Background
Interleukin-12 (IL-12) is a proinflammatory cytokine that bridges innate and adaptive immunity by stimulating the differentiation of T helper 1 cells and enhancing the cytolytic activity of natural killer (NK) and T cells. Historically, IL-12 has demonstrated potent anti-tumor activity in syngeneic mouse models and promising efficacy in humans. However, development of IL-12-based agents has been limited by severe systemic toxicities in the clinical setting. We created XTX301, an investigational tumor-activated, half-life extended engineered IL-12, to reduce toxicity and improve the therapeutic index of IL-12. XTX301 masking domain is designed to pharmacologically inactivate IL-12 in non-tumor tissue, while enabling generation of an active IL-12 moiety upon cleavage by proteases enriched in the tumor microenvironment (TME).
Methods
We created mXTX301, a murine surrogate for XTX301, and its unmasked control molecule for in vivo pharmacology assessments, as human IL-12 does not bind to mouse IL-12 receptors. We assessed the anti-tumor activity of mXTX301 in cancer mouse models with diverse immune phenotypes, including MC38, CT26, B16F10, and MB49. Flow cytometry and bulk RNA sequencing analysis were performed to elucidate the mechanism of action of mXTX301. XTX301 and mXTX301 cleavage was assessed ex vivo in human and mouse tumor samples. In addition, mXTX301 tumor specific activation was evaluated in the MC38 tumor model. mXTX301 anti-tumor activity in combination with immunotherapies including checkpoint blockade were also tested in a mouse model.
Results
mXTX301 was well-tolerated compared to the unmasked control molecule and resulted in anti-tumor activity across multiple syngeneic mouse tumor models with diverse immune phenotypes. mXTX301 also demonstrated efficacy in mice with large (~400 mm3) MC38 tumors that have highly immunosuppressive TME and typically respond poorly to treatment. mXTX301 anti-tumor activity was associated with increases in the IFN- cell signaling pathway and activation of NK and T cells. Tumor selective activation of XTX301 and mXTX301 was observed ex vivo in human tumor and plasma samples, and in vivo and ex vivo in mouse, respectively. Data in preclinical models also suggest the potential for XTX301 to be combined with other immunotherapies to improve anti-tumor activity.
Conclusions
In preclinical models, mXTX301, a tumor activated, half-life extended IL-12, demonstrated remodeling of the TME, anti-tumor activity, and improved tolerability compared to a systemically active IL-12, indicating that XTX301 has potential for activity in both hot and cold tumors while also improving the therapeutic index of IL-12 treatment. XTX301 is being evaluated in a Phase I clinical trial (NCT05684965).