surface expression of extracellular domain of the TanCAR in its entirety was confirmed using HER2/IL-13Rα2 specific methods on flowcytometry. Functionality of TanCAR T cells was evaluated using s...

Chimeric Antigen Receptor Modified T Cells Directed Against CD19 (CTL019 cells) Have Long-Term Persistence and Induce Durable Responses In Relapsed, Refractory CLL

BackgroundChimeric antigen receptors (CARs) combine the antigen recognition domain of an antibody with intracellular signaling domains into a single chimeric protein. CD19 is an ideal target for CARs since expression is restricted to normal and malignant B cells. Inclusion of the CD137 (4-1BB) signaling domain results in potent antitumor activity and in-vivo persistence of anti-CD19 CAR-modified T cells in mice. Lentiviral transduction into T cells facilitates strong surface expression of the CAR. We reported anti-tumor activity of CAR-modified autologous T cells targeted to CD19 (CTL019 cells) in 3 patients (pts) with CLL with relatively short follow up (Porter, et al NEJM 2011; Kalos et al Sci Trans Med 2011). We now report on outcomes and longer follow up from our pilot study treating 14 pts with relapsed, refractory CLL. MethodsAutologous T cells collected by leukapheresis were transduced with a lentivirus encoding anti-CD19 scFv linked to 4-1BB and CD3-ζ signaling domains. Gene-modified T cells were expanded and activated ex-vivo by exposure to anti-CD3/CD28 beads. Pts had to have relapsed or persistent disease after at least 2 previous treatments (1 prior therapy for patients with p53 mutation) and progressed at least within 2 years of their last therapy. All pts received lymphodepleting chemotherapy ending 3-5 days before T cell infusion. The target dose of cells was 5 x 109 mononuclear cells with an expected transfection efficiency of 10-40% (total CTL019 dose 5x108 – 2 x 109 total cells). Cell infusions were planned over 3 days (10% on day 1, 30% of day 2, and 60% on day 3) but were held for fevers or other toxicity. Results14 patients were treated on this pilot study including 12 men and 2 women with a median age of 67 (51-78). Pts had received a median of 4 prior therapies (1-10) and 6 pts had a mutation of p53. All pts had active disease at the time of CTL019 cell infusion. Lymphodepleting chemotherapy was Fludarabine/cyclophosphamide (3), pentostatin/cyclophosphamide (5), or bendamustine (6). A median of 7.5 x 108 total cells (range 1.7-50), corresponding to 1.4 x 108(range 0.14-5.9) genetically modified cells were infused over day 0, 1 and 2.There were no infusional toxicities >grade 2 though 6 pts developed fevers within 24 hrs of infusion #1 (3) or #2 (3) and did not receive additional CTL019 cells. Median follow-up as of July 15, 2013 was 9.4 mo (4-35) for all pts and 16 mo (5-35) for the 8 responding pts. 3 patients (21%) achieved a CR (follow-up 11, 34, and 35 mo), 5 (36%) achieved a PR (med follow up 11 mo, range 5-27 mo) and 6 (43%) had no response, for an overall major response rate of 57%. 2 of 5 pts with a PR progressed 4 mo after infusion with CD19+ CLL, and no patient with a CR has relapsed.Comparing responders to non-responders, there has been no association between response and patient age (66 vs 67 yrs), number of prior therapies (median 4 each), cell dose (7.5 vs 11.5 x 108MNC), or p53 mutation (3/8 vs 3/6, p>0.9), implying that within the dose ranges studied, there is no obvious dose:response relationship.All responding pts developed a delayed cytokine release syndrome (CRS), concurrent with peak T cell expansion, and was manifested by fever, and variable degrees of nausea, anorexia, myalgias, and transient hypotension and hypoxia. Detailed cytokine analysis showed marked increases from baseline values of IL6, IFN-γ, and IL2R, while no significant elevation in systemic levels of TNFα or IL2 were observed. The CRS required intervention in 5 patients. Treatment was initiated for hemodynamic or respiratory instability and was rapidly reversed in all cases with corticosteroids in 1 pt and the IL6-receptor antagonist tocilizumab (4 pts); 3 of these 4 pts also received 1 or 2 doses of corticosteroids. Persistence of CTL019 cells has been detected by flow cytometry in all 6 pts with ongoing responses 5-35 months after infusion, and all patients have sustained B cell aplasia without any unusual infectious complications. ConclusionsCTL019 cells are autologous T cells genetically engineered to express an anti-CD19 scFv coupled to 4-1BB/CD3-ζ signaling domains. These cells can undergo robust in-vivo expansion and can persist for at least 3 yrs. CTL019 therapy is associated with a significant CRS that responds rapidly to anti-cytokine treatment. CTL019 cells can induce potent and sustained responses (8/14) for patients with advanced, relapsed and refractory CLL regardless of p53 mutation status. Disclosures:Porter:Novartis: Patents & Royalties, Research Funding; Genentech: Spouse employment, Spouse employment Other. Off Label Use: CTL019 cells to treat CLL. Kalos:Adaptive biotechnologies: Member scientific advisory board , Member scientific advisory board Other; Novartis corporation: CART19 technology, CART19 technology Patents & Royalties. Grupp:Novatis: Research Funding. Lledo:Novartis: Research Funding. Chew:Novartis: Patents & Royalties. Zheng:Novartis: Patents & Royalties. Levine:Novartis: cell and gene therapy IP, cell and gene therapy IP Patents & Royalties. June:Novartis: Patents & Royalties, Research Funding.

A phase II, dose-optimization trial of autologous T cells genetically engineered to express anti-CD19 chimeric antigen receptor (CART-19) in patients with relapsed or refractory (r/r) CD19+ chronic lymphocytic leukemia (CLL).

TPS7132 Background: The poor prognosis and lack of effective treatment options for patients with r/r CLL highlight the need for novel therapies in this setting. CD19 is a promising anticancer target because it is broadly expressed on normal and malignant B cells through several stages of maturation but absent on pluripotent stem cells. CART-19 (CTL019) therapy involves adoptive transfer of autologous T cells genetically modified via lentiviral transduction to express chimeric antigen receptors (CAR) designed to target CD19+ cells. CART-19 cells express a CD19 antigen recognition domain combined with intracellular signaling domains, CD137 (4-1BB) and CD3-zeta, which mediate cytolytic T-cell activity. In patients with r/r CLL, CART-19 therapy showed potent antileukemic activity with long-term persistence of transduced cells at doses from 1.4 × 107 to 1.1 × 109 CART-19 cells. As of August 2012, 3 of 8 evaluable CLL patients achieved CR (two > 24 months and one > 5 months) and remain in CR with detectable CART-19 cells. Two patients had PR lasting 3 and 5 months, and 3 patients did not respond (Porter et al. NEJM. 2011; Kalos et al. Sci Transl Med. 2011; Porter et al. ASH 2012). Here, we describe a study to determine the optimal dose of CART-19 cells (NCT01747486). Methods: Adults with relapsed or persistent CLL or SLL after ≥ 2 previous therapies will undergo leukapheresis to obtain T cells, which will be stimulated, expanded, and lentivirus transduced ex vivo to express the CD19/4-1BB/CD3-zeta CAR. Patients will undergo lymphodepletion chemotherapy prior to infusion of CART-19 cells. In stage I of the II-stage trial, 30 patients will be randomized 1:1 and receive either 1-5 × 108 or 1-5 × 107 CART-19 T cells. The optimal dose in stage I will be selected based on clinical responses, feasibility, and tolerability. In stage II, 8 additional patients will be enrolled into the selected dose cohort. Study objectives are to determine the efficacy (CR rate within 3 months) and safety (CTCAE v 4.0) of each dose, in vivo CART-19 expansion, and manufacturing feasibility. Three patients have been enrolled as of January 2013. Clinical trial information: NCT01747486.

Pilot study of redirected autologous T cells engineered to contain anti-CD19 attached to TCRζ and 4-1BB signaling domains in patients with chemotherapy-resistant or -refractory CD19+ leukemia and lymphoma.

TPS10072 Background: Outcomes remain poor for patients (pts) with relapsed or refractory (r/r) B-cell malignancies such as acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia (CLL). CD19 is an attractive therapeutic target because it is widely expressed on normal and malignant B cells throughout B-cell maturation but not on pluripotent stem cells or non–B-cell tissues. We have developed chimeric antigen receptor T cells to target CD19+ cells (CART-19 or CTL019). This approach involves patient-derived T cells that are genetically modified via lentiviral transduction to express a CD19 antigen recognition domain attached to intracellular signaling domains (TCRζ and 4-1BB) that mediate T-cell activation. A recent study in CLL showed that CART-19 therapy had potent activity with responses in 5/8 evaluable pts (3 CR, 2 PR). Pts achieving CR (two > 24 months and one > 5 months) remain in CR with detectable CART-19 cells (Porter et al. ASH 2012). Here we describe a study of CART-19 therapy in pediatric pts with r/r leukemia and lymphoma (NCT01626495). Methods: Pts eligible for this single-arm, open-label study are aged 1 to 24 years with r/r CD19+ B-cell malignancies, without a transplant option or having relapsed after allogeneic stem cell transplantation (ASCT). Pts will undergo leukapheresis to obtain T cells, which will be stimulated, expanded, and transduced ex vivo to express the chimeric antigen receptor. Pts may receive lymphodepleting chemotherapy prior to CART-19 infusion. Study objectives: determine the safety and feasibility of administering CART-19 therapy to pediatric pts, assess duration of in vivo survival of CART-19 cells, and measure antitumor response. There are 2 cohorts in the study: Cohort 1 includes pts who have not undergone ASCT and are not currently eligible for a transplant, and cohort 2 includes pts who relapsed after ASCT. Cells are collected from the recipient, not the donor, which allows for this approach to be used in pts s/p cord blood transplant, and which we hypothesize will reduce the risk of GVHD as a result of toleration of the T cells in the recipient. To date, 7 pts have been enrolled. Clinical trial information: NCT01626495.

Abstract 4574: Use of CD19-targeted Chimeric Antigen Receptor-modified T (CART19) cells in ALL and CLL produce transient cytokine release syndrome (CRS), macrophage activation syndrome (MAS) and durable responses.

Abstract CART19 combines an antiCD19 scFv antibody antigen recognition domain with intracellular signal 1 (CD3z) and signal 2 (4-1BB) domains into a single chimeric protein, which results in remarkable antitumor activity with massive in vivo expansion and long-term persistence of CART19 cells in NSG mice. We reported dramatic anti-tumor activity of CART19 T cells in 3 pts with CLL with shorter follow up (Porter, et al NEJM 2011). We now report on outcomes, longer follow up, and complete responses in CLL and ALL from 12 pts treated with CART19. 12 pts with relapsed/refractory leukemia have received CART19: 10 adults with CLL and 2 children with ALL (1 s/p cord blood transplant); all had high disease burdens. A median of 7.5x108 total cells (range 1.7-50; 1.5x108 CAR+ [0.1-5.9]) were infused. Of 10 CLL pts, 1 is not yet evaluable, 7 had a CR or PR, including 3 sustained CRs and 2 complete BM responses. Both ALL patients had a CR, one sustained for 6mo. While there was no acute infusional toxicity, all responding pts also developed CRS. All had high fevers, as well as grade 3 or 4 hypotension/hypoxia. CRS preceded the peak of CART19 cells, and then increased in intensity until the peak (D10-70 post infusion). One ALL pt experienced grade 4 hypotension and respiratory failure and steroid therapy produced no improvement. On D9, with high levels of TNFa and IL6 (peak increases above baseline: IFNg=6040x; IL6=988x; IL2=163x and TNFa=17x), we gave TNFa and IL6 antagonists etanercept and tocilizumab. This resulted in resolution of fever and hypotension within 12hr and rapid wean off the ventilator. This had no apparent impact on CART19 cell expansion or efficacy, with a peak of CAR T cells (2539 CAR+ cells/uL; 77% of CD3 cells) occurred on D11, and D23 BM showed CR with negative MRD, compared to 65% ALL in her on-study BM. Clinical assessment of patients with major responses showed all had evidence of MAS including dramatic elevations of ferritin, peaking at 44,000 to 605,000 around the time of peak T cell proliferation. Subsequently, 3 CLL patients have also been treated with tocilizumab, also with prompt and striking resolution of symptoms. Interestingly, both ALL pts showed detectable CART19 cells in the CSF in each post-treatment sample (40-78% CAR+). CART19 cells and B cell aplasia have persisted in patients who achieved a CR out to the longest follow up (beyond 2yrs in 2 CLL pts)CART19 T cells may produce massive in-vivo expansion, long-term persistence, and potent and sustained responses, but can also cause significant CRS with features suggestive of MAS that responds rapidly to cytokine blockade. Prior to the CART19 proliferative phase IL6 blockade may interfere with proliferation and effector function, but if given at a point where cell proliferation is underway, it may ameliorate the symptoms that correlate with robust clinical responses. Citation Format: Stephan A. Grupp, Michael Kalos, David M. Barrett, David T. Teachey, Bruce Levine, Michael Milone, David Porter, Carl H. June. Use of CD19-targeted Chimeric Antigen Receptor-modified T (CART19) cells in ALL and CLL produce transient cytokine release syndrome (CRS), macrophage activation syndrome (MAS) and durable responses. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4574. doi:10.1158/1538-7445.AM2013-4574

Abstract 3966: Development of a fully-human chimeric antigen receptor against PSCA for the treatment of pancreatic cancer.

Abstract Pancreatic cancer represents the fourth leading cause of cancer-related death in developed countries. Despite remarkable advances in the understanding of its molecular pathophysiology, treatment options remain scarce, highlighting the need for novel therapies. Adoptive cell transfer (ACT) of lymphocytes engineered ex vivo to express chimeric antigen receptors (CAR) has come of age as a safe and clinically efficacious treatment for advanced cancers. We sought to develop a CAR specific for prostate stem cell antigen (PSCA), a glycoprotein with restricted expression in normal tissues that is over-expressed in pancreatic cancer cells starting at early stages of malignant transformation. In order to establish the optimal CAR design, we generated constructs containing antigen-recognition domains derived from mouse or human antibodies, and intracellular domains containing one or two co-stimulatory domains, in addition to CD3zeta. We compared multiple constructs and found that a CAR based on human monoclonal antibody Ha1-4.117 had greater reactivity in vitro. Using a murine model of ACT we observed that CAR-engineered human lymphocytes induced an antitumor effect on HPAC xenografts. Interestingly, unlike what has been described for other CARs, a second generation PSCA CAR (containing CD28 signaling domain) induced a more potent antitumor effect than its third generation counterpart (containing CD28 and 41BB domains). Our results provide evidence to support PSCA as a target antigen for immunotherapy of pancreatic cancer, using adoptively transferred autologous lymphocytes engineered to express an Ha1-4.117-based CAR. Citation Format: Daniel Abate-Daga, Kiran H. Lagisetty, Luca Gattinoni, Zhiya Yu, Nicholas P. Restifo, Steven A. Rosenberg, Richard A. Morgan. Development of a fully-human chimeric antigen receptor against PSCA for the treatment of pancreatic cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3966. doi:10.1158/1538-7445.AM2013-3966

Chimeric Antigen Receptor (CAR)-Specific Monoclonal Antibody to Detect CD19-Specific T Cells in Clinical Trials

Clinical trials targeting CD19 on B-cell malignancies are underway with encouraging anti-tumor responses. Most infuse T cells genetically modified to express a chimeric antigen receptor (CAR) with specificity derived from the scFv region of a CD19-specific mouse monoclonal antibody (mAb, clone FMC63). We describe a novel anti-idiotype monoclonal antibody (mAb) to detect CD19-specific CAR+ T cells before and after their adoptive transfer. This mouse mAb was generated by immunizing with a cellular vaccine expressing the antigen-recognition domain of FMC63. The specificity of the mAb (clone no. 136.20.1) was confined to the scFv region of the CAR as validated by inhibiting CAR-dependent lysis of CD19+ tumor targets. This clone can be used to detect CD19-specific CAR+ T cells in peripheral blood mononuclear cells at a sensitivity of 1∶1,000. In clinical settings the mAb is used to inform on the immunophenotype and persistence of administered CD19-specific T cells. Thus, our CD19-specific CAR mAb (clone no. 136.20.1) will be useful to investigators implementing CD19-specific CAR+ T cells to treat B-lineage malignancies. The methodology described to develop a CAR-specific anti-idiotypic mAb could be extended to other gene therapy trials targeting different tumor associated antigens in the context of CAR-based adoptive T-cell therapy.

Abstract IA16: Engineered T cell therapies for cancer.

Abstract Chimeric antigen receptors (CARs) combine an antigen recognition domain of a specific antibody with a intracellular signaling domains into a single engineered protein. CD19 is an ideal target for CARs since expression is restricted to normal and malignant B cells. In preclinical studies, inclusion of the CD137 (4-1BB) signaling domain results in remarkable antitumor activity and in vivo persistence of anti-CD19 CARs. We have previously reported dramatic in-vivo expansion, persistence and anti-tumor activity of CAR modified autologous T cells targeted to CD19 (CART19 cells) in 3 patients with CLL with relatively short follow up (Porter, et al NEJM 2011; Kalos et al Sci Trans Med 2011). Currently, 11 patients have been treated with CART19 cells. There were 9 adults (median age 65 years (range 51-78)) treated for relapsed, refractory and high risk CLL and two pediatric cases treated for relapsed refractory pre-B cell ALL. CLL patients had received a median of 5 prior regimens excluding single agent rituxumab (range 2-10) and all had active disease at the time of cell infusion. The pediatric patients with ALL had chemotherapy refractory relapse, and one was resistant to allogeneic transplantation and CD19 directed blinotumomab. A median of 7.5 x 10^8 total cells (range 1.7-50) corresponding to 1.45 (range 0.14-5.9 x 108) genetically modified cells were infused. In the patients in complete remission, CAR modified CART19 cells are detectable in the peripheral blood by flow cytometry at the most recent follow up for at least 2 years. All patients in CR have B cell aplasia. No patient with CR has progressed. Citation Format: Carl H. June. Engineered T cell therapies for cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr IA16.

Chimeric Antigen Receptor T Cells Directed Against CD19 Induce Durable Responses and Transient Cytokine Release Syndrome in Relapsed, Refractory CLL and ALL

Chimeric Antigen Receptor T Cells Directed Against CD19 Induce Durable Responses and Transient Cytokine Release Syndrome in Relapsed, Refractory CLL and ALL

A High Molecular Weight Melanoma-Associated Antigen–Specific Chimeric Antigen Receptor Redirects Lymphocytes to Target Human Melanomas

Immunotherapy, particularly the adoptive cell transfer (ACT) of tumor-infiltrating lymphocytes (TIL), is a very promising therapy for metastatic melanoma. Some patients unable to receive TIL have been successfully treated with autologous peripheral blood lymphocytes (PBL), genetically modified to express human leukocyte antigen (HLA) class I antigen-restricted, melanoma antigen-reactive T-cell receptors; however, substantial numbers of patients remain ineligible due to the lack of expression of the restricting HLA class I allele. We sought to overcome this limitation by designing a non-MHC-restricted, chimeric antigen receptor (CAR) targeting the high molecular weight melanoma-associated antigen (HMW-MAA), which is highly expressed on more than 90% of human melanomas but has a restricted distribution in normal tissues. HMW-MAA-specific CARs containing an antigen recognition domain based on variations of the HMW-MAA-specific monoclonal antibody 225.28S and a T-cell activation domain based on combinations of CD28, 4-1BB, and CD3zeta activation motifs were constructed within a retroviral vector to allow stable gene transfer into cells and their progeny. Following optimization of the HMW-MAA-specific CAR for expression and function in human PBL, these gene-modified T cells secreted cytokines, were cytolytic, and proliferated in response to HMW-MAA-expressing cell lines. Furthermore, the receptor functioned in both CD4(+) and CD8(+) cells, was non-MHC restricted, and reacted against explanted human melanomas. To evaluate this HMW-MAA-specific CAR in patients with metastatic melanoma, we developed a clinical-grade retroviral packaging line. This may represent a novel means to treat the majority of patients with advanced melanoma, most notably those unable to receive current ACT therapies.