Fig. 1. Engineering ProIL2 design.

a Pipeline for designing and screening the most optimal ProIL2 designs. ProIL2 designs were screened using HEK-Blue^TM IL-2 reporter cell assay, MMP cleavage assay for 4 h, and tumor inoculation and growth measurement as described in the “Methods”. b Multiple designs of ProIL2 were tested. The Fc arms, listed from N terminus to C terminus in each entry, of each proposed design are shown. HEKBlue^TM IL-2 reporter cell assay was used to determine the EC50 in nM of functional activity of each ProIL2 design. Fold block of IL-2 activity was measured by dividing the EC50 of each ProIL2 design to that of SumIL2-Fc. To compare between experiments, SumIL2-Fc EC50 and construct EC50 were normalized to the displayed EC50 value. Each ProIL2 design was cleaved by MMPs and run on SDS-PAGE, where the amount of cleaved vs uncleaved ProIL2 product was quantified. MC38 s.c. tumor-bearing mice were injected i.p. with PBS and an equimolar dose of the listed ProIL2 design 9 days after tumor inoculation. 12 days after treatment, tumor volumes of PBS-treated and ProIL2-treated mice were measured, and the decrease in tumor volume in ProIL2-treated mice on this day was quantified. Tumor growth delay is defined as the difference in days it takes for treatment vs PBS groups to reach an average MC38 tumor volume of 150 mm³. Abbreviations: IL2Rβ = IL-2 receptor beta, IL2Rβ* = truncated version of IL-2 receptor beta, MMP = 10-mer cleavable MMP substrate, IL2Rα = IL2 receptor alpha, WTIL2 = wild-type IL-2, IL2RβD1 = IL-2 receptor beta domain 1, IL2RβD2 = IL-2 receptor beta domain 2. Data represent mean ± s.e.m. (n = 4 individual mice per group). Source data are provided as a Source Data file.