Ozoralizumab is a 38 kDa humanized trivalent bispecific construct consisting of two anti-TNFα NANOBODIES^® and anti-HSA NANOBODY^® that was generated at Ablynx by a previously described method (23). Llamas were immunized with human TNFα and human muscle extract, which is rich in HSA, to induce the formation of anti-TNFα VHH and anti-HSA VHH. Both the anti-TNFα VHH and anti-HSA VHH were humanized by a complementary determining regions (CDR) grafting approach in which the CDR of the gene encoding llama VHH was grafted onto the most homologous human VHH framework sequence. Since binding to serum albumin prolongs the half-life of VHH (23, 26, 27), an anti-HSA VHH which efficiently binds murine serum albumin as well was incorporated into the two anti-TNFα VHHs. The three components were fused using a flexible Gly-Ser linker.

TNFα-sensitive mouse fibrosarcoma cells (L929 cells) were seeded in 96-well plates at a density of 5 × 10³ cells per well and grown for 48 hours as previously described (24, 28, 29). Various concentrations of ozoralizumab or clinical-glade adalimumab (Eisai, Japan) and 0.3 ng/mL human TNFα (FUJIFILM Wako Pure Chemical Corporation, Japan) were incubated in fresh Dulbecco’s Modified Eagle Medium (DMEM) for 15 minutes at room temperature and then added to designated wells containing L929 cells. The L929 cells were incubated for 24 hours at 37°C in this preincubated-medium with 1 μg/mL actinomycin D and then incubated for 10 minutes at room temperature with cell proliferation reagent CellTiter-Glo^® 2.0 (Promega Corporation, United States). Cell viability was measured by using the CellTiter-Glo assay according to the manufacturer’s protocol (30). The 50% inhibitory concentration (IC₅₀) of each TNF inhibitor was determined by plotting titration curves in the XLfit software package (IDBS, United Kingdom).

Tg197 human TNF transgenic mice (31) were bred and maintained in the animal facilities of Biomedcode Hellas SA under pathogen-free conditions. The animals were housed in standard plastic cages with wood chip bedding. The animal facility was maintained on an inverted 12/12-hour light/dark cycle at a constant room temperature of 22°C ± 2°C and approximately 60% relative humidity. Access to food pellets and filtered water were provided ad libitum. The test facility is accredited by the Hellenic Republic Directorate of Agricultural and Veterinary Policy (DAVP) of the Attica Region. All of the testing conditions conformed to the Presidential Decree applicable in Greece, which is the implementation of the EEC Directive 2010/63/EEC and approved by the DAVP of the Attica Region.

The Tg197 mouse is a well-established animal model of arthritis that can be used to investigate the pathogenesis and treatment of rheumatoid disease in humans (31, 32). Tg197 mice develop chronic polyarthritis with a 100% incidence starting at 3–4 weeks of age as a result of the overexpression of bioactive human TNF. The animal studies were conducted at Biomedcode Hellas SA, Greece. In the first study, 3-week-old mice were divided into 3 groups, each consisting of 10 heterozygous Tg197 male and female mice. The mean body weights and the genders of the animals were equally distributed among the 3 groups. The animals in the vehicle group were subcutaneously injected with phosphate-buffered saline (PBS) twice weekly from the start of the study at 3 weeks of age until euthanized at 11 weeks of age. The animals in the adalimumab group and ozoralizumab group were subcutaneously injected with 1 mg/kg anti-TNFα antibody twice weekly from the start of the study at 3 weeks of age until the end of the study at 16 weeks of age. Adalimumab (Abbvie) was purchased locally by Biomedcode Hellas SA. Serum samples were collected at 6, 8, 10 (all groups), 12, 14, and 16 (adalimumab group and ozoralizumab group) weeks of age. The animals in the vehicle group were euthanized at 11 weeks of age because of the increased mortality exhibited by the untreated Tg197 mice at that age.

The second study was conducted on 3-week-old Tg197 mice. The mice were divided into two groups: a vehicle group of 10 mice and an adalimumab group of 32 mice. The animals in the vehicle group were subcutaneously injected with PBS twice weekly until euthanized at 11 weeks of age, and the animals in the adalimumab group were subcutaneously injected with 1 mg/kg adalimumab twice weekly for 4 weeks. The 9 mice that had failed to generate ADAs against adalimumab at 6 weeks of age were eliminated from the study, and the ADA-positive animals in the adalimumab group were divided into two groups with equally distributed mean body weights and arthritis scores. The mice in one group continued to receive 1 mg/kg adalimumab (continued group, n = 13), and the mice in the other group were switched to administration of 1 mg/kg ozoralizumab (switched group, n = 10) at 7 weeks of age. The animals in the continued group and the switched group were subcutaneously injected with anti-TNFα antibody twice weekly until the end of the study at 12 weeks of age. The animals in the vehicle group were euthanized at 11 weeks of age because of the increased mortality exhibited by the untreated Tg197 mice at that age.

The analyses of ADAs against adalimumab or ozoralizumab and serum concentrations of anti-TNFα antibodies were performed 96 hours and 48 hours after antibody administration in the first study and the second study, respectively.

Heterozygous 6-week-old Tg197 mice were gender-balanced and assigned to two groups for the pharmacokinetic study of adalimumab and ozoralizumab (35–37). The animals in the adalimumab group (n = 4) and the ozoralizumab group (n = 4) were subcutaneously injected with adalimumab or ozoralizumab both at 1 mg/kg. Blood samples were collected at 1, 6, 24, 48, 72, and 96 hours after administration of the anti-TNFα antibodies. Serum was isolated by centrifugation at 3,800 g at 4°C for 8 minutes, followed by transfer of the supernatants to tubes that were further centrifuged at 15,300 g at 4°C for 8 minutes. Adalimumab and ozoralizumab concentrations in the serum samples were measured by the enzyme-linked immunosorbent assays (ELISAs) described below, and the pharmacokinetic parameters of the anti-TNFα antibodies were calculated by using the WinNonlin version 8.0 software program (Certara, United States).

Serum adalimumab and ozoralizumab concentrations were measured by ELISA (37). Immobilizer streptavidin F96 clear plates (Thermo Fisher Scientific, United States) were coated with 200 ng/mL biotinylated human TNFα for 1 hour at room temperature. The wells were washed with PBS containing 0.05% Tween 20 buffer (PBST) and then blocked for 1.5 to 3 hours at room temperature with PBST containing 1% bovine serum albumin (BSA). The serum samples of Tg197 mice in each study were 200-fold diluted with PBST containing 1% BSA. After washing the plates with PBST, the diluted serum samples were added to the wells, and the plates were allowed to stand overnight at 4°C. Adalimumab was detected with anti-human IgG-horseradish peroxidase (HRP) goat antibody (Jackson ImmunoResearch Laboratories, United States), and ozoralizumab was detected with anti-ozoralizumab rabbit antibody (Generated by Toray Research Center, Japan) followed by anti-rabbit IgG-HRP goat antibody (Bio-Rad Laboratories, United States). HRP activity was measured by using SureBlue™ 3,3’,5,5’-Tetramethylbenzidine (TMB) 1-Component Microwell Peroxidase Substrate (SeraCare Life Sciences, United States) according to the manufacturer’s protocol, and absorbance at 450 nm was measured with an ARVO X3 plate reader (PerkinElmer, United States). A standard curve was plotted for adalimumab and ozoralizumab in each plate in order to calculate the serum concentrations.

ADAs against adalimumab and ozoralizumab were measured by ELISA (37). The wells of 96-well microplates (Corning, United States) were coated with 1 μg/mL adalimumab or 500 ng/mL ozoralizumab overnight at room temperature and then washed with PBST. The wells were blocked for 1 hour at room temperature with PBST containing 4% BSA and then washed with PBST. Serum samples diluted 50-fold with PBST containing 1% BSA were added to the wells, and the plates were incubated for 1.5 hours at room temperature. After washing the wells with PBST, 3 μg/mL biotinylated adalimumab or 1 μg/mL biotinylated ozoralizumab was added to the wells, and the plates were incubated for 1.5 hours at room temperature. After washing the wells with PBST, HRP streptavidin (Vector Laboratories, United States) was added to the wells, and the plates were incubated for an additional 1.5 hours at room temperature. HRP activity was measured as described above in section 2.6 Measurement of serum concentrations of adalimumab and ozoralizumab. Response ratios of the diluted serum samples were calculated as the ratio of the absorbance of each sample to that of the negative control (serum of non-treated mice). The serum samples were examined for the presence of ADAs by performing a screening assay. Serum samples whose response ratio was more than twice that of the negative control (cut-off point) were considered ADA-positive. The titers of the positive samples were calculated by using a titering assay. The maximum dilution factor of each ADA-positive serum sample was calculated as its relative titer.

Neutralizing ADAs raised in the Tg197 mice injected with adalimumab or ozoralizumab were evaluated as follows. The wells of immobilizer streptavidin F96 clear plates (Thermo Fisher Scientific, United States) were coated with 200 ng/mL biotinylated human TNFα for 1 hour at room temperature. The wells were then washed with PBST and blocked for 1.5 to 3 hours at room temperature with PBS containing 1% BSA, and the wells were washed again with PBST. The serum samples were diluted 1,000-fold with PBS containing 1% BSA and mixed with 0.15 ng/mL anti-TNFα antibody (this concentration was equivalent to the approximately one thousandth of serum concentration of the mice 6–72 hours after a 1 mg/kg subcutaneous administration of anti-TNFα antibody). After pre-incubation for 90 minutes at room temperature, the mixtures were added to designated wells and incubated overnight at 4°C. The wells were then washed with PBST, and the amounts of active adalimumab and ozoralizumab were measured as described above in section 2.6 Measurement of serum concentrations of adalimumab and ozoralizumab. The neutralizing effect of the serum was estimated based on the decrease in active anti-TNFα antibodies.

Bioactive anti-TNFα antibodies remaining in the serum samples of the Tg197 mice were measured by a TNFα-induced cytotoxicity assay using L929 mouse fibroblast cells. After incubating 0.03 ng/mL human TNFα and a 1,000-fold diluted serum sample from 12-week-old mice in the switched group in fresh DMEM for 15 minutes at room temperature, it was added to designated wells containing L929 cells (38). Cell seeding and a cell viability assay were performed as described above in section 2.2 In vitro L929 cell cytotoxicity assay.

Formation of immune complexes by human TNFα and anti-TNFα antibodies was evaluated by an Ouchterlony double-diffusion assay. The central well of a rosette pattern of wells in an Ouchterlony gel plate (Thermo Fisher Scientific, United States) was loaded with 0.6 nmol human TNFα (39). Samples of anti-TNFα antibodies (0.6, 0.2, and 0.067 nmol) were applied to wells surrounding the center wells and allowed to diffuse overnight at 37°C under humid conditions. Protein precipitates formed by interactions between anti-TNFα antibodies and human TNFα were visualized in a bright field.

The body weight scores of the mice were analyzed parametrically by performing an ANOVA multiple comparison test and Dunnet’s post-hoc analysis. In vivo macroscopic and histopathological arthritis scores were compared by performing the Kruskal-Wallis test followed by Mann-Whitney U-test with Bonferroni correction. Tukey-Kramer multiple comparison tests were used to compare the data obtained for the neutralizing effects on anti-TNFα antibodies by ELISA and the cell-based assay.

Ozoralizumab consists of two anti-TNFα NANOBODIES^® and one anti-HSA NANOBODY^®. Hegen et al. found that human TNFα and monkey TNFα bind to ozoralizumab with very similar association and dissociation rates, resulting in nearly identical equivalent K_D values of 20.2 pM and 16.1 pM, respectively (Poster presented at the Annual Meeting of The American College of Rheumatology; November 6, 2011; McCormick Place Convention Center in Chicago). The L929 cell cytotoxicity assay is well known as a bioassay for TNFα and its biological inhibitors (24, 28, 29). The concentration of human TNFα used in this assay was 0.3 ng/mL and was chosen to yield nearly 80% cytotoxicity. Ozoralizumab neutralized human TNFα with an IC₅₀ of 5.7 pM ( Figure 1 ), whereas adalimumab neutralized human TNFα with an IC₅₀ of 80.9 pM. These results indicate that the TNFα-neutralizing potency of ozoralizumab is superior to that of adalimumab in the L929 cell cytotoxicity assay.

ADA formation is considered one of the main reasons for the loss of therapeutic efficacy of anti-TNFα antibodies (8, 9). The Tg197 human TNF transgenic mice constitutively produced human TNFα, and then spontaneously developed severe arthritis ( Figure 2A ). We evaluated ADA formation against each of the anti-TNFα antibodies and the efficacy of long-term administration of these anti-TNFα antibodies against the TNFα-mediated polyarthritis in Tg197 mice. The anti-TNFα antibodies doses based on mice weight were adjusted considering the clinically used doses and the difference in body weight between humans and mice. In general, RA patients are injected 40 mg or 80 mg (0.6 mg/kg or 1.3 mg/kg for adult men weighing 60 kg) of adalimumab subcutaneously. In phase 3 clinical study of ozoralizumab, patients were injected 30 mg or 80 mg (0.5 mg/kg or 1.3 mg/kg for adult men weighing 60 kg) of ozoralizumab subcutaneously. Thus, the Tg197 mice were subcutaneously injected with a 1 mg/kg dose of ozoralizumab or adalimumab twice a week starting at 3 weeks of age. As shown in Figure 2A , both antibodies significantly and comparably reduced the arthritis scores compared with those in the vehicle group, indicating marked suppression of disease progression (ozoralizumab group: 0.76, p < 0.001; adalimumab group: 0.89, p < 0.01, vs. vehicle in both groups). There was premature mortality in the vehicle group of Tg197 mice starting at 9 weeks of age ( Supplementary Figure 1A ). Premature mortality also occurred in the adalimumab group starting at 11 weeks of age, and its incidence reached 50% at 16 weeks of age. Importantly, no premature mortality was observed in the ozoralizumab group, at least up to 16 weeks of age. Body weight increase was impaired in the vehicle group, but there was a sustained increase in body weight in the ozoralizumab group and adalimumab group ( Supplementary Figure 1B ).

Joint damage was assessed histopathologically in the hind paws of the Tg197 mice in the vehicle group, ozoralizumab group, adalimumab group, and 3-week-old control group. The synovial lining of the Tg197 mice of vehicle group became markedly thickened because of synovial cell proliferation and inflammatory cell infiltration, and this proliferative mass, the pannus, invaded and progressively destroyed articular cartilage and bone, leading to irreversible destruction of joint structure and function. The histological changes characteristic of chronic inflammatory arthritis in the hind paws of the Tg197 mice were first noted at 3 weeks of age. Importantly, there was a significant reduction (p < 0.01) in the mean histopathologic score in the Tg197 mice that had been treated with ozoralizumab for 13 weeks in comparison with the vehicle group ( Figures 2B, C ).

A link between the presence of ADAs and both decreased serum drug concentrations and clinical efficacy of anti-TNFα antibodies has been well documented (8, 9). ADA titers have been reported to be associated with low levels or an absence of anti-TNFα antibodies in serum samples and to result in a weakened therapeutic response and even exacerbation of the underlying disease (40). To investigate whether ADAs reflect reductions in the serum levels of antibodies administered and result in a compromised therapeutic response, we measured ADA levels in serum samples obtained from Tg197 mice after administration of anti-TNFα antibodies. ADA formation against each of the anti-TNFα antibodies in the groups treated with ozoralizumab or adalimumab twice a week was monitored every two weeks until 16 weeks of age ( Tables 1A, B ). ADAs against adalimumab were detected in the serum obtained at 6 weeks of age, and the ADA level (log titer) rose dramatically, reaching an 80% incidence in the 16-week-old mice in the adalimumab group. Remarkably, ADAs against ozoralizumab were detected in hardly any of the mice in the ozoralizumab group, at least up to 13 weeks after the start of administration. Taken together, our findings demonstrate that ozoralizumab has strong ability to neutralize TNFα activity, without being immunogenic, during long-term administration to Tg197 mice.

To exclude a possible difference in pharmacokinetics between ozoralizumab and adalimumab, we monitored the serum concentrations of these antibodies in Tg197 mice after a single subcutaneous dose of 1 mg/kg of ozoralizumab or adalimumab. The results showed comparable serum concentration-time curves for ozoralizumab and adalimumab ( Figure 3 ). The pharmacokinetic parameters obtained for ozoralizumab and adalimumab were: peak concentration (C_max), 265 ng/mL and 257 ng/mL, respectively; time to peak concentration (t_max), 6 hours and 24 hours, respectively; and elimination half-life (t_1/2), 12 hours and 19 hours, respectively. Moreover, after twice weekly administration of adalimumab, the serum adalimumab concentrations of the Tg197 mice at 16 weeks of age were below the lower limit of quantification (<10 ng/mL, data not shown), indicating that the emergence of ADAs can cause serum drug levels to drop to sub-therapeutic levels.

Ozoralizumab has a unique structure that differs from the structure of IgG-type antibodies and fails to induce ADAs after repeated administration. We investigated whether ozoralizumab is capable of circumventing ADA-induced secondary failure after repeated administration of adalimumab by switching from administration of adalimumab to ozoralizumab in Tg197 transgenic mice. ADAs against adalimumab were detected in 23 of 32 mice treated with adalimumab during the first 3 weeks, and the log titers of ADA against adalimumab in the 6-week-old mice ranged from <0.477 to 1.91 (data not shown). The mice that were positive for adalimumab-induced ADAs at 6 weeks of age were divided into 2 groups: a continued group, in which adalimumab administration was continued for an additional 6 weeks, and a switched group, in which adalimumab was switched to ozoralizumab administration for the next 6 weeks. The in vivo arthritis scores of the mice in the vehicle group increased rapidly from 3 weeks of age onward and reached 1.73 at 11 weeks of age ( Figure 4A ). At 11 weeks of age, the arthritis scores of both the continued group and switched group were significantly lower than in the vehicle group ( Figure 4A ). The increases in histopathological scores were also significantly lower in both the continued group and switched group in comparison with the scores in the vehicle group ( Figures 4B, C ). Importantly, the arthritis scores of the switched group were much lower than in the continued group at 11 weeks of age and 12 weeks of age (1.04 vs. 1.36, p < 0.01 and 1.01 vs. 1.57, p < 0.001, respectively), indicating that switching to ozoralizumab markedly suppressed disease progression in comparison with continued administration of adalimumab. Furthermore, at 8 weeks of age, one week after the switching to ozoralizumab, the arthritis scores were already lower in the switched group compared to the continued group (0.70 vs. 0.84, p < 0.05), indicating rapid suppression of disease progression within a short period. It is also noteworthy that throughout the period of administration of ozoralizumab the arthritis scores in the switched group were steadily suppressed, whereas the arthritis scores in the continued group gradually and progressively increased until the end of the study.

We next investigated whether ozoralizumab is capable of exerting neutralizing activity even after ADA formation induced by repeated administration of adalimumab. To do so, we tested the neutralizing activity of the serum containing adalimumab-induced ADAs against exogenously added anti-TNFα antibodies by ELISA. In this experiment, serum samples collected from the vehicle group and adalimumab group of mice at 6 weeks of age were incubated with exogenously added adalimumab or ozoralizumab, and then the remaining binding activity of the anti-TNFα antibodies to immobilized TNFαwas measured by ELISA ( Figure 5A ). The TNFα-binding activity of adalimumab and ozoralizumab was unaffected by the serum from the vehicle group, whereas the remaining TNFα-binding activity of the adalimumab added to the serum containing ADAs against adalimumab was almost negligible (4.3%), indicating that most of the adalimumab was neutralized by the ADAs. By contrast, 94.8% of the TNFα-binding activity of ozoralizumab remained in the serum under the same conditions, demonstrating that ADAs against adalimumab do not neutralize ozoralizumab.

We further confirmed the difference in inhibitory effect on the cytotoxicity of circulating anti-TNFα antibodies in the serum samples obtained from the continued group and switched group of 12-week-old mice. The serum samples of the continued group failed to suppress cytotoxicity induced by human TNFα, cell viability was significantly increased in the serum samples from the switched group, ( Figure 5B ). The results thus demonstrated that the ozoralizumab in the serum of the switched group retained its potency to inhibit TNFα activity and that the serum of the mice that had received repeated administrations of adalimumab had lost its inhibitory activity against TNFα.

It was important to determine whether, ozoralizumab is effective in suppressing TNFα-induced arthritis once ADAs have generated after repeated administration of adalimumab. To do so, we investigated ADA levels against adalimumab and ozoralizumab in the mice in the continued group and switched group at the end of the study ( Table 2 ). ADAs against adalimumab were detected in the switched group as well as the continued group even though adalimumab administration had been discontinued in the switched group during the final 6 weeks ( Table 2A ). Remarkably, no ADAs against ozoralizumab were detected in most of the mice in the switched group (8 out of 10, Table 2B ), even though ADAs against adalimumab remained high in the serum of these mice at the end of study. The log titer of the ADAs against ozoralizumab detected in the switched group was almost negligible (<0.477, Table 2B ). Thus, even though ozoralizumab was administered to the mice that had generated ADAs against adalimumab, ADAs were not generated against ozoralizumab, and as a result switching to ozoralizumab was capable of continuing to suppress the arthritis. Furthermore, the serum adalimumab concentrations in most of the continued group were below the lower limit of quantification (<10 ng/mL, Table 2A ). However, the serum concentration of ozoralizumab in the switched group was 12.1–1690 ng/mL ( Table 2B ), consistent with the marked difference in ADA formation in response to adalimumab and ozoralizumab.

Formation of a large immune complex has been reported to be critical for developing high immunogenicity (9). IgG-type antibodies can form a very large complex by strongly interacting with two different trimeric TNFα molecules (41). To investigate whether ozoralizumab also forms a large complex, we performed the Ouchterlony double-diffusion assays with different antibody to TNFα concentration ratios. Adalimumab formed a large enough immune complex with TNFα to be detected as a precipitable line in the assay, whereas no precipitation line was observed with ozoralizumab in agarose gels, suggesting that it does not form a large immune complex with TNFα ( Figure 6 ).

The results showed that ozoralizumab does not form a large immune complex that might lead to ADA formation, and suggest that this difference is one of the reasons that ozoralizumab is functionally distinct from adalimumab.