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. 2009;11(6):R195.
doi: 10.1186/ar2897. Epub 2009 Dec 23.

Active immunization to tumor necrosis factor-alpha is effective in treating chronic established inflammatory disease: a long-term study in a transgenic model of arthritis

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Active immunization to tumor necrosis factor-alpha is effective in treating chronic established inflammatory disease: a long-term study in a transgenic model of arthritis

Laure Delavallée et al. Arthritis Res Ther. 2009.

Abstract

Introduction: Passive blockade of tumor necrosis factor-alpha (TNF-alpha) has demonstrated high therapeutic efficiency in chronic inflammatory diseases, such as rheumatoid arthritis, although some concerns remain such as occurrence of resistance and high cost. These limitations prompted investigations of an alternative strategy to target TNF-alpha. This study sought to demonstrate a long-lasting therapeutic effect on established arthritis of an active immunotherapy to human (h) TNF-alpha and to evaluate the long-term consequences of an endogenous anti-TNF-alpha response.

Methods: hTNF-alpha transgenic mice, which spontaneously develop arthritides from 8 weeks of age, were immunized with a heterocomplex (TNF kinoid, or TNF-K) composed of hTNF-alpha and keyhole limpet hemocyanin after disease onset. We evaluated arthritides by clinical and histological assessment, and titers of neutralizing anti-hTNF-alpha antibody by enzyme-linked immunosorbent assay and L929 assay.

Results: Arthritides were dramatically improved compared to control mice at week 27. TNF-K-treated mice exhibited high levels of neutralizing anti-hTNF-alpha antibodies. Between weeks 27 and 45, all immunized mice exhibited symptoms of clinical deterioration and a parallel decrease in anti-hTNF-alpha neutralizing antibodies. A maintenance dose of TNF-K reversed the clinical deterioration and increased the anti-hTNF-alpha antibody titer. At 45 weeks, TNF-K long-term efficacy was confirmed by low clinical and mild histological scores for the TNF-K-treated mice. Injections of unmodified hTNF-alpha did not induce a recall response to hTNF-alpha in TNF-K immunized mice.

Conclusions: Anti-TNF-alpha immunotherapy with TNF-K has a sustained but reversible therapeutic efficacy in an established disease model, supporting the potential suitability of this approach in treating human disease.

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Figures

Figure 1
Figure 1
Clinical evaluation of human tumor necrosis factor-alpha transgenic (TTg) mice immunized with tumor necrosis factor kinoid (TNF-K) or phosphate-buffered saline (PBS) or treated with infliximab (IFX). TTg mice were immunized with TNF-K or PBS emulsified in ISA-51 adjuvant or were IFX-treated. All mice were monitored for clinical signs of arthritis and for weight for 18 or 36 weeks. (a) TTg mice received three primary injections at 15, 16 and 19 weeks of age (open arrows) of TNF-K (n = 15, open and closed diamonds) or PBS (n = 8, squares). At 32 weeks of age (shaded arrow), TTg mice received a maintenance dose (md) of TNF-K (n = 7, open diamonds) or an injection of PBS emulsified in ISA-51 adjuvant (n = 8, closed diamonds). Eight TTg mice (circles) received weekly intraperitoneal injections of IFX (bold arrows) from week 15 for a period of 12 weeks (until 27 weeks of age). (b) The weight gain of all groups is represented. Results are expressed as mean ± standard error of the mean. *P < 0.05 versus PBS.
Figure 2
Figure 2
Examples of histological evaluation of tumor necrosis factor-alpha transgenic (TTg) mice immunized with tumor necrosis factor kinoid (TNF-K) or phosphate-buffered saline (PBS). Histological sections (magnification × 40) of the knees of TNF-K- or PBS-treated mice were prepared (see Materials and methods) and colored with hematoxylin and eosin (a, b) to observe synovial inflammation or with safranin-O (c, d) to observe cartilage degradation. For the histological sections of TTg mice immunized with TNF-K, inflammation (a) and destruction (c) were scored at 0; for the control group, inflammation (b) and destruction (d) were scored at 2. Black arrows show thickness and inflammatory infiltration of synovial membrane in (b) and a normal appearance in (a). White arrows show depletion of proteoglycan (a marker for cartilage destruction) in (d) and a normal full-red staining in (c).
Figure 3
Figure 3
Clinical score time trend. The severity of disease evolution over time was analyzed using Spearman rank correlation. We correlated clinical scores with the age of the mice, expressed in weeks, and divided the study into two periods of time. (a) Correlation between week 21 and week 32 for all of the immunized mice (n = 15). We observed an aggravation of disease in all mice immunized with tumor necrosis factor kinoid (TNF-K) a couple of weeks after the last immunization. (b) Correlation between week 33 and week 45 for immunized mice not receiving the maintenance dose (md). We observed an aggravation of the severity of the disease. (c) Correlation between week 33 and week 45 for immunized mice receiving the maintenance dose. After the maintenance dose at 32 weeks of age, we observed an amelioration of the scores. (d) Correlation between week 28 and week 45 for infliximab-treated mice. The injections were stopped at week 27, and we observed an aggravation of the disease over time thereafter. CI, confidence interval.
Figure 4
Figure 4
Evaluation of anti-human tumor necrosis factor-alpha (anti-hTNF-α) antibody production in TNF-α transgenic (TTg) mice immunized with TNF kinoid (TNF-K). TTg mice were immunized at 15, 16, and 19 weeks of age (open arrows) with TNF-K. (a) Enzyme-linked immunosorbent assay of anti-hTNF-α antibodies. (b) The neutralizing capacity of the anti-hTNF-α antibody was evaluated on L929 cells and is expressed as the mean of the reciprocal of the serum dilution that neutralizes 50% of hTNF-α activity (NC50). Closed histograms represent mice that did not receive the TNF-K maintenance dose (TNF-K without md) at 32 weeks of age (shaded arrow). Open histograms represent mice that did receive it (TNF-K with md). Results are expressed as mean ± standard error of the mean. *P < 0.05.
Figure 5
Figure 5
B-memory response after tumor necrosis factor kinoid (TNF-K) immunization. Thirty-six human tumor necrosis factor-alpha (hTNF-α) transgenic mice were immunized with TNF-K at 7 (day 0), 8 (day 7), and 11 (day 28) weeks of age. Bleeding was done every month from 12 weeks of age (day 38) until sacrifice. When we observed a decline of the anti-hTNF-α neutralizing antibody titer (closed symbols), we injected intraperitoneally (arrow) native hTNF-α (10 ng, n = 10, diamonds) (a, b), native hTNF-α (100 ng, n = 9, squares) (c, d), keyhole limpet hemocyanin (KLH) (10 μg, n = 10, circles) (e, f), or phosphate-buffered saline (equivalent volume, n = 10, triangles) (g, h). We studied the anti-KLH antibody titer (open symbols) and neutralizing anti-hTNF-α antibody titer (closed symbols) for 10 weeks (70 days). Each single plot represents the antibody titer of one mouse. The bold line represents the mean antibody titer at each time point. *P < 0.001 versus day 149; **P < 0.0001 versus day 171; #P < 0.05 versus day 178; ##P < 0.05 versus day 149. NC50, mean of the reciprocal of the serum dilution that neutralizes 50% of hTNF-α activity.

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