Multiple Tolerization Reduces Antibody Binding Against Tolerogen Cells: Implications for the Production of Monoclonal Antibodies

Abstract

We report an immunization technique that can update the production of monoclonal antibodies (mAbs): the multiple tolerization subtractive immunization (MTSI). A total of 10 BALB/C mice were used. Animals in group 1 received one inoculation of RWPE-1 cells (nontumoral), followed by cyclophosphamide, and then received serial inoculations of nonirradiated PC3 cells (tumoral). Animals in group 2 received our MTSI protocol, as follows: one inoculation of RWPE-1 cells, followed by cyclophosphamide (Cy). This whole tolerization step was repeated three other times, with 14-day intervals between the last Cy exposure and the next RWPE-1 cell inoculation. Finally, the animals received the same nonirradiated PC3 cell exposure as group 1. Blood was taken from each animal, and their polyclonal sera individually tested against the nontumoral RWPE-1 cells in flow cytometry. We found out that, after the MTSI was employed, the serum of the immunized animals, in group 2, contained considerably less antibodies that reacted against the tolerogenic cells, compared with the serum of the animals that underwent regular subtractive immunization. We showed that, by repeating the tolerization cycles, the polyclonal antibodies produced by mice have a reduced specificity toward common/immunodominant epitopes present at nontumoral cells, and thus this technique can be readily used by others in studies involving murine mAb protocols.

Multiple Tolerization Subtractive Immunization (MTSI) Protocol: Effects on Mice and Monoclonal Antibody Specificity

“…In a previous study, published by our group, we demonstrated, through flow cytometry assays, that the reactivity percentage of polyclonal antibodies (pAbs) towards non-tumor prostatic cells lowers as the tolerization steps are repeated. Two groups of immunized mice were used: i) the first that received only one tolerization step and ii) the other one that received four tolerization steps ( 16 ).…”

“…In a previous study, published by our group, we demonstrated, through flow cytometry assays, that the reactivity percentage of polyclonal antibodies (pAbs) towards non-tumor prostatic cells lowers as the tolerization steps are repeated. Two groups of immunized mice were used: i) the first that received only one tolerization step and ii) the other one that received four tolerization steps (16). Now, to increase the performance of the described MTSI technique (16), we have now performed three different immunization schedules, using different dosages of cyclophosphamide (Cy) (200, 150 and 100 mg/kg BW).…”

“…Two groups of immunized mice were used: i) the first that received only one tolerization step and ii) the other one that received four tolerization steps (16). Now, to increase the performance of the described MTSI technique (16), we have now performed three different immunization schedules, using different dosages of cyclophosphamide (Cy) (200, 150 and 100 mg/kg BW). In the present study, we confirmed the extensive deleterious effects on mice that received 200 mg/kg BW and 150 mg/kg BW of CY, including progressive weight loss, hair loss, and prominent body curvature.…”

“…This step was repeated three other times, with 15-days interval. Finally, on day 68 mice were immunized with immunogen cells (PC-3, 2 × 10 6 cells/mouse), which was repeated four other times, with a 10-days interval between each immunization ( 15 , 16 ). Three different MTSI protocols were performed: i) one with 200 mg/kg body weight (BW) of CY; ii) the second with 150 mg/kg BW; iii) another with 100 mg/kg BW.…”

“…After tolerization, mice are immunized with the immunogen. Thus, it is expected that an effective cellular and humoral immune response directed to immunogen-specific antigens will be activated, leading to the generation of antibodies focused on "hidden" or weakly antigens, associated with virulence, immune evasion or metastatic process of malignant cells (14)(15)(16).…”