Fig 2: Human CA-9-targeted chimeric MINPs inhibit RAG/hCA-9 tumor growth by activating tumor-infiltrating CD8+ T cells and NK cells in immunocompetent mice. (A) Ex vivo fluorescence images of RAG/hCA-9 tumors preserved 48 h after i.v. administration of Cy5-labeled α-CA-9 NPs plus Cy5-labeled α-4-1BB/α-CD27 NPs, or Cy5-labeled α-CA-9/α-4-1BB/α-CD27 NPs (n = 5, except for the non-treatment control group where n = 3). (B) Frequencies of active IFN-γ+ tumor-infiltrating NK cells. (C) Frequencies of active IFN-γ+ tumor-infiltrating CD8+ T cells. (D) frequencies of tumor-infiltrating TEM cells. (E) The TEM-to-TCM ratio. The phenotypes of tumor-infiltrating lymphocytes were determined by the FACS method 4 days after 2 targeted or non-targeted MINP treatments (n = 5). (F) Representative immunohistochemistry images of RAG/hCA-9 tumors after treatment with free combinational antibodies or hCA-9-targeted MINPs. The chimeric MINPs were functionalized using anti-human CA-9, anti-mouse 4-1BB, and/or CD27 agonistic antibodies in these in vivo studies. Data are presented as mean ± SEM. All p-values were analyzed using two-way ANOVA with Tukey’s HSD multiple comparisons post-hoc test

Fig 3: Design and characterization of hCA-9-targeted MINPs. (A) Schematic illustration of the design of hCA-9-targeted MINPs and their proposed mechanism of action against hCA-9-expressing RCC cells. (B) TEM images of azide-functionalized PEG-PLGA NPs (N3 PEG-PLGA NPs) and α-CA-9/α-4-1BB/α-CD27 NPs (chimeric MINPs, functionalized with anti-human α-CA-9 antibody and anti-mouse 4-1BB and CD27 agonistic antibodies). This subfigure was created using BioRender. (C) Intensity-average diameter distribution curves and zeta potential distribution curves recorded for N3 PEG-PLGA NPs and α-CA-9/α-4-1BB/α-CD27 NPs, as determined through DLS and aqueous electrolysis methods. (D) Representative fluorescence histograms recorded for various MINPs stained with fluorescently labeled hCA-9, mouse 4-1BB (m4-1BB), and mouse CD27 (mCD27). (E) Representative fluorescence histograms of 786-0, A498, and Caki-1 cells after incubation with different targeted rhodamine-labeled MINPs (n = 3). (F) Representative FACS density plots of wild-type RAG cells and hCA-9-transfected RAG/hCA-9 cells after incubation with targeted and non-targeted rhodamine-labeled MINPs. The hCA-9 gene was transfected with the green fluorescent protein (GFP) gene into the parental RAG cells. Consequently, the RAG/hCA-9 cells became fluorescent in the GFP channel. (G) FACS density plots of CD8+ T cell-gated mouse splenocytes and NKp46+ NK cell-gated mouse splenocytes after incubation with different rhodamine-labeled MINPs (n = 3)

Fig 4: Human CA-9-targeted chimeric MINPs mediate mouse CD8+ T cells and NK cells to kill hCA-9-expressing RCC cells in vitro. (A) In vitro lysis of free antibody- and chimeric MINP-pretreated calcein-loaded RCC cells after coculture with CD8+ T cells for 6 h. The effector cell-to-target (E: T) ratio was 5:1. (B) In vitro lysis of free antibody- and chimeric MINP-pretreated calcein-loaded RCC cells after coculturing for 6 h with mouse NK cells. The E: T ratio was 5:1. (C) Relative viability of free antibody- and chimeric MINP-pretreated RCC cells after coculture for 72 h with mouse CD8+ T cells, as determined through an MTS assay (E: T ratio = 5:1). (D) Relative viability of free antibody- and chimeric MINP-pretreated RCC cells after coculture for 72 h with mouse CD8+ T cells, as determined through an MTS assay (E: T ratio = 5:1). The chimeric MINPs were functionalized with anti-human CA-9, anti-mouse 4-1BB, and/or CD27 agonistic antibodies in these in vitro studies. (E) Representative CLSM images of free antibody- and chimeric MINP-pretreated calcein-loaded RAG/hCA-9 cells after coculture for 6 h with CD8+ T cells or NK cells. Calcein-free RAG/hCA-9 cells and calcein-loaded RAG/hCA-9 cells were used as negative and positive controls, respectively, for the calcein channel. The E: T ratio was 5:1. The merge channel images are the same as those shown in Fig. S12. Data are presented as mean ± SEM. All p-values were analyzed using two-way ANOVA with Tukey’s HSD multiple comparisons post-hoc test

Fig 5: The MINP platform mimics plate-bound agonistic antibodies to crosslink and activate 4-1BB and/or CD27 receptors on mouse CD8+ T cells and NK cells in vitro. (A) Frequencies of CD69+ activated mouse CD8+ T cells after incubation for 6 h with different formulations of α-CA-9, α-4-1BB, and/or α-CD27 (n = 4). (B) Frequencies of CD69+ activated mouse NKp46+ NK cells after incubation for 6 h with different formulations of α-CA-9, α-4-1BB, and/or α-CD27 (n = 4). (C) Frequencies of IFN-γ+ activated mouse CD8+ T cells after incubation for 72 h with different formulations of α-CA-9, α-4-1BB, and/or α-CD27 (n = 4). (D) Frequencies of IFN-γ+ activated mouse NK cells after incubation for 72 h with different formulations of α-CA-9, α-4-1BB, and/or α-CD27 (n = 4). Anti-human CA-9, anti-mouse 4-1BB, and CD27 agonistic antibodies were used in these studies. Data are presented as mean ± SEM. All p-values were analyzed using two-way ANOVA with Tukey’s HSD multiple comparisons post-hoc test