Kevin Bielamowicz

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.

HER2-Specific Chimeric Antigen Receptor–Modified Virus-Specific T Cells for Progressive Glioblastoma: A Phase 1 Dose-Escalation Trial

Importance Glioblastoma is an incurable tumor, and the therapeutic options for patients are limited. Objective To determine whether the systemic administration of HER2-specific chimeric antigen receptor (CAR)–modified virus-specific T cells (VSTs) is safe and whether these cells have antiglioblastoma activity. Design, Setting, and Participants In this open-label phase 1 dose-escalation study conducted at Baylor College of Medicine, Houston Methodist Hospital, and Texas Children’s Hospital, patients with progressive HER2-positive glioblastoma were enrolled between July 25, 2011, and April 21, 2014. The duration of follow-up was 10 weeks to 29 months (median, 8 months). Interventions Monotherapy with autologous VSTs specific for cytomegalovirus, Epstein-Barr virus, or adenovirus and genetically modified to express HER2-CARs with a CD28.&zgr;-signaling endodomain (HER2-CAR VSTs). Main Outcomes and Measures Primary end points were feasibility and safety. The key secondary end points were T-cell persistence and their antiglioblastoma activity. Results A total of 17 patients (8 females and 9 males; 10 patients ≥18 years [median age, 60 years; range, 30-69 years] and 7 patients <18 years [median age, 14 years; range, 10-17 years]) with progressive HER2-positive glioblastoma received 1 or more infusions of autologous HER2-CAR VSTs (1 × 106/m2 to 1 × 108/m2) without prior lymphodepletion. Infusions were well tolerated, with no dose-limiting toxic effects. HER2-CAR VSTs were detected in the peripheral blood for up to 12 months after the infusion by quantitative real-time polymerase chain reaction. Of 16 evaluable patients (9 adults and 7 children), 1 had a partial response for more than 9 months, 7 had stable disease for 8 weeks to 29 months, and 8 progressed after T-cell infusion. Three patients with stable disease are alive without any evidence of progression during 24 to 29 months of follow-up. For the entire study cohort, median overall survival was 11.1 months (95% CI, 4.1-27.2 months) from the first T-cell infusion and 24.5 months (95% CI, 17.2-34.6 months) from diagnosis. Conclusions and Relevance Infusion of autologous HER2-CAR VSTs is safe and can be associated with clinical benefit for patients with progressive glioblastoma. Further evaluation of HER2-CAR VSTs in a phase 2b study is warranted as a single agent or in combination with other immunomodulatory approaches for glioblastoma.

Adoptive cell therapies for glioblastoma.

Glioblastoma (GBM) is the most common and most aggressive primary brain malignancy and, as it stands, is virtually incurable. With the current standard of care, maximum feasible surgical resection followed by radical radiotherapy and adjuvant temozolomide, survival rates are at a median of 14.6 months from diagnosis in molecularly unselected patients (1). Collectively, the current knowledge suggests that the continued tumor growth and survival is in part due to failure to mount an effective immune response. While this tolerance is subtended by the tumor being utterly “self,” it is to a great extent due to local and systemic immune compromise mediated by the tumor. Different cell modalities including lymphokine-activated killer cells, natural killer cells, cytotoxic T lymphocytes, and transgenic chimeric antigen receptor or αβ T cell receptor grafted T cells are being explored to recover and or redirect the specificity of the cellular arm of the immune system toward the tumor complex. Promising phase I/II trials of such modalities have shown early indications of potential efficacy while maintaining a favorable toxicity profile. Efficacy will need to be formally tested in phase II/III clinical trials. Given the high morbidity and mortality of GBM, it is imperative to further investigate and possibly integrate such novel cell-based therapies into the current standards-of-care and herein we collectively assess and critique the state-of-the-knowledge pertaining to these efforts.

Autologous HER2 CMV bispecific CAR T cells are safe and demonstrate clinical benefit for glioblastoma in a Phase I trial.

Glioblastoma (GBM) remains incurable with current standard-of-care therapies. Adoptive T cell transfer holds the promise to improve outcomes for GBM patients. We report on the results of the Phase I clinical study, NCT01109095, administering autologous CMV.pp65 T cells grafted with a second generation HER2 chimeric antigen receptor (CAR) with a CD28.zeta signaling domain to patients with progressive GBM. Seventeen CMV-seropositive patients with radiologically progressive HER2+ GBM were enrolled. The median age was 49 years (range 11 to 71; 6 children; 11 adults). Children enrolled had significantly larger tumor volumes at infusion. A cell product was successfully generated for all patients from a peripheral blood draw (maximum 90mL). A median of 67% (range: 46-82) of T cells expressed the HER2 CAR, and exhibited a median 985.5 (range 390 to 1292) CMV.pp65 reactivity in an IFN-γ Elispot assay (SFC/105 T cells). Infusions of 1x106/m2-1x108/m2 were well tolerated without severe adverse events or cytokine release syndrome. HER2 CMV T cells were detected in the peripheral blood for up to 12 weeks post infusion, as judged by rtPCR of a CAR-specific amplicon. Out of 16 evaluable patients, 8 had progressive disease, 8/16 patients had objective responses: 1 patient had a partial response with a ~62% reduction in tumor volume lasting 8 months, 7 patients had stable disease for more than 6 weeks (of these 5 were durable >10 weeks) and 3 subjects are currently with a follow up 24 to >30 months, after T cell infusion. The median survival was 11.6 months from infusion and 24.8 months from diagnosis. The median survival for adults was 30 months from diagnosis. We conclude that systemically administered HER2 CAR CMV bispecific T cells are safe. A durable clinical benefit was observed in ~38% of patients.

Overexpression and constitutive nuclear localization of cohesin protease Separase protein correlates with high incidence of relapse and reduced overall survival in glioblastoma multiforme

Separase, an enzyme that cleaves the chromosomal cohesin during mitosis, is overexpressed in a wide range of human epithelial cancers of breast, bone and prostate (Meyer et al., Clin Cancer Res 15(8):2703–2710, 2009). Overexpression of Separase in animal models results in aneuploidy and tumorigenesis. We have examined the expression and localization of Separase protein in adult and pediatric glioblastoma and normal brain specimens. Immunofluorescence microscopy and Western blot analysis showed significant overexpression of Separase in all adult and a subset of pediatric glioblastoma cells. Tumor status and patient survival strongly correlate with the mislocalization of Separase into the nucleus throughout all stages of the cell cycle. Unlike exclusively nuclear localization in mitotic control cells, glioblastoma samples have a significantly higher number of resting (interphase) cells with strong nuclear Separase staining. Additionally, patient survival analysis demonstrated a strong correlation between overexpression of Separase protein in adult glioblastoma and a high incidence of relapse and reduced overall survival. These results further strengthen our hypothesis that Separase is an oncogene whose overexpression induces tumorigenesis, and indicate that Separase overexpression and aberrant nuclear localization are common in many tumor types and may predict outcome in some human malignancies.

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

Abstract A019: Chimeric antigen receptors (CARs) as a low-impact treatment of pediatric ependymomas

Ependymoma is the third most common paediatric brain tumor and can occur anywhere along the neuroaxis, the most common location being the posterior fossa (PF). Surgery followed by radiation therapy (>4 years of age) is to date the most effective treatment regimen; chemotherapy is not part of the current standard of care as multiple clinical trials have failed to show any survival benefits. Despite extensive characterization of ependymoma, few credible oncogenes and tumor suppressor genes have been identified, in fact, up to 50% of PF ependymoma cases exhibit a balanced and bland genomic profile. Consequently, actionable mutations are not currently identifiable in the vast majority of cases. We are investigating candidate chimeric antigen receptor (CAR)- strategies to treat posterior fossa-A ependymoma (PFA) — a subtype that has the worst associated prognosis and is found predominately in infants. Probing of our extensive ependymoma gene expression dataset has identified several potential CAR target antigens, including HER2, EPHA2 and IL13Rα2, these have been further verified with tissue microarrays (TMAs). Using our orthotopic xenograft models of PFA ependymoma, we are confident from our preliminary analysis that HER2-positive PFA ependymomas respond to HER2-specific CAR treatment. Furthermore, the use of a TRI-CAR, tailored to target cells expressing EPHA2, IL13Rα2 and HER2, both collectively and independently, increases the efficacy and specificity as a therapy for both primary and recurrent ependymoma. To ensure we are using the most effective negative controls when confirming the therapeutic response of our CARs, we are silencing our genes of interest in our patient-derived ependymoma lines using shRNA and CRISPR-technology for HER2, EPHA2 and IL13Rα2. Whole-genome and whole-exome sequencing has demonstrated, that in comparison to PFB ependymomas, PFA tumors have more methylated CpG sites, and more genes that are transcriptionally silenced by CpG hypermethylation. To reduce this silencing and potentially improve therapeutic response to our CARs, we are using Azacitadine in combination with our CAR therapies. In parallel with our pre-clinical trials, we are performing safety trials to assess the administration of intravenous versus intrathecal HER2 CAR delivery. Patients presenting with a brain tumor frequently present with a leaky blood-CSF and blood-Brain barrier, therefore we aim to observe whether CARs injected intrathecally can be found within the blood and vice versa. Using a HER2 Copy Number Assay, we observed two significant results: firstly, HER2 CARs injected intrathecally, in the presence of a HER2-positive brain tumor, remain within the central nervous and are not found within the blood; secondly, HER2 CARs administered intraventrically in the presence of a HER2-positive brain tumor, migrate to the CSF (P = 0.009). Results from this project have the potential to create a paradigm shift in our approach to the treatment of ependymoma, including the initiation of a clinical trial for children with PFA ependymoma. This technology has the real potential to improve cure rates whilst also dramatically improving the quality of life for surviving patients by reducing the impact that current treatments have on normal brain development. Citation Format: Laura K. Donovan, Kevin J. Bielamowicz, Alex Manno, Nabil Ahmed, Michael D. Taylor. Chimeric antigen receptors (CARs) as a low-impact treatment of pediatric ependymomas [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 A019.

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

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