Abstract 1091: Distinct mechanisms of orthotopic tumor control by alphavbeta3and HER2 CAR Ts support development of a bispecific CAR T cell therapy for pediatric rhabdomyosarcoma
Authors: Lulu Amanda M. , Park Nagyeong , Caruso Joseph J. , Cobb Dustin A. , Lee Daniel W. 
Published: March 2024

Goal: Evaluate αvβ3 and HER2 as antigens for a bispecific chimeric antigen receptor T (CAR T) cell therapy in pediatric rhabdomyosarcoma.

Background: Current treatments are ineffective against relapsed/refractory or metastatic rhabdomyosarcoma (RMS). Antigen loss or modulation on tumor cells commonly leads to tumor escape across diverse cancer types after CAR T therapy targeting different antigens. We hypothesize that simultaneously targeting two RMS antigens with a bispecific CAR T will reduce the likelihood of tumor escape. The integrin alphavbeta3 (αvβ3) plays a role in cancer progression, metastasis and neovascularization in several malignancies. HER2 also contributes to cancer progression and metastasis.

Methods: Control of intramuscular RMS tumors by αvβ3 or HER2 CAR Ts was assessed in NSG mice, with non-specific CD19 CAR Ts as controls. Tumor burden was assessed weekly by bioluminescence imaging. Persistence and phenotype of CAR Ts were evaluated by flow cytometry. CAR T cell infiltration, tumor antigen expression and residual disease were determined by immunohistochemistry.

Results: Six of six pediatric patient-derived RMS cell lines express αvβ3 and/or HER2. αvβ3 and HER2 CAR Ts kill RMS cells in vitro at low effector-to-target ratios. αvβ3 and HER2 CAR Ts independently control—and nearly eliminate—orthotopic RMS tumors. Fewer HER2 CAR Ts (5 × 106) are needed to control tumors than αvβ3 CAR Ts (10 × 106), but this is not due to lack of persistence or exhaustion of αvβ3 CAR Ts. Tumor control by αvβ3 CAR Ts is delayed (&gt;21 days) compared to that by HER2 CAR Ts (7 days), and is robust, despite initial tumor progression following treatment. Importantly, despite strong tumor control by both CAR Ts, at 7 weeks post-treatment residual tumor nests are evident by histology. At this time, CAR Ts are still abundant, are not exhausted and express memory markers, suggesting that the remaining tumor cells are no longer susceptible to CAR T killing. We are currently evaluating the hypothesis that the remaining tumor cells have down-regulated antigen expression. Delayed tumor control by αvβ3 CAR Ts was ameliorated by engineering αvβ3 overexpression in RMS tumors.

Conclusions: αvβ3 and HER2 CAR Ts control orthotopic RMS tumors by distinct mechanisms with changes in αvβ3 antigen expression, likely due to tumor progression, one prevalent factor. This is consistent with our prior reports of αvβ3 CAR Ts preventing breast cancer metastases, which naturally upregulate αvβ3 compared to the primary tumor. Despite near elimination of tumors, residual nests of tumor cells are likely to rebound as CAR T resistant tumors, similar to what is seen in patients treated with monospecific CAR Ts. These data support the incorporation of αvβ3 and HER2 CAR Ts into a bispecific CAR T approach to mitigate antigen escape. Our data also support investigation into the role of αvβ3 in RMS.

Citation Format: Amanda M. Lulu, Nagyeong Park, Joseph J. Caruso, Dustin A. Cobb, Daniel W. Lee. Distinct mechanisms of orthotopic tumor control by alphavbeta3and HER2 CAR Ts support development of a bispecific CAR T cell therapy for pediatric rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1091.