Chemical conjugation of ADX40 to A20-Id is required to achieve antitumor effects. (A) Immunization with ADX40 conjugated to the irrelevant mouse IgG 16.5H2 (gray line) did not enhance mouse survival compared with the vehicle control PBS (black line). (B) In addition, admixing ADX40 with A20-Id (light gray line) was not sufficient to induce antitumor immune responses leading to tumor protection. Chemical conjugation of ADX40 to A20-Id (dark gray line) resulted in significantly increased overall survival compared with admixed ADX40 and A20-Id and PBS (black line). (C) In vitro culture of splenocytes with ADX40-A20_FITC and 20C2-A20_FITC showed specific uptake of ADX40-A20 _FITC through the CD40 receptor, with a significantly increased fluorescence on B cells (black bars), DCs (open bars), and macrophages (hashed bars) compared with isotype control. (D) MHC class II expression was significantly enhanced on B cells (black bars) in response to uptake of ADX40-A20_FITC compared with the isotype control. Panels C and D represent 1 experiment with n = 3 of a total of 4 individual experiments carried out. Background fluorescence (cells cultured with concanavalin A) has been subtracted from plotted values in panels C and D. MFI indicates mean fluorescent intensity. *P < .05; **P < .005; and ***P < .0005. (E) Confocal image (40× magnification) showing cells from left draining lymph node (20C2-A20_FITC), in which there is no green fluorescence. (F) Confocal image (40× magnification) of cells from the right draining lymph node (ADX40-A20 _FITC) showing the presence of green fluorescence in the cytoplasm of lymph cells, indicating specific A20-Id uptake by these cells.