Immune response with long-term memory triggered by amorphous aggregates of misfolded anti-EGFR V HH -7D12 is directed against the native V HH -7D12 as well as the framework of the analogous V HH -9G8.

We previously demonstrated that amorphous aggregates of misfolded V _HH -7D12 antibodies (V _HH -Mis), a potential anti-EGFR drug, can generate a robust serum IgG response. Here we investigate the immunogenic nature, especially the specificity of the immune response induced by V _HH -Mis. To this end, we used two natively folded and 77% identical anti-EGFR V _HH s (V _HH -7D12 and V _HH -9G8) that possess a common framework but distinct complementarity determining regions (CDRs). In 60% of mice immunized with V _HH -Mis, the anti-V _HH -7D12 IgG titer was stronger than the anti-V _HH -9G8 titer (Group-1). In the remaining mice (40%; Group-2), the anti-V _HH -7D12 and anti-V _HH -9G8 titer were almost identical. We rationalized these results by hypothesizing that mice in Group-1 produced IgG mostly against the V _HH -7D12's CDRs, whereas in Group-2 mice, they targeted the V _HH 's framework. The IgG specificity against V _HH -7D12 and V _HH -9G8 was essentially unchanged over 17 weeks in both groups. Further, in all mice (Group-1&2) re-immunized with native V _HH -7D12, the IgG titer against V _HH -7D12 increased sharply but not against V _HH -9G8. On the other hand, none of the three Group-1 mice re-immunized with native V _HH -9G8 showed immunogenicity against V _HH -7D12 nor V _HH -9G8. Whereas, in Group-2 mice (three/three) re-immunized with V _HH -9G8, the IgG titers against both V _HH s increased but slowly. Flow-cytometric studies showed that V _HH -Mis immunized mice generated a higher number of effector and central memory T-cells. Overall, these observations indicate that amorphous aggregates made of a misfolded V _HH can induce serum IgG against its natively folded self and analogous V _HH s having a similar framework but distinct CDRs. Furthermore, a robust long-term immune response with memory was established against its natively folded self but with a nil-to-moderate immune response against natively folded V _HH analogs.
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