Fig. 1: Molecular weight and collagen affinity are key determinants of IL-2 efficacy.
From: Maximizing response to intratumoral immunotherapy in mice by tuning local retention
a Schematic of yeast library displaying mutagenized-LAIR (white) as Aga2 (gray) fusion proteins. LAIR expression was detected using a fluorescent antibody against c-myc epitope (green) and binding to biotinylated collagen-related peptide (CRP) was detected using fluorescent streptavidin (SA-AF647, red). b Successive flow assisted cells sorts isolating LAIR mutants with diminished binding to biotinylated CRP. c Equilibrium binding titration of biotinylated CRP on enriched yeast from panel b (mean + s.d.; n = 8) compared to wild-type LAIR-displaying yeast (n = 1). Binding was measured by median fluorescence intensity of SA-AF647 by flow cytometry. d LAIR and LAIRR41A E43A, henceforth LAIRx, binding to collagen measured by enzyme-linked immunosorbent assay (ELISA) (mean + s.d., n = 6 for LAIR and n = 3 for LAIRx). e Collagen binding affinity (Kd, equilibrium dissociation constant) of IL-2 fusion proteins in small and large molecular weight formats (kD, kilodaltons) measured by ELISA (mean + s.d; n = 3 for all groups except n = 6 for LAIR-IL2). f Dose-dependent proliferation of CTLL-2 cells exposed to IL-2 fusion proteins (mean + s.d.; n = 3) g Schematic of B16F10 tumor study timeline (top) and treatment components (bottom). Mice were inoculated with 1 × 106 B16F10 cells subcutaneously in the right flank on day 0. Treatments were administered on day 6 and 12. TAA, tumor-associated antigen; i.tu., intratumoral; i.p., intraperitoneal. h, i Tumor growth (left, mean + s.d.) and survival (right) over time (n = 10 mice for the PBS (i.tu.) and PBS (i.tu.) + TA99 (i.p.) groups, and n = 7 mice for all other groups). Tumor area is shown until a mouse in the group is euthanized. Statistical significance of each treated group’s tumor area and survival (top right corner) was calculated by using a one-tailed Student’s t-test and log-rank Mantel-Cox test, respectively, versus the PBS (i.tu.) + TA99 (i.p.). Other survival comparisons (legend adjacent) were generated by a log-rank Mantel-Cox test. *P < 0.03; **P < 0.002; ***P < 0.0002; ****P < 0.0001; n.s. not significant. Source data for panel c–f and h, i are provided in the Source Data file. Panels a and g created with BioRender.com.
