# Development of Anti-OSCAR Antibodies for the Treatment of Osteoarthritis
## 3 Results
### In Vivo Efficacy of the Affinity-Matured Anti-OSCAR Antibodies  
The DMM OA model mimics the OA that is commonly induced in humans by clinical meniscal injury [30; 31]. It is a commonly used model of OA and thus was used to test the therapeutic efficacy of B4K-C11, B4L-E2, D11-B9, and their parent antibodies. The anti-OSCAR antibodies or the positive control hOSCAR-Fc were injected intra-articularly twice a week for 8 weeks, starting 1 week after the DMM surgery. After euthanizing the mice at 9 weeks, the joints were subjected to histological analysis. The parental B4 and D11 antibodies inhibited the cartilage degradation with comparable efficacy to hOSCAR-Fc, as indicated by the OARSI grade [33] (Supplementary Figure S2A,B). The B4K-C11, B4L-E2, and D11-B9 antibodies also significantly reduced the OARSI grades relative to the isotype control group (Figure 3A,C). All antibodies also markedly reduced other DMM-induced OA signs, namely, subchondoral bone plate sclerosis and loss of hyaline cartilage (Figure 3B,C). Notably, D11 and its D11-B9 variant inhibited OA more effectively than B4 and its variants. Moreover, immunohistochemical analysis of the cartilage tissues confirmed that antibody-mediated inhibition of OSCAR downregulated the OA-associated TRAIL expression by articular chondrocytes while upregulating their OPG expression (Supplementary Figure S3). Furthermore, TUNEL assays of the cartilage tissues showed that the anti-OSCAR antibodies significantly reduced chondrocyte death. In particular, D11 and D11-B9 completely suppressed OA chondrocyte apoptosis (Figure 4 and Supplementary Figure S2C). Collectively, these results show that the anti-OSCAR antibodies significantly inhibit OSCAR functions, particularly its ability to promote chondrocyte apoptosis and thereby induce OA. \begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**Legend** & **Parameters** & **D11** & **D11-B9** & **B4** & **B4K-C11** & **B4L-E2** \\ \hline \multirow{3}{*}{hOSCAR} & \(k_{\text{on}}\) (M\({}^{-1}\)s\({}^{-1}\)) & 9.13 \(\times\) 10\({}^{4}\) & 1.01 \(\times\) 10\({}^{5}\) & 8.99 \(\times\) 10\({}^{4}\) & 1.54 \(\times\) 10\({}^{5}\) & 1.38 \(\times\) 10\({}^{5}\) \\  & \(k_{\text{off}}\) (s\({}^{-1}\)) & 5.12 \(\times\) 10\({}^{-4}\) & 4.15 \(\times\) 10\({}^{-5}\) & 2.92 \(\times\) 10\({}^{-4}\) & 3.37 \(\times\) 10\({}^{-5}\) & 1.21 \(\times\) 10\({}^{-5}\) \\  & \(K_{\text{D}}\) (nM) & 5.61 & 0.41 & 3.25 & 0.22 & 0.088 \\  & \(k_{\text{on}}\) (M\({}^{-1}\)s\({}^{-1}\)) & 9.13 \(\times\) 10\({}^{4}\) & 9.13 \(\times\) 10\({}^{5}\) & 9.13 \(\times\) 10\({}^{5}\) & 9.13 \(\times\) 10\({}^{5}\) \\  & \(k_{\text{off}}\) (s\({}^{-1}\)) & 2.54 \(\times\) 10\({}^{-4}\) & 2.59 \(\times\) 10\({}^{-5}\) & 3.89 \(\times\) 10\({}^{-4}\) & 6.79 \(\times\) 10\({}^{-5}\) & 2.47 \(\times\) 10\({}^{-5}\) \\  & \(K_{\text{D}}\) (nM) & 2.87 & 0.22 & 4.61 & 0.40 & 0.23 \\ \hline \hline \end{tabular}
\end{table} 
Table 4: Binding kinetics of the anti-OSCAR antibodies, as determined by biolayer interferometry. \begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**EC\({}_{\text{50}}\) (nM)** & **D11** & **D11-B9** & **B4** & **B4K-C11** & **B4L-E2** \\ \hline hOSCAR & \textgreater{}1000 & 36.8 & 590 & 6.74 & 1.33 \\ mOSCAR & 151 & 0.93 & 414 & 1.30 & 7.20 \\ \hline \hline \end{tabular}
\end{table} 
Table 3: EC50 values of the anti-OSCAR antibodies for binding to human and mouse OSCAR. Figure 3: The anti-OSCAR B4 and D11 antibodies and their affinity-matured variants block OA in a murine model. 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 and stained with safranin-O to determine the OARSI grade, subchondral bone plate (SBP) thickness, and the ratio of hyaline cartilage (HC) to calcified cartilage (CC). (**A**) Representative images of the safranin-O-stained sections.