Single Chimeric Antigen Receptor Research Articles

190. Adaptive CAR T Cell Design

Chimeric antigen receptor (CAR) T cell therapy has recently emerged as an attractive approach for the treatment of CD19-expressing hematological malignancies. However extending the success of this strategy to other targets has proven to be more complicated that simply replacing the scFv. To address this issue we have implemented a form of adaptive CAR design whereby a series of sequential modifications are made to a single domain and subsequently tested in vitro and in vivo to assess activity. We illustrate the utility of such a strategy using our published CAR-PSCA (Anurathapan U. et al, Mol Ther. 2014) as a template (CAR-PSCA v1.0) with subsequent iterations coded as versions 2.0, 3.0, 4.0 and 5.0. We now demonstrate how modifications made to a single CAR structural domain can result in enhanced (i) T cell migration, (ii) antigen recognition, and (iii) cell phenotype, ultimately producing superior anti-tumor effects. First, with CAR v2.0 and v3.0 we were able to improve T cell migration, which was evident in NSG mice engrafted s.c. with Capan-1 and treated i.v. with FFluc+ T cells. Ten days post CAR administration we saw a 2 log increase in the T cell signal at the tumor site (4.5±2.3×105 p/s vs 4.8±0.5×107 p/s vs 4.0±1.1×107 p/s, CAR v1.0, v2.0 and v3.0 respectively). Subsequently to enhance in vivo T cell persistence, we next generated CAR v4.0, which resulted in a less differentiated T cell phenotype (Tnaive: 1.8±0.6% to 19.2±4.0%, TCM: 10.4±1.4% to 14.1±3.0%, TEM: 83.5±1.2% to 53.7±6.9% and TEMRA: 4.3±0.9% to 12.9±1.7% - CAR v3.0 and CAR v4.0, respectively). When administered to Capan-1-engrafted NSG mice CAR v4.0 T cells exhibited enhanced in vivo longevity as measured using bioluminescence imaging (7.3±4.6×107 p/s CAR v3.0 vs 2.8±1.7×108 p/s CAR v4.0 T cells - day 35 post-administration). Finally, antigen recognition of CAR-PSCA was further improved in v5.0 where a final modification to the same domain produced superior anti-tumor effects against a PSCA-dim target tumor cell line (DU145) in a 6hr 51Cr-release assay (20.7±5.8% vs 48.4±5.2%, CAR v4.0 vs CAR v5.0, 40:1 E:T). Overall, therefore, implementation of this adaptive design produced a CART cell product with enhanced in vivo anti-tumor activity. This was clearly illustrated when we compared the tumor volume of NSG mice treated with CAR v1.0 or CAR v5.0 T cells (1309±143 mm3 vs 510±53 mm3 on Day 66). Specific details of the modifications conducted in this adaptive CAR design will be presented.

Preclinical Development of Bispecific Chimeric Antigen Receptor Targeting Both CD19 and CD22

Acute lymphoblastic leukemia (ALL) is the most common childhood malignancy. Despite advances in upfront therapy, relapsed and refractory ALL remains a primary cause of cancer-related mortality in children. Immunotherapy using genetically engineered T cells expressing chimeric antigen receptors (CARs) targeting CD19 has demonstrated dramatic clinical results in children and young adults with pre-B cell ALL with a 70-90% complete remission rate in multiple clinical trials. However, not all patients respond and at least 10% of patients will relapse due to loss of the targeted CD19 epitope. CD22 is another pan-B cell surface antigen that is broadly expressed on ALL blasts and has been targeted successfully using immunoconjugates. We have recently developed a highly active CD22 CAR currently being tested in a clinical trial. Conceptually, dual targeting both CD19 and CD22 on Pre-B ALL with a single bispecific CAR may result in enhanced avidity and increased T cell activation which may result in additive or synergistic responses compared to targeting a single antigen. In addition, since there are heterogeneous expression levels of CD19 and CD22 on leukemic blasts, dual targeting may also provide a more broadly active therapy. Finally, simultaneously targeting of both CD19 and CD22 on the surface of pre-B cell ALL may reduce the likelihood of antigen escape due to downregulation or deletion. We describe the preclinical development of two novel CD19 and CD22 bispecific CARs assembled with CD19 and CD22 single chain fragment variable regions (scFv) binding domains on a single construct. T cells transduced the CD19/CD22 bispecific CARs have comparable CAR surface expression when compared to T cells expressing either the CD19 or CD22 CAR. T cells expressing the bispecific CAR are active in vitro against ALL cell lines expressing both CD19 and CD22 as measured by interferon gamma production and chromium release. In vitro activity is maintained against K562 cells expressing either CD19 or CD22 alone. Interestingly, the order and the linker between the CD19 and CD22 scFvs in the CAR construct has a major impact on the efficacy of the dual CAR with a membrane-distal CD22 binding scFv demonstrating improved efficacy. Finally, in xenograft models both bi-specific CARs efficiently eradicate ALL cell lines and patient derived xenografts. In vivo treatment of a Crisper-knockdown-CD19-negative leukemia line and a CD19-negative patient-derived xenograft is ongoing. In summary, CD19/CD22 bispecific CARs demonstrate robust pre-clinical activity against pre-B cell ALL and provides a strategy to improve the clinical efficacy of CAR therapy. DisclosuresNo relevant conflicts of interest to declare.

Abstract B74: Co-targeting the tumor and its associated vasculature in glioblastoma

Abstract Background: Glioblastoma (GBM) is the most common and most aggressive primary brain tumor in adults. With the current standard treatment, survival is poor with substantial treatment related toxicities underscoring the need for new targeted therapies. Preclinical and early clinical data demonstrate the efficacy of chimeric antigen receptor (CAR)-grafted T-cells as a novel approach against cancer. Previous work from our lab has demonstrated that HER2-specific T cells can induce tumor regression in an orthotopic xenogeneic model. However, tumors recurred in a subset of mice treated with HER2-specific T cells. It is now evident that tumors are heterogeneous cellular complexes whose growth is dependent upon reciprocal interactions between the tumor and its heterogeneous microenvironment. In particular, glioblastomas are highly vascularized tumors, known for their invasive phenotype. Tumor Endothelium Marker 8 (TEM8) is upregulated in tumor angiogenesis and conserved in mice, thus making it a target which can be studied pre-clinically for both safety and efficacy. Objective: The purpose of this study is to determine whether co-targeting the tumor antigen, HER2 and Tumor Endothelium Marker 8 (TEM8) using bispecific CAR T cells will result in improved tumor control in GBM. Methods: In order to generate TEM8 and HER2 bispecific CAR T cells we employed two methods. For the first method we designed a TEM8 CAR, to be coexpressed along with a separate HER2 CAR on the same T cells (TEM8-HER2 BiCAR T cells). For the second method we designed a bispecific tandem CAR (TEM8-HER2 TanCAR) in which TEM8 and HER2 specific single chain variable fragments have been placed in-tandem and are connected by a glycine-serine linker to make a single CAR to be expressed on T cells. CAR constructs were assembled on clone manager, synthesized, then sequence verified and force expressed on T cells using a retroviral system. CAR T cells were expanded in IL-2 in parallel with the control T cells from the same donor. Flow cytometry was then used to confirm CAR expression on T cells and binding specificity. ELISA and flow-based cytotoxicity assays were used to assess selectivity and killing potential of TEM8 and HER2 bispecific T cells. Results: We have successfully cloned in the TEM8 and TEM8-HER2 TanCAR constructs and generated TEM8-HER2 BiCAR T cells TEM8-HER2 specific TanCAR T cells, respectively. Up to 70% of T cells expressed the specific CAR on the cell surface. TEM8 and HER2 specific CAR T cells selectively recognize and kill TEM8 and HER2 positive targets and release the immuno-stimulatory cytokines interferon-gamma and interleukin-2 in vitro. Further, cytokine release was augmented when bispecific T cells were simultaneously exposed to both target targets. Conclusion: Heterogeneous nature of GBM favors co-targeting the tumor and its microenvironment. We have successfully generated the first TEM8 specific CAR T cell product to specifically target the tumor associated vasculature. Co-targeting tumor and its endothelium using bispecific CAR T cells could enhance T cell activation and can potentially be used to improve tumor control and have therapeutic application in GBM patients. Citation Format: Tiara T. Byrd, Kristen Fousek, Zakaria Grada, Kevin Aviles-Padilla, Kevin Bielamowicz, Stephen Gottschalk, Bradley St Croix, Bradley Fletcher, Meenakshi Hegde, Nabil Ahmed. Co-targeting the tumor and its associated vasculature in glioblastoma. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B74. doi:10.1158/1538-7445.CHTME14-B74

Abstract LB-264: Two is better than one: Adoptive T cell therapy targeting tumor antigens and the tumor endothelium potentiates T cell activation and cytolytic activity

Abstract Background: T cells can be genetically modified to express chimeric antigen receptors (CAR) molecules on the surface. These CAR-bearing T cells combine the specificity of an antibody with the killing properties of a T cell for a targeted immunotherapy approach. Previously our lab has targeted the tumor antigen HER2, using HER2 specific CAR T cells. While these CAR T cells induced tumor regression in an orthotopic xenogeneic model, a subset of mice went on to recur. Tumor endothelium marker 8 (TEM8), is a tumor endothelium associated antigen that is conserved in mice and humans. Purpose: The purpose of this study is to determine whether CAR T cells specific for both TEM8 and HER2 would display increased cytolytic activity and/or activation. Procedure: We designed in silico, a single CAR molecule composed of TEM8 and HER2 specific exodomains in tandem. These domains were linked together via a glycine serine linker and shared a CD28 and CD3-zeta chain signaling domain, to form a TEM8-HER2 TanCAR. The construct was then synthesized, cloned into a retroviral vector and used to transduced T cells. New results: TanCARs were expressed on >90% of primary T cells; up 20% from a previous report of 70%. When tested against TEM8 and HER2 double negative, single positive and double positive cell lines TanCAR T cells were able to distinctly recognize and TEM8 and HER2 positive targets as indicated by standard 4 hour 51Cr release assays. Particularly, when TanCAR cells were cultured with TEM8 and HER2 double positive cell lines versus single positive (TEM8+ or HER2+) there was a 10-14% increase in cytolytic activity. Increased T cell activation was also indicated by the release of the immune-stimulatory cytokines interferon-gamma and interleukin-2 in in vitro coculture assays. Forced expression of TEM8 on the same HER2 positive cell line resulted in a >3 fold increase in release of interferon-gamma. Further, IL-2 release was only detected when TanCAR T cells were exposed to cell lines positive for both antigens. Conclusion: The distinct recognition and increased cytolytic activity and T cell activation demonstrated by TEM8-HER2 specific TanCAR T cells upon exposure to two target antigens warrants further investigation. This single CAR molecule could potentially have therapeutic benefit in targeting not only the tumor, but associated vasculature in HER2 positive solid tumors. Citation Format: Tiara Byrd, Kristen Fousek, Zakaria Grada, Kevin Aviles-Padilla, Kevin Bielamowicz, Stephen Gottschalk, Bradley St Croix, Bradley Fletcher, Meenakshi Hegde, Nabil Ahmed. Two is better than one: Adoptive T cell therapy targeting tumor antigens and the tumor endothelium potentiates T cell activation and cytolytic activity. [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-264. doi:10.1158/1538-7445.AM2014-LB-264

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

Quantitative single-cell functional characterization of CD19-specific chimeric antigen receptor T cells (P4344)

Abstract Adoptive cell therapy (ACT) utilizing chimeric antigen receptor (CAR) T cells rendered specific for CD19 have demonstrated significant anti-tumor effects in patients with CD19+ chronic lymphocytic leukemia (CLL). In spite of the clinical promise of ACT in achieving complete responses, their efficacy remains unpredictable and new approaches are needed to a priori define the therapeutic potential of T-cell based therapies. In our current work, we characterize the in vitro functionality of CD19-specific (CD19RCD28) CAR+ T cells propagated using artificial antigen presenting cells, by employing a novel methodology single-cell nanowell screening (SNS).We show that CAR+ T cells exert specific cytotoxicity against NALM6 cells (31 ± 8 %) when co-incubated at a 1:1 ratio in nanowell containers. Furthermore, single CAR+ T cells were capable of engaging and killing multiple targets; 17 ± 8% of T cells killed two target cells within the 6 hour window of observation. Hierarchical clustering indicated that interferon-gamma (IFNγ) secretion is not correlated to cytotoxicity of T cells. CAR+ T cells that secreted IFNγ upon target ligation did not undergo activation-induced cell death (AICD) whereas T cells that engaged in repeated killing showed an increased propensity to undergo AICD (p = 0.04). In summary, our SNS based methodology allows the deep functional characterization of clinical grade CAR+ T cells and can be used to determine in vitro functions of CAR+ T cells.

Abstract 4745: Quantitative single-cell functional characterization of CD19-specific CAR+ T Cells for immunotherapy.

Abstract Adoptive cell therapy (ACT) utilizing chimeric antigen receptor (CAR) T cells rendered specific for CD19 have demonstrated significant anti-tumor effects in patients with CD19+ chronic lymphocytic leukemia (CLL) that were considered untreatable, including the use of stem-cell transplantation. In spite of the clinical promise of ACT in achieving complete responses, their efficacy remains unpredictable and new approaches are needed to address a priori define the therapeutic potential of T-cell based therapies. In our current work, we characterize the in vitro functionality of CD19-specific (CD19RCD28) CAR+ T cells propagated using artificial antigen presenting cells expressing membrane bound IL-21, by employing a novel methodology single-cell nanowell screening that determines their cytotoxic ability and cytokine secretion capability at single-cell resolution. We show that CAR+ T cells exert specific cytotoxicity against NALM6 cells (31 ± 8 %) when co-incubated at a 1:1 ratio in nanowell containers. Furthermore, single CAR+ T cells were capable of engaging and killing multiple targets; 17 ± 8% of T cells killed two target cells and 9 ± 3% killed three target cells within the 6 hour window of observation. In parallel, microengraving was used to determine the cytokine secretion profile of these same cells. Hierarchical clustering of the two functions indicated that interferon-gamma (IFNγ) secretion is not correlated to cytotoxicity or the ability of T cells to kill multiple target cells. Simultaneously, monitoring apoptosis on CAR+ T cells allowed us to quantify their activation-induced cell death (AICD). CAR+ T cells that secreted IFNγ upon target ligation did not undergo AICD whereas T cells that engaged in repeated killing showed an increased propensity to undergo AICD (p = 0.04). Finally, automated time-lapse microscopy of hundreds of these effector target interactions allowed us to quantify the kinetics of effector mediated lysis and effector cell velocity profiles before and after lysis. These analyses demonstrate that individual CAR+ T cells demonstrate vastly different individual killing profiles, at least in vitro. In summary, our SNS based methodology allows the deep functional characterization of clinical grade CAR+ T cells and can be used to determine in vitro functions of CAR+ T cells that correlate with clinical efficacy. Citation Format: Navin Varadarajan, Ivan Liadi, Gabrielle Romain, Harjeet Singh, Laurence J.N. Cooper. Quantitative single-cell functional characterization of CD19-specific CAR+ T Cells for immunotherapy. [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 4745. doi:10.1158/1538-7445.AM2013-4745