# Development of Anti-OSCAR Antibodies for the Treatment of Osteoarthritis
## 4 Discussion
Consequently, D11 was subjected to focused CDR-H3 randomization. The B4 parent antibody already had nanomolar dissociation constants for hOSCAR and mOSCAR, and affinity maturation improved this to sub-nanomolar levels. This was also observed with D11 for mOSCAR, and notably, the affinity maturation of D11 completely restored its response to hOSCAR-Fc: The EC50 improved from >1000 nM for D11 to 0.93 nM for the D11-B9 variant. Interestingly, the CDR-H3 of D11-B9 not only bore amino acid substitutions but was also one amino acid shorter than its parental sequence. Both of these changes probably significantly altered the CDR conformation [38], thereby markedly improving the affinity of the antibody. The fact that the light chain shuffling approach improved the affinity of B4 but not D11, coupled with the considerable difference in the CDR-H3 sequence between D11-B9 and D11, suggests that the target binding activity of D11 relies comparatively more on VL to compensate for the suboptimal CDR-H3 sequence. In the in-vivo OA model, B4, D11, and their affinity-matured variants effectively blocked OA pathogenesis. Particularly, D11 and its variant D11-B9 performed better than B4 and its variants. Given the overall similar affinities of these antibodies, it is likely that this efficacy difference reflects differences between D11 and B4 in the epitopes they recognize or their mode of binding. Notably, however, the in vivo efficacies of the parent antibodies did not differ significantly from those of the affinity-matured clones despite the >10-fold lower \(K_{\rm D}\) values of the latter. We speculate that the local concentration of intra-articularly injected antibody was high enough to saturate the OSCAR molecules on the chondrocytes, even if the antibody (e.g., B4 and D11) only had moderate affinity. Affinity maturation of D11 was still necessary to rescue the affinity toward human OSCAR (see above). Higher affinity antibodies might also prove more efficacious at a lower dose or with other routes of administration. It is noted that the hOSCAR-Fc decoy receptor, and not mOSCAR-Fc, was used as a positive control in the cell-based assays as well as the animal studies, even though a murine chondrocyte cell line (ATDC5) and a mouse disease model, respectively, were used in these experiments. This is because hOSCAR-Fc was already successfully evaluated in these settings [14] with potential clinical applications in mind, for which its human-derived sequence would be advantageous with lower immunogenicity. While the antibody-targeting of OSCAR is a promising strategy for the treatment of OA, the approach is not without limitations. OA is a chronic, non-lethal disease, and an exceptional long-term safety profile is required for OA therapy. Because OSCAR is also expressed in osteoclasts, monocytes, and other immune and non-immune cell types [17; 39], targeting and neutralizing OSCAR might result in unexpected and undesirable on-target, off-tissue effects. Also, as with most other OA therapies, anti-OSCAR therapy is unlikely to reverse the extensive cartilage damage of late-stage disease. Further studies are needed to better understand the advantages and limitations of OSCAR blockade and its mode of action and to develop anti-OSCAR agents as OA therapy.