Protein engineering for cancer therapy

[摘要] The immunosuppressive effects of CD4⁺CD25⁺ regulatory T cells (Tregs) interfere with anti-tumor immune responses in cancer patients. In the first part of this work, we present a novel class of engineered Interleukin-2 (IL-2) analogues that antagonize the IL-2 receptor, for inhibiting Treg suppression. These antagonists are engineered for high affinity to the IL-2 receptor a subunit and low affinity to either the [beta] or [gamma] subunit, resulting in a signaling-deficient IL-2 analogue that sequesters the IL-2 receptor a subunit from wild type IL-2. Using this design, human and mouse IL-2 antagonists were generated with inhibition constants ranging from 200 pM to 5 nM in vitro. Genetic fusions with IgG2a Fc enhanced serum half-life up to 30 hours. In order to study the effects of IL-2 antagonism, Fc fragments with disrupted effector functions were used. Fc-antagonist fusions bound to but could not deplete peripheral Tregs. They downregulated CD25 on Tregs, but could not perturb Treg function in a syngenic tumor model, presumably due to the high sensitivity of the IL-2 receptor and a high threshold for antagonism in vivo. In the second part of this work, we present a novel multi-agent protein-based system for targeted siRNA delivery that provides potential advantages over other nanoparticle- and proteinbased delivery vehicles. In the first agent, the double stranded RNA binding domain (dsRBD) of human protein kinase R is used as an siRNA carrier, in fusion proteins that target epidermal growth factor receptor (EGFR). Targeted dsRBD proteins deliver large amounts of siRNA to endosomal compartments in an EGFR expressing cell line, but efficient gene silencing is limited by endosomal escape. The use of a second agent that contains the cholesterol dependent cytolysin, perfringolysin 0, enhances endosomal escape of siRNA. Targeted delivery of perfringolysin 0 induces gene silencing in a dose-dependent and EGFR-dependent manner. However, cytotoxicity of the cytolysin creates a narrow therapeutic window. Multiepitopic EGFR binders that induce EGFR clustering are explored as tools for enhancing gene silencing efficiency. Interestingly, they not only enhance gene silencing potency but also protect against toxicity from EGFR-targeted cytolysins, thus significantly widening the therapeutic window of this method.

[发布机构] Massachusetts Institute of Technology