# Development of Anti-OSCAR Antibodies for the Treatment of Osteoarthritis
## 4 Discussion  
Our previous study showed that blocking OSCAR with intra-articular injections of a soluble protein containing the extracellular domains of OSCAR (hOSCAR-Fc) reduces cartilage destruction in OA [14]. However, hOSCAR-Fc exerts these effects by binding to collagen, which is ubiquitous and highly abundant in the body. Thus, the risk of undesirable side effects may be high. By contrast, monoclonal antibodies that bind with high specificity and affinity to OSCAR and thereby block its pro-chondrolytic signaling are likely to constitute a safer approach. This approach to drug development is also readily facilitated by well-established technologies for engineering, optimizing, and producing monoclonal antibodies, which have made monoclonal antibodies particularly suited to drug development. Thus, in the present study, we sought to develop OSCAR-neutralizing antibodies. The extracellular Ig-like 1 and 2 domains of the human and mouse OSCAR proteins show moderate sequence identity (77.6%). Since species cross-reactivity is desirable for the preclinical and clinical development of antibody-based drugs, we panned scFv libraries against both human and mouse OSCAR in alternating rounds. Binding to mouse OSCAR is necessary for experiments, including cell-based assays, efficacy tests in animal models, and toxicological and pharmacokinetics studies, while affinity to human OSCAR is required for  
Figure 4: The anti-OSCAR antibodies ameliorate OA by blocking articular chondrocyte apoptosis. Wild-type mice were subjected to DMM or sham surgery and then injected intra-articularly twice a week for 8 weeks with each anti-OSCAR antibody (\(n=6\) per group). The mice were sacrificed 9 weeks after surgery, and the articular cartilage in their joints was sectioned. The apoptotic articular chondrocytes were detected and quantified by TUNEL assay. (**A**) Representative images. Scale bar: 50 \(\mu\)m. (**B**) Quantitative analysis of the apoptotic chondrocyte numbers. Error bars represent mean \(\pm\) S.E.M. Two-way ANOVA followed by [PERSON]’s Multiple Comparison test was conducted. \({}^{**}p<0.01\), \({}^{***}p<0.001\) when anti-OSCAR antibody-treated groups were compared to the hlgG control group. clinical applications. This led to seven scFv clones that recognized both proteins. Once they were converted to IgG, however, two of the clones (D2 and D11) largely lost their ability to bind to hOSCAR, although they could still strongly recognize mOSCAR. Nonetheless, we continued to investigate these antibodies because we assumed that the lost binding activity could be regained by affinity maturation. Indeed, when D11 was affinity-matured (D2 was later abandoned due to stability issues), its variants regained the ability to recognize hOSCAR. After identifying the seven OSCAR-binding antibodies, we asked whether they could neutralize the cellular activities of OSCAR. Thus, we assessed their effect on the collagen-induced expression of OA-marker genes (_Mmp3_, _Mmp13_, and _Epsa1_) by the chondrogenic ATDC5 cell line. This cell line is of murine origin, thus allowing D11 and D2 to be tested as well. Five of the seven antibodies inhibited OSCAR-mediated expression of the OA marker genes, which suggested they might also have in vivo efficacy against OA. While these five antibodies exhibited comparable in vitro efficacies, only B4 and D11 were selected for further development based on their higher expression levels and better stability. They also effectively inhibited two other known cellular OSCAR functions, namely, (i) OSC peptide + IL-1B-induced expression of the OSCAR and TRAIL genes in murine articular chondrocytes [14; 19] and (ii) differentiation of murine osteoclast progenitors into mature osteoclasts [15; 16]. B4 and D11 were subjected to affinity maturation in order to enhance their efficacies and also to rescue the binding activity of D11 to hOSCAR, which was lost after its conversion to IgG. Light chain shuffling was attempted first and proved successful with B4 but not with D11.