Activated NK CAR Cells to Cure HIV

ABSTRACT Virus-specific CD8 T cells exert antiviral activity against HIV-1/SIV in vitro and in vivo. Yet, despite these responses in HIV-1-infected humans and SIV- infected macaques, they are unable to fully suppress virus replication. This is likely due to the majority viral replication occurring in CD4+ T cells within B-cell follicles in secondary lymphoid tissues; where virus-specific CD8 T cells are relatively few in number. In fact, we found that in vivo effector virus-specific CD8 T cell to target SIV RNA+ cell ratios (E:T) were over 40-fold lower inside compared to outside of B cell follicles in lymphoid tissues. These findings indicate that B cell follicles are an immune privileged site in which low levels of virus-specific CD8 T cells permit ongoing viral replication. Furthermore, we found that few virus-specific CD8 T cells express the follicular homing molecule CXCR5, likely explaining their low levels in B cell follicles. These data suggest that the inability of virus specific CD8 T cells to fully suppress virus replication may be due to a deficiency of these T cells in B-cell follicles. These findings have led us to our central hypothesis that targeting HIV-specific immunotherapy to B cell follicles will lead to durable remission of HIV infection. In support of this hypothesis we have shown that increased levels of virus-specific CD8 T cells in B cell follicles is associated with lower viral loads. Although many immunotherapies utilize patient T cells to generate CAR-T therapies, there are special consideration in the treatment of HIV. One major short comings of CAR-T approaches is the fact that T cells need to be autologous due to the risk of graft versus host disease (GvHD), requiring complicated/expensive manufacturing processes of patient cells. This is also challenging in the HIV setting as the patient T cells are already compromised and processing cells which may contain active virus is risky. Alternatively, Natural Killer (NK) cells are highly suited for allogeneic use as they do not cause GvHD and thus hold significant clinical potential as an off-the-shelf cellular product. Thuts, we propose to evaluate NK immunotherapy that targets virus-specific CAR NK cells (expressing CD4-MBL-CAR and CXCR5) to B cell follicles. Moreover, we will use CRISPR/Cas9 to knockout negative regulators of NK cell function, such as PD1, which we have previously shown to enhance NK cell function. Our long-term goal is to develop an intervention that will lead to durable remission of HIV infection using CAR NK cells. To test our hypotheses, we propose the following aims. 1) Develop reagents and methods to generate human and rhesus macaque CAR/CXCR5/PD1KO NK cells. 2) Determine the ability of CAR/CXCR5/PD1KO NK cells to migrate into B cell follicles of SIV-infected rhesus macaques and to induce and maintain viral suppression. Our proposed studies targeting CAR NK cells to follicles will have a broad impact on the field by providing insights into cell trafficking, persistence, and pre-conditioning regimens for NK immunotherapy. Moreover, our methods for engineering rhesus macaque NK cells will enable studies assessing the therapeutic use of NK cells in preclinical NHP models. Moreover, these studies could result in an effective strategy to induce long-term sustained remission of HIV.