Tumor-activating IL2 rejuvenates intratumoral TIM3-CD8+T cell responses through targeting TIM3+CD8+T cell
TIM3, a T-cell inhibitory receptor, is expressed on exhausted T cells in tumor microenvironment (TME). Anti-TIM3 antibody therapy could alleviate the suppression of tumor-infiltrating lymphocytes (TILs) in IL-2 dependent fashion. We hypothesize that high expression of TIM3 and limited IL-2 in TME facilitate immune evasion.  To test that, we engineered anti-TIM3-pro-IL2 to selectively deliver IL-2 to TIM3high TILs by TIM3 antibody and reduce its toxicity with a cis delivery mechanism. Since IL-2 could reduce its activity at acidic pH inside TME, we also screened a low pH-selective IL-2 mutein (IL2V2) with enhanced affinity for IL-2Rß.  We then integrated pro-IL-2 into the anti-TIM3 antibodies in two different forms: TIM3-Ra-MMPs-IL2V2 (TIM3-ProIL2V2) as IL-2 release-form vs TIM3-IL2V2-MMPs-Ra as cis-form after binding to TIM3high T cells, ensuring the need of targeted delivery to TIM3-expressing TILs. Surprisingly, released IL2V2 from TIM3-Ra-MMPs-IL2V2 proved superior in anti-tumor immunity, but not cis delivery form TIM3-IL2V2-MMPs-Ra. Mechanistically, TIM3-ProIL2V2 not only reactivated TIM3+ TILs but also facilitated the activation and expansion of TIM3- T cells which in turn supported a sustained source of TIM3+ effector. TIM3-ProIL2V2 could control multiple tumor models including human tumor in humanized mice, confirming the hypothesis. TIM3-ProIL2V2 activates and expands TIM3-CD8+ T cells to overcome current major unmet medical need: anti-PD-1/L1 resistance.  This strategy illustrates the potential of a tailored, low pH-resistant IL2 variant in invigorating both TIM3-negative and positive CD8+ T cells, offering a promising avenue for treating resistant tumors with reduced toxicity. Overall design: Since the antitumor effect of TIM3-ProIL2V2 depended on pre-existing CD8+ T cells, we further isolated intratumoral CD8+ T cells and investigated the transcriptional signatures at the single-cell level following TIM3-ProIL2V2 monotherapy.