Engineering and characterization of targeted proteins into DCIR mAb. (A) Five constructs are shown. (I) Diagram of mouse IgG1 cross-linked to the target antigen FluMP. (II-III) Diagram of chimeric mAbs (IgG4).doc conjugated to coh.antigen (FluMP [II] or MART-1 [III]). (IV-V) Diagram of chimeric fusion mAb IgG4-antigen (MART-1 [IV] or HIV gag p24 [V]). (B) Staining of HLA-A201–FluMP complexes on CD34⁺-derived DCs unpulsed (control DCs, gray histogram), or pulsed with 50nM DCIR-targeted FluMP. Cells were activated with 5 μg/mL anti-CD40 mAb (12E12, Baylor Research Institute; BIIR) and stained after 24 hours with phycoerythrin-labeled tetramerized anti–HLA-A201–FluMP Fab (M1D12).³⁹  (C) Cross-presentation of FluMP to CD8⁺ T cells by autologous HLA-A201⁺CD34⁺–derived LCs that were cultured with 8nM (top panel) or 0.8nM (bottom panel) of anti–DCIR.doc-coh.FluMP or IgG4.doc-coh.FluMP conjugate mAbs. Dot plots show the proportions of HLA-A201–FluMP(58-66) peptide tetramer-positive CD8⁺ T cells after 10 days. (D) Proportions of HLA-A201–FluMP(58-66) tetramer-positive CD8⁺ T cells induced by DCs that were pulsed for 18 hours with 8nM anti–DCIR.doc-coh.FluMP or control IgG2a.doc-coh.FluMP conjugate mAbs, washed and cultured with autologous CD8⁺ T cells for 10 days. Graphs show the proportions of HLA-A201–FluMP(58-66) tetramer-positive CD8⁺ T cells, mean ± SD; n = 3.