Fig. 2 |. Linker optimization for PROTAV-OVA to elicit potent T cell response in mice.
a, Representative synthesis route for PROTAVs via EDC-NHS conjugation. b, The chemical structures of a list of linkers screened for PROTAV-OVA. c, Deconvoluted ESI-MS spectra of OVA and PROTAV-OVA with an EG4 linker. d, UV-vis absorbance spectra of pomalidomide, OVA, and PROTAV-OVA (i.e., pomalidomide-OVA conjugates). The presence of pomalidomide-characteristic absorption (peak at 420 nm) in PROTAV-OVA suggests its successful synthesis. e, Timeline of T cell response study for PROTAV-OVA in mice. C57BL/6 mice (6–8 weeks; n = 5) were immunized with PROTAVs and controls, individually, by s.c. administration at tail base (day 0, day 14). CpG (2 nmole) adjuvant was mixed with OVA or PROTAV-OVA (10 μg). PBMCs were collected for T cell response analysis at indicated time points. f, g, H-2Kb/SIINFEKL tetramer staining results (day 21, day 35) (f) and intracellular cytokine staining (day 35) (g) showing the fraction of SIINFEKL-specific T cell response among PBMC CD8+ T cells elicited by PROTAV-OVA candidates with various linkers. PROTAV-OVA with an EG4 linker consistently elicited one of the most potent T cell responses. h, Nano ITC results and fitted curve of the binding kinetics between PROTAV-OVA with CRBN protein, with OVA as a control. Kd of PROTAV-OVA and CRBN binding was determined to be 1.2 μM using a one site-specific binding model. OVA and CRBN ITC data did not fit into the binding modeling, suggesting negligible binding. i-j, H-2Kb/SIINFEKL tetramer staining results (day 21, day 35) (i) and intracellular cytokine staining (day 35) (j) showing the fraction of SIINFEKL-specific T cell response among PBMC CD8+ T cells elicited by PROTAV-OVA synthesized with four different feeding molar ratios of pomalidomide-EG4-NHS: OVA. Data represent mean ± s.e.m.; statistical analysis was conducted using one-way ANOVA with Bonferroni post-test.