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CAR T cell therapy of cancers promises to revolutionize oncology by harnessing the powers of synthetic biology and immunotherapy in a single agent. CARs are synthetic receptors composed of an extracellular antigen binding domain and one or more intracellular signaling domains which act in concert to activate the T cell upon antigen recognition. CARs targeting B cell associated CD19 demonstrated robust in vivo cytolytic activity, expansion, and persistence upon antigen exposure paving the way for clinical application of this technology and ultimately FDA approval for pediatric and young adult acute lymphoblastic leukemia as well as patients with relapsed or refractory diffuse large B cell lymphoma. However, these successes have not yet been replicated in the arena of solid tumors. Unlike hematologic malignancies, solid tumors present numerous challenges in the form of an immunosuppressive tumor microenvironment. In this chapter, we will highlight clinical application of CAR T cells in solid tumors, discuss hurdles that have impeded CAR T cell function in these malignancies, and propose methods to overcome these limitations.

CAR T cell therapy of cancers promises to revolutionize oncology by harnessing the powers of synthetic biology and immunotherapy in a single agent. CARs are synthetic receptors composed of an extracellular antigen binding domain and one or more intracellular signaling domains which act in concert to activate the T cell upon antigen recognition. CARs targeting B cell associated CD19 demonstrated robust in vivo cytolytic activity, expansion, and persistence upon antigen exposure paving the way for clinical application of this technology and ultimately FDA approval for pediatric and young adult acute lymphoblastic leukemia as well as patients with relapsed or refractory diffuse large B cell lymphoma. However, these successes have not yet been replicated in the arena of solid tumors. Unlike hematologic malignancies, solid tumors present numerous challenges in the form of an immunosuppressive tumor microenvironment. In this chapter, we will highlight clinical application of CAR T cells in solid tumors, discuss hurdles that have impeded CAR T cell function in these malignancies, and propose methods to overcome these limitations.

Novel tumor-targeting drug conjugates, BLT-F2 (1) and BLT-S-F6 (2), bearing a fluorotaxoid as the warhead, a mechanism-based self-immolative disulfide linker, and biotin as the tumor-targeting module, were designed and synthesized as 19F NMR probes. Fluorine atoms and CF3 groups were strategically incorporated into the conjugates to investigate the mechanism of linker cleavage and factors that influence their plasma and metabolic stability by real-time monitoring with 19F NMR. Time-resolved 19F NMR study on probe 1 disclosed a stepwise mechanism for release of a fluorotaxoid, which might not have been detected by other analytical methods. Probe 2 was designed to bear two CF3 groups in the taxoid moiety as “3-FAB” reporters for enhanced sensitivity and a polyethylene glycol oligomer insert to improve solubility. The clean analysis of the linker stability and reactivity of drug conjugates in blood plasma or cell culture media by HPLC and 1H NMR is troublesome, due to the overlap of key signals/peaks with background arising from highly complex ingredients in biological systems. Accordingly, the use of 19F NMR would provide a practical solution to this problem. In fact, our “3-FAB” probe 2 was proven to be highly useful to investigate the stability and reactivity of the self-immolative disulfide linker system in human blood plasma by 19F NMR. It has also been revealed that the use of polysorbate 80 as excipient for the formulation of probe 2 dramatically increases the stability of the disulfide linker system. This finding further indicates that the tumor-targeting drug conjugates with polysorbate 80/EtOH/saline formulation for in vivo studies would have high stability in blood plasma, while the drug release in cancer cells proceeds smoothly.