Immunogenicity of single-chain antibodies: germlining of a VHH lowers T-cell activation from epitopes in FR2 and CDR regions

Single-chain antibodies (scAbs), derived from camelid antibodies, have gained attention as therapeutic candidates due to their small size and perceived low immunogenicity, but recent studies have reported immune responses to several scAbs. To better understand their immunogenicity, we investigated the T-cell responses induced by VHH76, a VHH-Fc engineered to target the SARS-CoV-2 RBD, along with its humanized and germlined variants. The humanized variant contains six human substitutions, while the germlined variant was obtained by screening of a combinatorial library of the VHH76 sequences, comprising human and wild-type substitutions at 12 different positions. The germlined variant finally contains 16 human substitutions. All VHH76 variants triggered CD4 T-cell responses from healthy donors, with the germlined VHH76 showing significantly reduced T-cell stimulation. Two epitope regions were identified: one overlapping CDR3 and another spreading from CDR1 to CDR2. Additional human substitutions at the VHH-conserved positions in FR2 compromised the biological properties of the germlined VHH76 and did not seem to reduce clearly the risk of T-cell response. In conclusion, using a sensitive T-cell assay, we showed that T cells specific for VHH76 variants were detected in the blood of healthy donors and that the frequency of responding T cells diminished with germlining. While epitopes in CDR3 are linked to VHH76 specificity, modifying the conserved FR2 region presents challenges for reducing VHH76 immunogenicity. This study contributes to the understanding of VHH76 immunogenicity and offers insights into strategies to mitigate immune responses.