KLRC1 knockout overcomes HLA-E-mediated inhibition and improves NK cell antitumor activity against solid tumors.

Introduction: Natural Killer (NK) cells hold the potential to shift cell therapy from a complex autologous option to a universal off-the-shelf one. Although NK cells have demonstrated efficacy and safety in the treatment of leukemia, the limited efficacy of NK cell-based immunotherapies against solid tumors still represents a major hurdle. In the immunosuppressive tumor microenvironment (TME), inhibitory interactions between cancer and immune cells impair antitumoral immunity. KLRC1 gene encodes the NK cell inhibitory receptor NKG2A, which is a potent NK cell immune checkpoint. NKG2A specifically binds HLA-E, a non-classical HLA class I molecule frequently overexpressed in tumors, leading to the transmission of inhibitory signals that strongly impair NK cell function. Methods: To restore NK cell cytotoxicity against HLA-E+ tumors, we have targeted the NKG2A/HLA-E immune checkpoint by using a CRISPR-mediated KLRC1 gene editing. Results: KLRC1 knockout resulted in a reduction of 81% of NKG2A+ cell frequency in ex vivo expanded human NK cells post-cell sorting. In vitro, the overexpression of HLA-E by tumor cells significantly inhibited wild-type (WT) NK cell cytotoxicity with p-values ranging from 0.0071 to 0.0473 depending on tumor cell lines. In contrast, KLRC1 KO NK cells exhibited significantly higher cytotoxicity when compared to WT NK cells against four different HLA-E+ solid tumor cell lines, with p-values ranging from<0.0001 to 0.0154. Interestingly, a proportion of 43.5% to 60.2% of NKG2A- NK cells within the edited NK cell population was sufficient to reverse at its maximum the HLA-E-mediated inhibition of NK cell cytotoxicity. The expression of the activating receptor NKG2C was increased in KLRC1 KO NK cells and contributed to the improved NK cell cytotoxicity against HLA-E+ tumors. In vivo, the adoptive transfer of human KLRC1 KO NK cells significantly delayed tumor progression and increased survival in a xenogeneic mouse model of HLA-E+ metastatic breast cancer, as compared to WT NK cells (p = 0.0015). Conclusions: Our results demonstrate that KLRC1 knockout is an effective strategy to improve NK cell antitumor activity against HLA-E+ tumors and could be applied in the development of NK cell therapy for solid tumors.

Cytokine Adjuvants IL-7 and IL-15 Improve Humoral Responses of a SHIV LentiDNA Vaccine in Animal Models.

HIV-1 remains a major public health issue worldwide in spite of efficacious antiviral therapies, but with no cure or preventive vaccine. The latter has been very challenging, as virus infection is associated with numerous escape mechanisms from host specific immunity and the correlates of protection remain incompletely understood. We have developed an innovative vaccine strategy, inspired by the efficacy of live-attenuated virus, but with the safety of a DNA vaccine, to confer both cellular and humoral responses. The CAL-SHIV-IN- lentiDNA vaccine comprises the backbone of the pathogenic SHIVKU2 genome, able to mimic the early phase of viral infection, but with a deleted integrase gene to ensure safety precluding integration within the host genome. This vaccine prototype, constitutively expressing viral antigen under the CAEV LTR promoter, elicited a variety of vaccine-specific, persistent CD4 and CD8 T cells against SIV-Gag and Nef up to 80 weeks post-immunization in cynomolgus macaques. Furthermore, these specific responses led to antiviral control of the pathogenic SIVmac251. To further improve the efficacy of this vaccine, we incorporated the IL-7 or IL-15 genes into the CAL-SHIV-IN- plasmid DNA in efforts to increase the pool of vaccine-specific memory T cells. In this study, we examined the immunogenicity of the two co-injected lentiDNA vaccines CAL-SHIV-IN- IRES IL-7 and CAL-SHIV-IN- IRES IL-15 in BALB/cJ mice and rhesus macaques and compared the immune responses with those generated by the parental vaccine CAL-SHIV-IN-. This co-immunization elicited potent vaccine-specific CD4 and CD8 T cells both in mice and rhesus macaques. Antibody-dependent cell-mediated cytotoxicity (ADCC) antibodies were detected up to 40 weeks post-immunization in both plasma and mucosal compartments of rhesus macaques and were enhanced by the cytokines.