Design of a Universal Chimeric Antigen Receptor-expressing T Cells and Associated Tumor Targeting Ligands for Controlled Destruction of Solid Tumors
Chimeric antigen receptor expressing T cells (CAR T cells) show substantial promise for the immunotherapy of cancer, both because of their remarkable tumor specificity and their robust tumor cytotoxicity. Clinical data to date demonstrate that CD19-targeted CAR T cells are effective in treating B cell lymphomas (i.e. yielding 70-93% complete responses). However, despite this highly promising demonstration of efficacy, current CAR T cell technologies are compromised by deficiencies that limit their expansion to other cancers. These limitations include: (1) an inability to control the intensity of CAR T cell activation, leading to cytokine release syndrome (CRS); (2) an incapacity to terminate the CAR T cell-mediated killing once the tumor has been completely eliminated; and (3) a failure to kill those cancer cells in an antigenically heterogeneous tumor that do not express the targeted antigen (i.e. with most tumors mutating rapidly, the emergence of malignant cells that lack the targeted antigen is almost inevitable) (Chapter 1). In order to address these limitations, we have designed a controllable and universal CAR T that in combination with the appropriate tumor-specific low molecular weight adaptor molecule should be able to kill virtually any cancer cell in a controlled and easily terminated manner. In this strategy, the CAR T cell is engineered to contain a single-chain variable fragment (scFv) that recognize and bind fluorescein with high affinity and specificity. Moreover, intracellular T cell signaling domains (i.e. CD137 and CD3ζ) were incorporated with aforementioned anti-fluorescein scFv for activation of CAR T cell (i.e. anti-fluorescein CAR T cell). A low molecular weight adapter (i.e. bispecific adapter), comprised of fluorescein linked to a tumor-specific ligand, is then added that can bridge between the anti-fluorescein CAR T cell and the tumor cell antigen to which the tumor-specific ligand binds. By mediating a tight association between the cancer cell and the CAR T cell, cancer cell killing and CAR T cell proliferation are induced. Because the bispecific adapter can be administered at any desired rate, and since these adapters are known to rapid excrete into the urine (t1/2<20min in>vivo) if they are not captured by the CAR T cell or the targeted receptor on the cancer cell, we demonstrated that the intensity of CAR T cell activation can be sensitively regulated (i.e. minimizing any CRS) and the target cell killing can be terminated as desired (Chapter 3). Most importantly, by simply administering the desired selection of bispecific adapters, we demonstrated that the anti-fluorescein CAR T cell can be enabled to kill multiple genetically distinct cancer cell clones expressing orthogonal antigens, without requiring a different re-designed CAR construct for each cancer cell variant. Moreover, treatment of antigenically heterogeneous tumors can be readily achieved by administration of a mixture of bispecific adapters, each with a fluorescein linked to the desired tumor-specific targeting ligand (Chapter 4). Taken together, these data demonstrate anti-fluorescein CAR T cell platform with a bispecific adapter can address current CAR T cells’ limitations without compromising their anti-tumor potency or specificity.
Low, Purdue University.
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