Fig 1: 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 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: The MINP platform reduces hepatotoxicity induced by agonistic α-4-1BB and α-CD25 antibodies in healthy immunocompetent mice. (A) Serum ALT, AST, and BUN levels recorded for healthy BALB/c mice 48 h after 4 i.v. administrations of free α-CA-9, α-4-1BB, and α-CD27, or α-CA-9/α-4-1BB/α-CD27 NPs (chimeric MINPs) (n = 7, except for mice treated with MINPs, where n = 8). (B) Representative H&E-stained images of the liver, lung, spleen, and kidney preserved from mice at the study endpoint. (C) Representative immunohistochemistry images of liver sections preserved at the study endpoint after different treatments. (D) Serum DyLight 650 fluorescent intensity 6 h after i.v. administration of DyLight 650 labeled α-CA-9, α-4-1BB, and α-CD27 or α-CA-9/α-4-1BB/α-CD27 NPs (functionalized with DyLight 650-labeled antibodies) (n = 5, except for the non-treatment control group, where n = 3). The chimeric MINPs were functionalized using anti-human CA-9, anti-mouse 4-1BB, and 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
Supplier Page from BioLegend for Recombinant Mouse TNFRSF9 (carrier-free)