# Development of Anti-OSCAR Antibodies for the Treatment of Osteoarthritis
## 3 Results
### Affinity Maturation of Anti-OSCAR Antibodies  
Given that D11 lost its ability to bind to hOSCAR when it was converted to IgG, we further optimized both the B4 and D11 antibodies by affinity maturation. The light chain shuffling approach was employed first. Thus, the lambda and kappa light chain variable domain (VL) repertoires of the OPAL-S scFv library [25] were amplified by PCR and then assembled with the heavy chain variable domain (VH) of B4 by overlap extension PCR, and the resulting PCR products were ligated to pComb3X phagemid vector to construct VL-shuffled scFv phagemid libraries that contained 3.3 \(\times\) 10\({}^{7}\) kappa light chain and 1.9\(\times\)10\({}^{7}\) lambda light chain transformations (Figure 2A). These libraries were then panned against  
Figure 1: Ability of candidate anti-OSCAR Abs to regulate OSCAR expression and pro-catabolic downstream signaling activity in chondrocytes. (**A**) Ability of seven anti-OSCAR hIgGs to bind human and mouse OSCAR-Fc on ELISA. (**B**) Schematic depiction of the assay used to identify antibodies that could inhibit the collagen-I-induced pro-catabolic downstream signaling of OSCAR in chondrocytes. (**C**) The _Oscar, Epus1, Mmp3,_ and _Mmp13_ mRNA levels in the ATDC5 cell line were determined by using quantitative RT-PCR. hFc: the human OSCAR-Fc fusion protein, which served as the positive control inhibitor. hIgG: the isotype control, which served as the negative control. Error bars represent mean \(\pm\) S.E.M. Two-way ANOVA followed by [PERSON]’s Multiple Comparison test was performed. \({}^{\bullet}p<0.05,\)\({}^{\bullet\bullet}p<0.01,\)\({}^{\bullet\bullet\bullet}p<0.001\) when the anti-OSCAR antibody- or hOSCAR-Fc-treated group was compared to the hIgG negative control group. OSCAR to yield B4 variants with improved affinity. However, the same approach failed to produce D11-derived clones with improved affinity. Therefore, we employed NNK-walking randomization of CDR-H3 for D11, in which two consecutive codons of the CDR-H3 sequence of D11 were randomized in an overlapping manner by using the NNK degenerate codon (N = any of the four deoxynucleotides; K = G or T) (Figure 2A) The resulting small libraries (each of which contained 400 unique amino acid sequences; 1.4 \(\times\) 10\({}^{5}\)-1.8 \(\times\) 10\({}^{6}\) transformants) were panned individually against OSCAR. Figure 2: Affinity maturation of the B4 and D11 anti-OSCAR antibodies. (**A**) Schematic depiction of the methods used to diversify the B4 and D11 parental antibodies. B4 was diversified by light chain shuffling, while D11 was diversified by two-codon NNK walking of the CDR-H3 (parental D11 CDR-H3 sequence is shown in red). (**B**) Serial-dilution ELISA to determine the affinity of the B4 and D11 parent clones and their affinity-matured variants for mouse (left) and human (right) OSCAR-Fc. (**C**) Biology interferometry analysis of the binding kinetics of the anti-OSCAR antibodies. Three rounds of panning against hOSCAR-Fc were performed with increasing string-gency. Thus, 1 ug/mL hOSCAR-Fc and five washes with PBST were used for the first two rounds, and 0.2 ug/mL hOSCAR-Fc, eight normal washes, and two extended washes involving 30 min incubation were used for the third round. The output clones were then screened by ELISA, and the binders were sequenced. Three unique binder sequences from the light chain shuffling of B4 and six from the CDR-H3 randomization of D11 were identified and subjected to further characterization. Two of the B4 variants had kappa light chains (B4K-C3 and B4K-C11), whereas the parental B4 clone and B4L-E2, the third B4 variant, had a lambda light chain. The CDR-L1s and CDR-L3s of B4K-C3 and B4K-C11 belong to the VK1 family, whereas the CDR-L2s were from the VK3 family. This reflects the fact that the light chain repertoire used in the chain shuffling was from a synthetic scFv library with a single scaffold and diversified CDRs [25] and implies that a single variable domain framework can accommodate CDRs of different germline family origins.