GI

the most substantial anti‐tumor effect for StCl‐GI_GII (P = 0.0344) (Figure 4a,b; Figure S13b).

this multivalent nanoconjugate induced potent antigen‐specific anti‐tumor immune responses compared to StCl‐GI (P = 0.0218), as shown by the frequency of splenic positive cells for tetramers that bind to T cell receptor‐specific for the gp100 peptide epitope (Figure 4h).

StCl‐GI_GII + αPD‐1 treatment supported animal survival up to forty‐five days post‐tumor inoculation, a value significantly higher compared to treatment with PBS or TAAs + αPD‐1 (P < 0.0001 in both cases) (Figure 5e).

Treatment with TAAs + αPD‐1 or StCl‐GI_GII + αPD‐1 significantly reduced tumor sizes when compared to PBS‐treated control mice at day twenty‐one (P = 0.0053 and P < 0.0001, respectively) (Figure 5b–d).

Treatment with TAAs + αPD‐1 or StCl‐GI_GII + αPD‐1 significantly reduced tumor sizes when compared to PBS‐treated control mice at day twenty‐one (P = 0.0053 and P < 0.0001, respectively) (Figure 5b–d).

At days fifteen and seventeen post‐treatment initiation, treatment with StCl‐GI_GII (P = 0.0037 and P < 0.0001) ... induced significantly reduced tumor growth compared to PBS (Figure 6c).

At days fifteen and seventeen post‐treatment initiation, treatment with ... StCl‐GI_GII + αPD‐1 (P = 0.0156 and P < 0.0001) induced significantly reduced tumor growth compared to PBS (Figure 6c).

At day 17 post‐treatment initiation, we observed a significant reduction in the individual tumor weight of mice treated with StCl‐GI_GII (P = 0.0086) or StCl‐GI_GII + αPD‐1 (P = 0.0092) (Figure S18d).

At day 17 post‐treatment initiation, we observed a significant reduction in the individual tumor weight of mice treated with StCl‐GI_GII (P = 0.0086) or StCl‐GI_GII + αPD‐1 (P = 0.0092) (Figure S18d).

the monovalent as well as the multivalent nanoconjugates significantly induced CD3+ T cell infiltration (P < 0.0001) within tumors compared to controls (Figure 4d).

the enhanced frequency of splenic CD4+ (P < 0.0001), CD8+ (P < 0.0001), and CD8+ IL2+ T cells (P < 0.0249), and CD8+/Treg ratio (P < 0.0001) (Figure S14d–g) compared to the other treated groups.

the enhanced frequency of splenic CD4+ (P < 0.0001), CD8+ (P < 0.0001), and CD8+ IL2+ T cells (P < 0.0249), and CD8+/Treg ratio (P < 0.0001) (Figure S14d–g) compared to the other treated groups.

PD‐1 expression by CD8+ T cells became significantly reduced in animals treated with StCl‐GI_GII + αPD‐1 compared to TAAs + αPD‐1 (P = 0.0006) or PBS (P = 0.0002) (Figure 5j).

number of anti‐tumor/pro‐inflammatory tumor‐associated macrophages (TAMs) (P = 0.0404) (Figure 5i) within the TME of mice treated with StCl‐GI_GII + αPD‐1 compared to mice treated with PBS or TAAs + αPD‐1.

we observed a higher percentage of human CD3+ T cells (P = 0.0232) in the TME of animals treated with StCl‐GI_GII + αPD‐1 compared to StCl‐GI_GII (P = 0.0232) (Figure 6d).

a higher number of human CD8+ T cells in the spleen following treatment with StCl‐GI_GII + αPD‐1 compared to StCl‐GI_GII (P = 0.0023) and PBS (P = 0.0001) (Figure 6e).

we did observe a significant reduction in PD‐1‐expressing CD3+ (P = 0.0160), and CD8+ T cells (P = 0.0018) in the spleen of animals treated with StCl‐GI_GII + αPD‐1 compared to StCl‐GI_GII and PBS (P = 0.0027 and P < 0.0001, respectively) (Figure 6g,h).

we did observe a significant reduction in PD‐1‐expressing CD3+ (P = 0.0160), and CD8+ T cells (P = 0.0018) in the spleen of animals treated with StCl‐GI_GII + αPD‐1 compared to StCl‐GI_GII and PBS (P = 0.0027 and P < 0.0001, respectively) (Figure 6g,h).

We observed the increased infiltration of CD3+ (P = 0.0378), CD4+ (P = 0.0206), and CD8+ (P = 0.0293) T cells into tumors of animals treated with StCl‐GI_GII + αPD‐1 (Figure 6i–k,n).

We observed the increased infiltration of CD3+ (P = 0.0378), CD4+ (P = 0.0206), and CD8+ (P = 0.0293) T cells into tumors of animals treated with StCl‐GI_GII + αPD‐1 (Figure 6i–k,n).

We observed the increased infiltration of CD3+ (P = 0.0378), CD4+ (P = 0.0206), and CD8+ (P = 0.0293) T cells into tumors of animals treated with StCl‐GI_GII + αPD‐1 (Figure 6i–k,n).

significantly increased Caspase‐3 expression compared to PBS‐treated animals (P < 0.0001) (Figure 6l–n).

CD4+ T cell (P = 0.0002) (Figure 6j) and Caspase‐3 (P < 0.0001) (Figure 6l) levels induced by this combination immunotherapy were significantly higher than the ones quantified in the tumor of animals treated only with the multivalent conjugate.

CD4+ T cell (P = 0.0002) (Figure 6j) and Caspase‐3 (P < 0.0001) (Figure 6l) levels induced by this combination immunotherapy were significantly higher than the ones quantified in the tumor of animals treated only with the multivalent conjugate.

we observed a significant decrease in Ki67 tumor expression for StCl‐GI_GII compared to the PBS group (P = 0.0134) (Figure 6m,n).

LDH levels increased in GI+GII+TLRl–treated mice due to antigen‐associated toxicity and significantly decreased in StCl‐GI_GII‐treated mice (P = 0.0105) (Figure S13c).

lower AST levels in the blood of mice treated with StCl‐GI_GII + αPD‐1 (P = 0.0018) (Figure S17b).

a decrease in AST after treatment with StCl‐GI_GII (P = 0.0848) and GI+GII+TLRl (P = 0.0001) (Figure S13c).

a decrease in AST after treatment with StCl‐GI_GII (P = 0.0848) and GI+GII+TLRl (P = 0.0001) (Figure S13c).

we found reduced PD‐L1 expression by BECs following the administration of each nanoconjugate, with StCl‐GI + StCl‐GII‐treated mice displaying the lowest value (P = 0.0355) (Figure S15d).

Tumors from StCl‐GI + StCl‐GII‐treated mice also displayed increased levels of IFN‐γ expressing BECs (P = 0.0122) (Figure S15e) and LECs (P = 0.0027) (Figure S15f) compared to animals treated with GI +GII in solution.

Tumors from StCl‐GI + StCl‐GII‐treated mice also displayed increased levels of IFN‐γ expressing BECs (P = 0.0122) (Figure S15e) and LECs (P = 0.0027) (Figure S15f) compared to animals treated with GI +GII in solution.

StCl‐GI_GII treatment increased the percentage of MHCI‐expressing LECs (P < 0.0001) while decreasing the percentage of MHCII‐ (P = 0.0236) and PD‐L1‐expressing LECs (P < 0.0001) (Figure S16a–d,h) in tumor‐draining lymph nodes when compared to the same tissues in untreated mice.

StCl‐GI_GII treatment increased the percentage of MHCI‐expressing LECs (P < 0.0001) while decreasing the percentage of MHCII‐ (P = 0.0236) and PD‐L1‐expressing LECs (P < 0.0001) (Figure S16a–d,h) in tumor‐draining lymph nodes when compared to the same tissues in untreated mice.

B16F10 melanoma‐bearing mice treated with TAAs + αPD‐1 and StCl‐GI_GII + αPD‐1 presented with tumors displaying a significantly reduced frequency of BECs (P = 0.0315 and P = 0.0469, respectively) (Figure 5m).

B16F10 melanoma‐bearing mice treated with TAAs + αPD‐1 and StCl‐GI_GII + αPD‐1 presented with tumors displaying a significantly reduced frequency of BECs (P = 0.0315 and P = 0.0469, respectively) (Figure 5m).

only mice treated with TAAs + αPD‐1 presented with a significantly increased percentage of LECs (P = 0.0417) (Figure 5n) when compared to PBS.

Ki67 analysis demonstrated the near absence of proliferation following treatment with TAAs + αPD‐1 (P = 0.0077) and StCl‐GI_GII + αPD‐1 (P = 0.0085) (Figure 5r).

Ki67 analysis demonstrated the near absence of proliferation following treatment with TAAs + αPD‐1 (P = 0.0077) and StCl‐GI_GII + αPD‐1 (P = 0.0085) (Figure 5r).