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. 2015 Feb 15;194(4):1695-701.
doi: 10.4049/jimmunol.1402324. Epub 2015 Jan 5.

Bortezomib reduces pre-existing antibodies to recombinant immunotoxins in mice

Michael L Manning  1 , Emily Mason-Osann  1 , Masanori Onda  1 , Ira Pastan  2
Affiliations

Bortezomib reduces pre-existing antibodies to recombinant immunotoxins in mice

Michael L Manning et al. J Immunol. .

Abstract

Recombinant immunotoxin (RIT) therapy is limited in patients by neutralizing Ab responses. Ninety percent of patients with normal immune systems make neutralizing Abs after one cycle of RIT, preventing repeated dosing. Furthermore, some patients have pre-existing Abs from environmental exposure to Pseudomonas exotoxin, the component of the RIT that elicits the neutralizing Ab response. Bortezomib is an U.S. Food and Drug Administration-approved proteasome inhibitor that selectively targets and kills plasma cells that are necessary for the neutralizing Ab response. We hypothesized that bortezomib may abrogate neutralizing Ab levels, making dosing of RIT possible in mice already immune to RIT. We immunized BALB/c mice with multiple doses of SS1P, a RIT whose Ab portion targets mesothelin. Mice with elevated Ab levels were separated into groups to receive saline, bortezomib, the pentostatin/cyclophosphamide (PC) regimen, or the bortezomib/PC (BPC) combination regimen. Four weeks after finishing therapy, plasma Ab levels were assayed, and bone marrow was harvested. The bortezomib and PC regimens significantly reduced Ab levels, and we observed fewer plasma cells in the bone marrow of bortezomib-treated mice but not in PC-treated mice. The BPC combination regimen almost completely eliminated Abs and further reduced plasma cells in the bone marrow. This regimen is more effective than individual regimens and may reduce Ab levels in patients with pre-existing neutralizing Abs to Pseudomonas exotoxin, allowing RIT treatment.

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Figures

FIGURE 1
FIGURE 1
B, PC, and BPC regimens all reduce anti-SS1P antibody levels (panels A, B, and C, respectively). Four weeks after therapy plasma was collected and anti-SS1P antibody levels were determined by ELISA. Results for individual mice and group averages are shown. Initial antibody levels for each mouse were normalized to 100% and antibody levels after treatment are scaled relative to initial levels. ELISAs were performed in triplicate. Error bars indicate SEM. The entire experiment was repeated once.
FIGURE 2
FIGURE 2
The B and PC regimens reduce anti-SS1P antibody levels and the BPC regimen nearly eliminates them. Each regimen lowered antibody levels relative to initial levels (percent anti-SS1P IgG reduction and P values are relative to saline in each experiment and indicated on respective graphs). The line represents the median. There was no difference in initial antibody levels between the B, PC and BPC groups. After treatment, the BPC regimen outperformed both the B and PC regimens (both P < 0.0001). Data shown is compilation of two independent experiments.
FIGURE 3
FIGURE 3
Lower levels of plasma cells detected in the bone marrow of experimental mice compared to control mice. Bone marrow cells were harvested 4 weeks after therapy and stained for CD19, B220/CD45R, CD38, and CD138. Expression was analyzed by flow cytometry. (A) Dot plots representative of experimental groups; CD38+/CD138+ plasma cells are gated in the black rectangle and percentage of total bone marrow cells is indicated above the gated cells. (B) Percentage of plasma cells detected in the bone marrow of control and experimental mice. The experiment was repeated on separate days with similar results and data was combined. Error bars indicate SEM. P values are relative to control (saline). *P < 0.05, ***P < 0.001.
FIGURE 4
FIGURE 4
Bortezomib reduced anti-SS1P antibody levels after a 9-month resting period. Immunized mice rested for 9 months before treatment with the B regimen. Antibody levels rose during this period (P < 0.05). Subsequent B treatment lowered antibody levels relative to levels before treatment (P < 0.05), but not to levels prior to the 9-month resting period (P = 0.41). Antibody levels were determined by ELISA, which was done in triplicate. The line represents mean and error bars indicate SEM.
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