Novel genetically-humanized mouse model established to evaluate efficacy of therapeutic agents to human interleukin-6 receptor

Abstract

For clinical trials of therapeutic monoclonal antibodies (mAbs) to be successful, their efficacy needs to be adequately evaluated in preclinical experiments. However, in many cases it is difficult to evaluate the candidate mAbs using animal disease models because of lower cross-reactivity to the orthologous target molecules. In this study we have established a novel humanized Castleman's disease mouse model, in which the endogenous interleukin-6 receptor gene is successfully replaced by human IL6R, and human IL6 is overexpressed. We have also demonstrated the therapeutic effects of an antibody that neutralizes human IL6R, tocilizumab, on the symptoms in this mouse model. Plasma levels of human soluble IL6R and human IL6 were elevated after 4-week treatment of tocilizumab in this mouse model similarly to the result previously reported in patients treated with tocilizumab. Our mouse model provides us with a novel means of evaluating the in vivo efficacy of human IL6R-specific therapeutic agents.

Figure 1. Generation of human IL6 receptor (IL6R) gene knock-in mouse.

(a) Schematic representation of the knock-in strategy for the hIL6R gene. A knock-in vector was constructed by inserting hIL6R cDNA with neo cassette flanked by two loxP sites into the mouse Il6ra genomic locus in the frame of a BAC genomic clone. A knock-in allele and a neo-deleted knock-in allele are also shown. Arrows indicate PCR primers (m10355 and m11166R) for genotyping. TC, terminal codon. (b) A representative result of genotyping to confirm the neo-depleted hIL6R knock-in allele and homozygosity of the hIL6R knock-in allele. Wild-type allele and knock-in allele were detected as signals of 0.8 kb and 4.2 kb, respectively, whereas knock-in allele after removing neo cassette was detected as a signal of 2.7 kb. M, DNA molecular marker. Numbers above the gel denote the mouse genotypes, (1) Il6ra^+/+, (2) Il6ra^hIL6R/+, (3) Il6ra^hIL6R/hIL6R and (4) Il6ra^{hIL6R(neo)/hILR6(neo)}. (c) Representative results of RT-PCR analysis for tissue distribution of Il6ra^+/+ (Wi) and Il6ra^hIL6R/hIL6R (Ho) mice. (d) Plasma levels of soluble hIL6R in Il6ra^+/+ (n = 14), Il6ra^hIL6R/+ (n = 16) and Il6ra^hIL6R/hIL6R mice (n = 13). (e) Species-specific ligand response was confirmed after intraperitoneal injection of mouse Il6 (mIL6) or human IL6 (hIL6) in Il6ra^+/+ and in Il6ra^hIL6R/hIL6R mice. Ligand responses were evaluated by the elevation of plasma SAA levels after injection of vehicle (n = 3), mIL6 (n = 4) and hIL6 (n = 3) in Il6ra^+/+ and those of vehicle (n = 2), mIL6 (n = 3) and hIL6 (n = 3) in Il6ra^hIL6R/hIL6R.

Figure 2. Treatment with an hIL6R-neutralizing antibody in humanized Castleman's disease model mice.

(a) Protocol for 4-week treatment with the anti-mouse Il6ra antibody, MR16-1, or the anti-human IL6R antibody, tocilizumab (TCZ). (iv.), intravenous injection; (sc.) subcutaneous injection. (b) Spleen weights of Il6ra^+/+-hIL6 transgenic mice and (c) Il6ra^hIL6R/hIL6R-hIL6 transgenic mice after 4-week treatment (n = 5 per group). Statistical significances were determined by nonparametric comparisons with control using the Dunn method for joint ranking. The data of each treatment group were compared with those of the respective hIL6 non-transgenic mouse group in each genotype of interleukin-6 receptor, Il6ra^+/+ (b) and Il6ra^hIL6R/hIL6R (c). *, p < 0.05, **, p < 0.01 and NS, not significant. Non Tg, hIL6 non-transgenic mice; Tg, hIL6 transgenic mice.

Figure 3. Spleen tissue of an Il6ra^hIL6R/hIL6R mouse (a, d), an Il6ra^hIL6R/hIL6R-hIL6 transgenic mouse (b, e), and a tocilizumab-treated Il6ra^hIL6R/hIL6R-hIL6 transgenic mouse (c, f).

Increase of plasma cells and increased numbers of white pulp in the Il6ra^hIL6R/hIL6R-hIL6 transgenic mouse (b, e) were ameliorated after 4-week treatment with tocilizumab (c, f). Plasma cells are shown in insert (b). Bars: (a–c), 100 μm; (d–f), 500 μm. W, white pulp.

Figure 4. Plasma levels of soluble hIL6R (a) and hIL6 (b) concentration after 4-week treatment with 0.1, 0.25 and 0.5 mg/body of TCZ in each genotype of mouse (n = 5 per group).

(a) Plasma soluble hIL6R concentrations were approximately 21 ng/ml in saline-treated Il6ra^hIL6R/hIL6R-hIL6 transgenic mice, whereas marked elevation of plasma soluble hIL6R levels, approximately 40–50 times higher than those of vehicle control, was observed after 4 weeks in TCZ-treated Il6ra^hIL6R/hIL6R-hIL6 transgenic mice. (b) Plasma hIL6 was detected at the level of 163 pg/ml in saline-treated Il6ra^hIL6R/hIL6R-hIL6 transgenic mice, whereas the hIL6 levels were markedly elevated to the levels of 936–1204 pg/ml after 4-week treatment of TCZ. ND, not detected. Non Tg, hIL6 non-transgenic mice; Tg, hIL6 transgenic mice.

Figure 5. Titers of plasma anti-drug antibodies after 4-week treatment with 0.1, 0.25 and 0.5 mg/body of TCZ in each genotype of mouse (n = 5 per group).

Extremely high levels of plasma anti-TCZ-antibody titers were detected in Il6ra^+/+-hIL6 transgenic mice, whereas those in TCZ-treated Il6ra^hIL6R/hIL6R-hIL6 transgenic mice were minimally detected. Non Tg, hIL6 non-transgenic mice; Tg, hIL6 transgenic mice.