Modulation of anti-tumor necrosis factor alpha (TNF-α) antibody secretion in mice immunized with TNF-α kinoid

Abstract

Tumor necrosis factor alpha (TNF-α) blockade is an effective treatment for patients with TNF-α-dependent chronic inflammatory diseases, such as rheumatoid arthritis, Crohn's disease, and psoriasis. TNF-α kinoid, a heterocomplex of human TNF-α and keyhole limpet hemocyanin (KLH) (TNF-K), is an active immunotherapy targeting TNF-α. Since the TNF-K approach is an active immunization, and patients receiving this therapy also receive immunosuppressant treatment, we evaluated the effect of some immunosuppressive drugs on the generation of anti-TNF-α antibodies produced during TNF-K treatment. BALB/c mice were injected intramuscularly with TNF-K in ISA 51 adjuvant. Mice were also injected intraperitoneally with one of the following: phosphate-buffered saline, cyclophosphamide, methylprednisolone, or methotrexate. Anti-TNF-α and anti-KLH antibody levels were assessed by enzyme-linked immunosorbent assay and the anti-TNF-α neutralizing capacity of sera by L929 bioassay. Our results showed that current treatments used in rheumatoid arthritis, such as methylprednisolone and methotrexate, do not significantly alter anti-TNF-α antibody production after TNF-K immunization. In contrast, the administration of cyclophosphamide (200 mg/kg) after immunization significantly reduced anti-TNF-α antibody titers and their neutralizing capacity.

Fig 1

Effect of chronic immunosuppressant treatment on anti-hTNF-α (A) and anti-KLH (B) Ab titers in sera of TNF-K-immunized mice, showing a significant production peak at day 60 and a significant decrease at day 70 for all groups (within-group variation, P < 0.001), except for anti-KLH antibodies in group A2. BALB/c mice received four TNF-K immunizations (days 0, 7, 28, and 49). Chronic injections of low doses of immunosuppressants started either 6 days before (A2 and B2) or 4 days after (A1 and B1) the first injection of TNF-K. Anti-KLH and anti-hTNF-α Abs in sera were quantified by ELISA. Results are expressed as the dilution factor giving half-maximal absorbance (medians and interquartile ranges are reported).

Fig 2

Effect of chronic immunosuppressant treatment on hTNF-α-neutralizing capacities in sera of TNF-K immunized mice, evaluated by an L929 bioassay. A significant production peak at day 60 and a significant decrease at day 70 were found for all groups (within-group variation, P < 0.001). Chronic injections of low doses of immunosuppressants starting either 6 days before (A2 and B2) or 4 days after (A1 and B1) the first injection of TNF-K did not induce significant reduction of neutralizing capacity. Neutralizing titers of individual mice are expressed as the reciprocal of the serum dilution that neutralizes 50% of hTNF-α activity (medians and interquartile ranges are reported).

Fig 3

Effect of short-term immunosuppressant treatment on anti-hTNF-α (A) and anti-KLH (B) Ab titers in sera of TNF-K-immunized mice, showing an overall lower anti-hTNF-α Ab production in the CYC group (P < 0.01) and a significant reduction in Ab levels at days 60 and 70 compared to all other groups (P < 0.001). Mice received three TNF-K immunizations (days 0, 7, and 28) before high-dose immunosuppressant administration. Mice received 2 (CYC) or 4 (MP, MTX, and PBS) doses between day 35 and day 44. Anti-KLH and anti-hTNF-α Abs in sera were quantified by ELISA. Results are expressed as the dilution factor giving half-maximal absorbance (medians and interquartile ranges are reported).

Fig 4

Effect of short-term immunosuppressant treatment on hTNF-α-neutralizing capacities in sera of TNF-K-immunized mice, evaluated by an L929 bioassay. An overall lower rate of production of anti-hTNF-α-neutralizing Ab was found in the CYC group (P < 0.05), with a significant decrease at day 60 and day 70, resulting in lower neutralizing capacity than in all other groups (P < 0.001). Neutralizing titers are expressed as the reciprocal of the serum dilution that neutralizes 50% of hTNF-α activity (medians and interquartile ranges are reported).