Figure 9: Targeting of adenosine antagonist-loaded ANCs to immune checkpoint PD1 improves NP delivery to circulating and LN-resident T cells and anti-tumor effects of systemic combination immunotherapy in 4T1 TNBC tumor model.

Representative flow cytometry plots (A) and quantification (B) of NP⁺ frequency of B220⁻ leukocytes 1 h post i.v. administration of aCD3- or isotype-ANC (AlexaFluor647-labeled NP; 40μg mAb dose) in TdLN, spleen, blood, and tumor tissues of day 6 4T1-bearing mice. C) Quantification of aCD3- or isotype-ANCs binding to B220⁺ leukocytes 1 h post i.v. administration. D) Tumor growth curves after combination immunotherapy administered i.p. on days 6, 9, 12, and 15 post-implantation of 4T1 tumors. Treatments included aPD1-ANCs (60 µg mAb) encapsulating SCH-58261 (2 µg) or isotype ANCs encapsulating SCH-58261 co-injected with free aPD1 mAb. E) Representative gating of TdLN lymphocytes on day 35 after 4T1 tumor implantation. LN metastases quantified as the fraction of analyzed total events within the shown lymphocyte gate. Gating controls include cells harvested from 4T1 tumors (Tumor) and cells from LNs of tumor-free mice (Tumor-free LN). F) Quantified levels of LN lymphocytes on day 35 from (E). Dotted line represents % lymphocytes in LN of non-tumor bearing mice. G) Spleen area. Dotted line represents spleen area in non-tumor bearing mice, statistics relative to non-tumor bearing mouse spleen size. H) Animal survival. Statistical analyses were done using two-tailed unpaired t-test (B-C), two- (D) or one-way (F-G) ANOVA with Tukey’s test. Log-rank (Mantel-Cox) test for survival curves (H). *p<0.05, **p<0.01, ****p<0.0001. n.s., not significant.