Structure-guided design of antibody CDRs to reduce their reactivity to treatment-emergent anti-drug antibodies

Immunogenicity prediction is widely used in the developability assessment of antibodies, and many marketed and clinical-stage therapeutics have a predicted T-cell epitope in the second complementary-determining region of their light chain (CDR2L). To investigate such CDR2Ls in more detail, we identified an antibody with a CDR2L for which a patient had developed treatment-emergent (TE) anti-drug antibodies (ADAs) in a clinical setting. With this, we establish the importance of predicted T-cell epitopes in CDR2L. In the course of deleting the T-cell epitope, we decided to aim for a solution that can be applied broadly to facilitate larger high-throughput discovery campaigns. For this purpose, we have developed a double-mutation scheme that targets AHo67 (Kabat51) and AHo68 (Kabat52) in the CDR2L. This 67G-68G mutation scheme was applied to all light chain sequences of a tri-specific single-chain diabody fused to a single-chain variable fragment (scMATCH3™) antibody for which TE ADAs had been observed. Analyses of patient sera showed that introduction of 67 G-68 G in CDR2L in combination with our previously described T101S-T146K (Kabat: T87S-T110K) framework mutations led to a scMATCH3 antibody with significantly reduced levels of both preexisting and TE ADA reactivities. For a diverse collection of single-chain variable fragments, application of the 67 G-68 G mutation scheme was experimentally seen to not substantially affect the functional or biophysical properties of the molecules, suggesting that this mutation scheme may be applicable to the improvement of therapeutic safety of antibodies of many types, with CDR2L-associated immunogenicity.