Kristen Fousek

Tandem CAR T cells targeting HER2 and IL13Rα2 mitigate tumor antigen escape

In preclinical models of glioblastoma, antigen escape variants can lead to tumor recurrence after treatment with CAR T cells that are redirected to single tumor antigens. Given the heterogeneous expression of antigens on glioblastomas, we hypothesized that a bispecific CAR molecule would mitigate antigen escape and improve the antitumor activity of T cells. Here, we created a CAR that joins a HER2-binding scFv and an IL13Rα2-binding IL-13 mutein to make a tandem CAR exodomain (TanCAR) and a CD28.ζ endodomain. We determined that patient TanCAR T cells showed distinct binding to HER2 or IL13Rα2 and had the capability to lyse autologous glioblastoma. TanCAR T cells exhibited activation dynamics that were comparable to those of single CAR T cells upon encounter of HER2 or IL13Rα2. We observed that TanCARs engaged HER2 and IL13Rα2 simultaneously by inducing HER2-IL13Rα2 heterodimers, which promoted superadditive T cell activation when both antigens were encountered concurrently. TanCAR T cell activity was more sustained but not more exhaustible than that of T cells that coexpressed a HER2 CAR and an IL13Rα2 CAR, T cells with a unispecific CAR, or a pooled product. In a murine glioblastoma model, TanCAR T cells mitigated antigen escape, displayed enhanced antitumor efficacy, and improved animal survival. Thus, TanCAR T cells show therapeutic potential to improve glioblastoma control by coengaging HER2 and IL13Rα2 in an augmented, bivalent immune synapse that enhances T cell functionality and reduces antigen escape.

The Evolution of T-cell Therapies for Solid Malignancies

Primary resistant, recurrent, and relapsed solid tumors are often nonresponsive to conventional antineoplastic therapies. Moreover, in responsive tumors, the therapeutic-to-toxic range of these interventions remains quite narrow, such that side effects of therapy are substantial. Targeted therapies, such as adoptive T-cell transfer, not only spare normal tissues but also use alternative killing mechanisms to which the tumor cells are usually not immune. Adoptive T-cell transfer for solid tumors faces unique challenges because of the inherent heterogeneity of tumor parenchyma, the complexity of the tumor microenvironment, and tumor occurrence in areas with limited therapeutic accessibility. In this review, we examine the recent evolution of various T-cell–based immunotherapeutics, the mechanisms of action behind their antitumor activity, their increasing complexity, and the prospect of building on previous successes in the treatment of solid tumors. Clin Cancer Res; 21(15); 3384–92. ©2015 AACR.

TEM8/ANTXR1-specific CAR T cells as a targeted therapy for triple-negative breast cancer

Triple-negative breast cancer (TNBC) is an aggressive disease lacking targeted therapy. In this study, we developed a CAR T cell-based immunotherapeutic strategy to target TEM8, a marker initially defined on endothelial cells in colon tumors that was discovered recently to be upregulated in TNBC. CAR T cells were developed that upon specific recognition of TEM8 secreted immunostimulatory cytokines and killed tumor endothelial cells as well as TEM8-positive TNBC cells. Notably, the TEM8 CAR T cells targeted breast cancer stem-like cells, offsetting the formation of mammospheres relative to nontransduced T cells. Adoptive transfer of TEM8 CAR T cells induced regression of established, localized patient-derived xenograft tumors, as well as lung metastatic TNBC cell line-derived xenograft tumors, by both killing TEM8+ TNBC tumor cells and targeting the tumor endothelium to block tumor neovascularization. Our findings offer a preclinical proof of concept for immunotherapeutic targeting of TEM8 as a strategy to treat TNBC.Significance: These findings offer a preclinical proof of concept for immunotherapeutic targeting of an endothelial antigen that is overexpressed in triple-negative breast cancer and the associated tumor vasculature. Cancer Res; 78(2); 489-500. ©2017 AACR.

A bispecific chimeric antigen receptor molecule enhances T cell activation through dual immunological synapse formation and offsets antigen escape in glioblastoma

Meeting abstracts Antigen escape tumor cell variants prevail in tumors recurring after treatment with chimeric antigen receptor (CAR) T cells with a single specificity. Recurrent tumors preserve alternative non-targeted tumor associated antigens. A bispecific CAR will mitigate antigen escape

A homing system targets therapeutic T cells to brain cancer

Successful T cell immunotherapy for brain cancer requires that the T cells can access tumour tissues, but this has been difficult to achieve. Here we show that, in contrast to inflammatory brain diseases such as multiple sclerosis, where endothelial cells upregulate ICAM1 and VCAM1 to guide the extravasation of pro-inflammatory cells, cancer endothelium downregulates these molecules to evade immune recognition. By contrast, we found that cancer endothelium upregulates activated leukocyte cell adhesion molecule (ALCAM), which allowed us to overcome this immune-evasion mechanism by creating an ALCAM-restricted homing system (HS). We re-engineered the natural ligand of ALCAM, CD6, in a manner that triggers initial anchorage of T cells to ALCAM and conditionally mediates a secondary wave of adhesion by sensitizing T cells to low-level ICAM1 on the cancer endothelium, thereby creating the adhesion forces necessary to capture T cells from the bloodstream. Cytotoxic HS T cells robustly infiltrated brain cancers after intravenous injection and exhibited potent antitumour activity. We have therefore developed a molecule that targets the delivery of T cells to brain cancer.Therapeutic T cells bearing ligands engineered to optimize adhesion and transmigration through the blood–brain barrier can be targeted to brain tumours.

Abstract A25: TEM8 specific CAR T cells induce regression of patient-derived xenograft and metastatic models of triple-negative breast cancer

Lacking marked expression of human epidermal growth factor receptor 2 (HER2), estrogen receptor (ER), and progesterone receptor (PR), triple-negative breast cancer (TNBC) is a breast cancer subtype in desperate need of targeted therapy options. Tumor endothelial marker 8 (TEM8), initially identified as a tumor endothelium marker in colon cancer, has been shown to be upregulated in TNBC. To confirm this, we stained primary TNBC tissues for TEM8; in all cases TEM8 was expressed with no expression in normal breast tissue. TEM8 is expressed by TNBC cell lines as indicated by flow cytometry and Western blot. We thus engineered chimeric antigen receptor (CAR) T cells to specifically target TEM8 in TNBC. TEM8 CAR T cells distinctly recognized TEM8, secreted immunostimulatory cytokines, and killed TEM8-positive TNBC cells in vitro. In vivo, the adoptive transfer of TEM8 CAR T cells induced regression against orthotopic patient-derived xenograft (PDX) models, including the aggressive claudin-low TNBC PDX, WHIM12. Systemic administration of TEM8 CAR T cells also induced regression against a lung metastasis TNBC model. In all models, treatment with TEM8 CAR T cells resulted in a survival advantage in mice compared to controls. Hence, TEM8 may serve as an attractive targeted immunotherapy of TNBC. Citation Format: Tiara Byrd, Kristen Fousek, Antonella Pignata, Christopher Szot, Heba Samaha, Lacey Dobrolecki, Htoo Zarni Oo, Poul Sorensen, Matthew Ellis, Michael Lewis, Meenakshi Hegde, Bradley Fletcher, Brad St. Croix, Nabil Ahmed. TEM8 specific CAR T cells induce regression of patient-derived xenograft and metastatic models of triple-negative breast cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A25.

Abstract 2312: TEM8/ANTXR1 specific T cells co-target tumor stem cells and tumor vasculature in triple-negative breast cancer

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with no approved targeted therapies. Tumor endothelial marker 8 (TEM8), initially identified as a marker of tumor endothelial cells in colorectal cancer and other solid tumors has recently been shown to be upregulated in TNBC and breast cancer stem cells (BCSCs). We investigated whether TEM8 specific chimeric antigen receptor (CAR) T cells recognize and kill both tumor endothelial cells as well as TNBC tumor cells. TEM8 specific CAR molecules were generated using single chain variable fragment derived from the monoclonal antibody, L2. L2 CAR T cells selectively recognized TEM8, secreted immunostimulatory cytokines and effectively killed both TEM8 positive TNBC and tumor endothelial cell lines. Moreover, L2 CAR T cells targeted breast cancer stem cells significantly reducing the number of mammospheres relative to non-transduced T cells. In vivo, adoptive transfer of L2 CAR T cells induced regression of established vascularized TNBC xenografts. Hence, TEM8 may serve as an attractive target for immunotherapy of TNBC. Citation Format: Tiara Byrd, Kristen Fousek, Antonella Pignata, Christopher Szot, Kevin Bielamowicz, Steven Seaman, Daniel Landi, Nino Rainusso, Poul Sorensen, Joachim Koch, Winfried Wels, Bradley Fletcher, Meenakshi Hegde, Brad St Croix, Nabil Ahmed. TEM8/ANTXR1 specific T cells co-target tumor stem cells and tumor vasculature in triple-negative breast cancer. [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 2312.

Abstract PD3-07: TEM8 specific CAR T cells serve as a novel targeted therapy for triple negative breast cancer and its supporting endothelium

Background: Triple Negative Breast Cancer (TNBC) refers to an aggressive subtype of breast cancer negative for HER2, estrogen and progesterone receptors. Lacking these receptors, individuals with TNBC do not benefit from many of the targeted therapies for breast cancer. Tumor endothelial marker 8 (TEM8), originally identified as a tumor endothelium associated antigen, has more recently been implicated in TNBC pathogenesis and as a marker of breast cancer stem like cells. Here we report that T cells expressing a TEM8-specific chimeric antigen receptor (CAR) serve as a novel approach to target both TNBC cells and its supporting endothelium. CARs combine the specificity of a monoclonal antibody with the signaling properties of a T cell. Methods: We designed two novel TEM8-specific CAR molecules. A CAR molecule containing an exodomain derived from the anti-TEM8 L2 antibody, followed by CD28 and CD3-zeta signaling domains (second generation CAR) and CD28, 41BB and CD3-zeta signaling domains (third generation CAR), respectively. Retroviral transduction was used to express the TEM8 CAR transgene constructs on HEK 293T cells, then on primary T cells. Results: Immunofluorescence staining revealed that in a panel of primary TNBC breast cancer samples, TEM8 was overexpressed in comparison to normal adjacent breast tissue (6/6). Costaining with the pan-endothelial cell marker CD31 revealed that this overexpression was not confined to the endothelial compartment, but also present on tumor parenchymal cells. The immortalized TNBC cell lines (MDA-MB-231, MDA-MB-436, MDA-MB-468 and Hs578T) expressed endogenous levels of TEM8 protein as revealed by western blot. Greater than 90% transduction of primary human T cells was achieved using both of our CAR constructs, as detected by flow cytometry. TEM8 specific T cells displayed significantly higher killing of TEM8 positive TNBC and tumor endothelial cells (2H11 and bEND.3) in standard four hour chromium release assays when compared to both non-transduced or irrelevant (CD19) CAR T cells and secreted immunomostimulatory cytokines upon encounter of TEM8 positive cells in coculture assays. In a vascularized xenograft model, MDA MB468 cells were injected subcutaneously with matrigel into athymic nude mice and followed via bioluminescence imaging over the course of two months. Established tumors were treated with either, second or third generation TEM8 specific T cells, HER2 specific T cells, non-transduced T cells or left untreated. Relative to non-transduced T cells, TEM8 specific second and third generation CAR T cells significantly delayed tumor growth by 36 days and 50 days, respectively. Conclusion: TEM8 specific CAR T cells could serve as a novel targeted therapy for TNBC and supporting endothelium. Citation Format: Byrd T, Fousek K, Pignata A, Szot C, Bielamowicz K, Wakefield A, Koch J, Landi D, Seaman S, Wels W, Fletcher B, Hegde M, St Croix B, Ahmed N. TEM8 specific CAR T cells serve as a novel targeted therapy for triple negative breast cancer and its supporting endothelium. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr PD3-07.

720. Triple-Negative Breast Cancer Cells and Tumor Endothelium Are Killed by Targeting Tumor Endothelial Marker 8 (TEM8)

Triple-negative breast cancer (TNBC) refers to a subset of breast cancers that are HER2, estrogen-receptor and progesterone-receptor negative. Associated with an aggressive phenotype and high incidence of recurrence, and devoid of the aforementioned receptors, there are currently no targeted therapies for TNBC. Tumor Endothelial Marker 8 (TEM8) is one of nine gene products up-regulated in the tumor vs. normal endothelium and is overexpressed in TNBC.

721. Safety of Multiple Doses of CAR T Cells

Chimeric antigen receptor (CAR) T cells have demonstrated promising results for cancers in pre-clinical models and early phase trials. However, some groups have observed serious adverse events (SAEs) and toxicities attributable to the administration of CART cells, the most clinically significant being cytokine release syndrome (CRS). In addition, a recent case report described a patient who developed an anaphylaxis reaction to CART cells, ultimately attributed to receipt of multiple doses of cells. As there are a number of ongoing clinical trials at our institution utilizing CAR T cells to treat various hematologic and solid malignancies and several patients have received multiple doses, we performed a retrospective review to assess whether early or late infusion toxicities were observed with subsequent infusions. We identified 45 of over 200 patients who received more than one dose of CAR T cells between January 2009 and December 2014. We assessed patient characteristics including type of malignancy, disease status at the time of subsequent infusions, cell product (autologous vs. allogeneic), presence or absence of prior lymphodepletion, number of infusions, dosing schedule, and the relationship to development of CRS. Each characteristic was examined for any correlation to developing a SAE. Our results indicate that at our institution, repeated CAR T cell infusions are well tolerated, with the majority of grade 3-4 adverse events being hematologic and electrolyte abnormalities. Furthermore, the majority of SAEs reported after repeated CAR T cell infusions were attributed as unrelated to the infusion itself. However, further evaluation of a larger cohort is necessary to determine whether an association between the timing of repeated infusions and CRS and other SAEs exists. These findings and our continued evaluation of patients receiving multiple infusions will help us to ensure the safety of administering multiple doses of CAR T cells in the future.