(193j) Combination Cancer Therapy Using Chimeric Antigen Receptor Engineered Natural Killer Cells As Drug Carriers

Cancer treatments typically include surgical removal, chemotherapy, radiation, or some combination of these therapies. While these methods can remove the majority of the tumor cells, tumor recurrence and metastasis remain a major obstacle in cancer treatment. Numerous nanoparticle-based active targeting approaches have emerged to enhance the intracellular concentration of drugs in tumor cells; however, efficient delivery of these systems to the tumor site while sparing healthy tissue remains elusive. Cancer immunotherapy has garnered much attention within the last decade, and more recently, the concept of using immune cells as vehicles to transport anticancer agents directly to the tumor site has gained traction. Natural killer (NK) cells are part of the innate immune system and mediate rapid, short-lived responses by releasing cytokines that directly lyse infected or abnormal cells including tumor cells. The NK92 cell line is identical to the parental NK line isolated from a leukemia patient. NK92 cells are well-documented and have antitumor effects against various types of cancer including melanoma, leukemia and breast cancer in both preclinical and clinical settings. NK cells are not specifically cytotoxic to certain antigen-expressing target cells, but their specificity and efficacy can be enhanced with CARs. CARs are antigen-specific engineered receptors that can be expressed on human immune cells. They are composed of an extracellular antigen-binding domain derived from an antibody fragment. This allows the CAR-expressing immune cells to bind to specific surface antigens overexpressed on cancer cells. Intracellular signaling domains within the CAR provide signals to the immune cell to attack the antigen-expressing cell. Typically, CARs are expressed in T cells, but recent studies show CARs are effective tumor-targeting domains in NK cells as well. The inclusion of CARs in NK92 cells increases homing, specificity, and efficacy of tumor killing.

We modified our CAR.NK cells with crosslinked multilamellar liposomal vesicles (cMLVs) containing a chemotherapeutic drug, paclitaxel (PTX). cMLVs are liposomes functionalized with thiol-reactive maleimide headgroups, which allows them to be stably conjugated to the thiol-rich NK cell surface. Previously, we have demonstrated that cMLVs can act as a novel agent for combinatory drug delivery by co-localizing two drugs with distinct physicochemical properties to a single site, inducing a synergistic anti-tumor effect in vitro and in vivo. Here, our work presents the combination of immunotherapy and chemotherapeutic drug delivery by utilizing CAR.NK cells as carriers for PTX-loaded crosslinked multilamellar liposomal vesicles (cMLV (PTX)) to enhance antitumor efficacy in Her2 and CD19 overexpressing cancer models.

We ensured that the conjugation of cMLVs to the CAR.NK cell surface does not affect the functionality of the CAR.NK cell itself. After confirming the functionality of our cMLV(PTX)-conjugated CAR.NK cells in vitro, we established a mouse xenograft model to observe the effects of the anti-CD19 CAR.NK cells in vivo. Mice treated with conjugated cMLV(PTX)-CAR.NK had the most dramatically slowed tumor growth of all groups. We performed ex vivo analysis of our mouse xenograft tumor model to support our hypothesis that CAR.NK cells facilitate PTX delivery into the tumor site. The conjugated group had significantly higher PTX concentrations within the tumor tissue compared to all other groups. Overall, our work demonstrates that the efficacy of chemotherapy can be enhanced in vitro and in vivo while reducing off-target toxicity by using CAR-engineered NK92 (CAR.NK) cells as carriers to guide drug-loaded nanoparticles to the target site.