Background Innate immunity plays a vital role in cancer immunotherapy. Natural killer cell engager (NKCE) has garnered significant attention in recent years. Conventional NKCEs have shown limited anti-tumor activity as monotherapy although reasonable safety profiles were observed. Therefore, the development of a next-generation NKCE modality is imperative to enhance its anti-tumor activity. IL-15 plays a crucial role in expanding and activating NK cells and has recently been explored as a building block in the design of NKCEs to enhance activity. However, wild type IL-15 with full potency demonstrated serious toxicities in clinical trials. In this study, we incorporated a potency-reduced variant of IL-15 (IL15v) into our proprietary NKp46 targeting NKCEs. This led to the development of a novel tetravalent NKCE platform comprising IL15v, anti-NKp46, anti-tumor-associated antigen (TAA), and a functional Fc region.

Methods The IL15v was generated as a complex consisting of IL-15 mutein (N65A) and IL-15Rα. The IL15v-NKCE was developed by fusing the IL15v to the C-terminus of Fc domain of NKCE. The activity of IL15v moiety on primary immune cells was assessed by measuring pSTAT5 activation using flow cytometry. NK activation and cytotoxicity against tumor cells were measured by in vitro cell based assay. In vivo anti-tumor efficacy and toxicity of IL15v-NKCE were evaluated in human NKp46 knock-in or wild-type tumor bearing mice.

Results In vitro studies demonstrated that the IL15v moiety is structured to selectively activate NK cells while exerting no impact on T cells. The addition of IL15v to NKCE enhances its cytotoxicity against tumor cells, prevents spontaneous NK apoptosis upon NK activation during tumor cell killing, and promotes proliferation of these immune cells. By selectively targeting NK cells, the IL15v-NKCE molecule exhibited good tolerability in mice at doses as high as 19mg/kg, while the wild-type IL-15 fusion protein only reached a maximal tolerated dose of 1mg/kg. Additionally, IL15v-NKCE exhibited superior in vivo anti-tumor efficacy compared to a conventional NKCE. Our study demonstrated that all four components, namely anti-NKp46, a competent Fc, IL15v, and anti-TAA, are essential for achieving maximal activity, indicating a functional synergy in the mechanism of IL-15 driven NK engagers. Furthermore, IL15v-NKCE demonstrated greater potency than conventional NKCE when combined with anti-PD-1 for cancer treatment in preclinical tumor models.

Conclusions By enhancing the anti-tumor efficacy of NKCE while mitigating IL-15-induced systemic toxicity, IL15v-NKCE presents significant potential for developing next generation anti-cancer therapies.

Ethics Approval All animal studies were conducted under approval of the Shanghai Yishang Biotech committee on animal care in accordance with local guidelines. Human PBMCs were purchased from SailyBio and the informed consent was obtained from all donors.