Abstract LB-254: A universal remedy for CAR T cell limitations
Authors: Lee Yong Gu , Chu Haiyan , Tenneti Srinivasarao , Kanduluru Ananda Kumar , Low Philip S. 
Published: July 2016

Abstract
Chimeric antigen receptor (CAR) T cells show significant potential for treating cancer due to their tumor-specific activation and ability to focus their killing activity on cells that express a tumor antigen. Unfortunately, this promising therapeutic technology is still limited by: (1) an inability to control the rate of cytokine release and tumor lysis; (2) the absence of an “off switch” that can terminate cytotoxic activity when tumor eradication is complete; (3) a failure to eliminate tumor cells that do not express the targeted antigen; and (4) a requirement to generate a different CAR T cell for each unique tumor antigen
In order to address these limitations, we have exploited a low molecular weight bi-specific adaptor molecule that must bridge between the CAR T cell and its targeted tumor cell by simultaneously binding to the chimeric antigen receptor on the CAR T cell and the unique antigen on the tumor. Using this bispecific adaptor, one can control CAR T cell cytotoxicity by adjusting the concentration and rate of administration of the adaptor. Because the half life of the adapter is &lt;20 minutes in vivo, termination of CAR T cell killing can be accomplished by cessation of adapter administration. Moreover, when heterogeneous tumors containing cells that express orthogonal antigens must be treated, the same CAR T cell can be targeted to multiple antigens by attachment of the same CAR ligand to the appropriate selection of tumor-specific ligands. Finally, when the targeted tumor antigen is also expressed at low levels on normal cells, tumor specificity can be achieved by adjusting the affinity of the tumor-specific ligand to enable CAR T cell engagement only when a highly multivalent interaction is possible.
To experimentally demonstrate the aforementioned benefits of using low molecular weight bispecific adaptors, CAR T cells were constructed by fusing an anti- fluorescein isothiocyanate (FITC) scFv to a CD3 zeta chain containing the intracellular domain of CD137 (i.e. CAR4-1BBZ T cells). Then, to enable their tumor-specific cytotoxicity, a bispecific adaptor molecule comprised of fluorescein linked to a small organic ligand with high affinity and specificity for a tumor-specific antigen (FITC-SMC) was synthesized. For these studies, the tumor-specific ligands were: i) folate for recognition of the folate receptor that is over-expressed on ∼1/3 of human cancers, ii) DUPA for binding to prostate specific membrane antigen that is over-expressed on prostate cancers, and iii) NK-1R ligand that is over-expressed on neuroendocrine tumors. The ability of the same clone of CAR4-1BBZ T cells to eliminate tumors expressing each of the above antigens was then demonstrated by administration of the desired FITC-SMC to mice injected with the CAR4-1BBZ T cells. Our data show that anti-tumor activity: i) is only induced when both CAR4-1BBZ T cells and the correct antigen-specific FITC-SMC are present, ii) anti-tumor activity and toxicity can be sensitively controlled by adjusting the dosing of FITC-SMC, and iii) treatment of antigenically heterogeneous tumors can be achieved by administration of a mixture of the desired FITC-SMCs. Taken together, these data show that many of the limitations of CAR T cell technology can be addressed by use of a bispecific adaptor molecule to mediate tumor cell recognition and killing.
Citation Format: Yong Gu Lee, Haiyan Chu, Srinivasarao Tenneti, Ananda Kumar Kanduluru, Philip S. Low. A universal remedy for CAR T cell limitations. [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 LB-254.