Abstract LB-109: Adaptor controlled CAR-T cell immunotherapy for treatment of folate receptor-alpha/beta positive solid and liquid tumors
Authors: Lu Yingjuan June , Chu Haiyan , Wheeler Leroy W. , Nelson Mellissa , Westrick Elaine M. , Vetzel Marilynn R. , Klein Patrick J. , Johnson Adam J. , Yokoyama Jason K. , Gustafson Joshua A. , Jensen Michael C. , Lee Yong-Gu , Low Philip S. , Leamon Christopher P. 
Published: August 2018

Personalized immunotherapy has reached a pivotal milestone with the recent approval of CD19-directed chimeric antigen receptor (CAR) T-cell therapies for certain B-cell malignancies. Other than on-target off-tumor autoimmunity, the commonly seen toxicity is severe cytokine release syndrome (sCRS) which is somewhat managed with tocilizumab and corticosteroids. Although the landscape of CAR-T cell development includes &gt;200 clinical trials in various tumor types, success against solid tumors remains a significant challenge. Much of the unmet medical need has been attributed to limited “ideal” tumor-specific targets, tumor heterogeneity, CAR-T cell infiltration/survival, and the presence of an immunosuppressive tumor microenvironment.

Due to frequent overexpression in many human cancers, the folate receptor (FR) has become an exploratory target for anti-FRα/β CAR-T cell therapies. We are developing a CAR-T adaptor molecule (CAM)-based therapy that uses the small-molecule bispecific ligand, EC17 (folate-FITC). In this setting, EC17 effectively “paints” FR+ tumors with the FITC antigen to attract anti-FITC CAR-T cells and induce an immune response. As previously confirmed in mice as well as in human clinical studies, EC17 penetrates solid tumors within minutes and is retained due to high affinity for the FR; whereas unbound EC17 rapidly clears from the blood and from receptor-negative tissues. When tested against human xenografts, EC17/CAR-T therapy showed consistent antitumor activity with low or no adverse reactions. However, sCRS was observed under defined experimental conditions. Further testing revealed that sCRS of grade ≥3 (out of a 0-5 scale) can be mitigated or even prevented using intermittent “on-and-off” dosing and/or dose titration of the EC17 CAM. But, under extreme conditions where dose cessation failed, we discovered that intravenous sodium fluorescein (NaFl) could be used as a fast-acting rescue agent to temporarily displace CAR-T cells from their targets and reverse the sCRS symptoms.

For translation into first-in-human testing, we selected a high-affinity anti-FITC CAR construct comprising (i) a humanized FITC scFv (E2; 0.75 nM Kd), (ii) an IgG4 hinge-CH2-CH3 spacer fused to a CD28-transmemeber domain, (iii) a second-generation 4-1BB:zeta-endodomain, and (iv) a cell-surface human EGFRt tag. Co-culture experiments were performed using this construct to establish EC17-dependent dose response and confirm target specificity as well as FR expression threshold using a panel of FR-positive cancer cell lines. Clinically relevant EC17 dosing regimens were evaluated using tumor-free and tumor-bearing mice to study CAR-T cell proliferation, cytokine production and the onset/mitigation of sCRS. We confirmed that EC17 administration in the presence of FR+ tumors was the key to drive in vivo CAR-T cell proliferation towards a more persistent phenotype. In addition, higher levels of CAR-T cells were detected in metastatic tumors versus adjacent normal tissues. Finally, EC17/CAR-T cell therapy demonstrated remarkable efficacy against some of the more aggressive and chemo-resistant FR+ tumors of various histology. In summary, our CAM-driven CAR-T cell approach provides antitumor activity with multiple safety control mechanisms.

Citation Format: Yingjuan June Lu, Haiyan Chu, Leroy W. Wheeler, Mellissa Nelson, Elaine M. Westrick, Marilynn R. Vetzel, Patrick J. Klein, Adam J. Johnson, Jason K. Yokoyama, Joshua A. Gustafson, Michael C. Jensen, Yong-Gu Lee, Philip S. Low, Christopher P. Leamon. Adaptor controlled CAR-T cell immunotherapy for treatment of folate receptor-alpha/beta positive solid and liquid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-109.