Tumor Cell-Surface Binding of Immune Stimulating Polymeric Glyco-Adjuvant via Cysteine-Reactive Pyridyl Disulfide Promotes Antitumor Immunity

Immune stimulating agents like Toll-like receptor 7 (TLR7) agonists induce potent antitumor immunity but are limited in their therapeutic window due to off-target immune activation. Here, we developed a polymeric delivery platform that binds excess unpaired cysteines on tumor cell surfaces and debris to adjuvant tumor neoantigens as an in situ vaccine. The metabolic and enzymatic dysregulation in the tumor microenvironment produces these exofacial free thiols, which can undergo efficient disulfide exchange with thiol-reactive pyridyl disulfide moieties upon intratumoral injection. These functional monomers are incorporated into a copolymer with pendant mannose groups and TLR7 agonists to target both antigen and adjuvant to antigen presenting cells. When tethered in the tumor, the polymeric glyco-adjuvant induces a robust antitumor response and prolongs survival of tumor-bearing mice, including in checkpoint-resistant B16F10 melanoma. The construct additionally reduces systemic toxicity associated with clinically relevant small molecule TLR7 agonists.

p(PDS) was dissolved in PBS and the full UV-Vis absorbance spectrum was recorded. Reducing S2 agent β-mercaptoethanol was added in 100x excess of PDS and allowed to react to completion (30 minutes). This verifies that the PDS groups are functionally active and can be used to quantify PDS content using a standard curve of 2-mercaptopyridine. Alternatively, the APC MFI of the whole TRP1 + or CD45 + populations was recorded. S3

Fig. S4. Intracellular thioredoxin-1 is detectable in B16F10 cells via flow cytometry.
B16F10 cells were removed from culture, washed free of media with PBS two times, then permeabilized using FIX&PERM Cell Permeabilization Kit (ThermoFisher) for intracellular antibody staining with AF594-conjugated anti-thioredoxin-1 (Novus Biologicals  B16F10 cells were removed from culture, washed free of media with PBS two times, then incubated with 1 mg/mL D-mannose on ice for 30 minutes. AZDye 674-labeled polymer was added directly to the mannose mixture to make a polymer solution at 80 µM fluorophore then incubated for an additional 90 minutes (n = 3, mean +/-SEM). Cells were collected by flow cytometry as described in methods, where MFI correlates to amount of polymer attached to the cells. Binding is confirmed to be independent of mannose pre-incubation, as the polymer MFI does not change. Statistical differences were determined by unpaired t-tests.

S6
Uptake is confirmed to be dependent on the incorporation of mannose and is not significantly affected by PDS-mediated binding. Statistical differences were determined by unpaired t-tests. 8week-old female BALB/c mice were inoculated with CT26 tumors as described in methods. On days 6, 9, and 12 post-inoculation, mice were injected intratumorally with 40 μg TLR7 monomer equivalent p(Man-TLR7-PDS) (n = 8) or vehicle control (PBS, n = 7) (mean +/-SEM). The mouse weights were recorded every three days. Mice were euthanized when the tumor volume exceeded 500 mm 3 and/or based on humane end-point criteria. S11
On days 6, 9, and 12 post-inoculation, mice were injected intratumorally with various concentrations of p(Man-TLR7-PDS) (n = 7, mean +/-SEM). The mouse weights were recorded every three days. Mice were euthanized when the tumor volume exceeded 500 mm 3 and/or based on humane end-point criteria. Statistical analyses were performed using Kruskal-Wallis tests followed by Dunn's multiple comparison for nonparametric data. All differences are non-significant.
Mice were euthanized when the tumor volume exceeded 500 mm 3 and/or based on humane endpoint criteria. Shown are individual (thin dotted lines) and mean (thick solid lines) tumor growth curves and survival plot. Arrows denote treatment days. Statistical differences were determined by pairwise log-rank (Mantel-Cox) tests.

Supplementary Methods
No unexpected or unusually high safety hazards were encountered conducting this work.

Monomer and polymer synthesis
All chemicals were reagent grade and purchased from Sigma Aldrich and used as received unless