Development of potent Affitin-based bispecific NK cell engagers for the therapy of MSLN-expressing cancers

Well-characterized tumor-associated antigens (TAAs) represent targets of interest in many anticancer therapeutic strategies. Although the use of monoclonal antibodies has led this field for years, the development of smaller molecules, with improved properties, holds great potential for specific anticancer applications. Affitins are small (7 kDa), thermostable, and high-affinity nonimmunoglobulin protein scaffolds derived from archaea. Using ribosome display and next-generation sequencing, we isolated the first Affitins specific for human mesothelin (hMSLN), a TAA overexpressed in many solid cancers and currently targeted in clinical trials. The homodimerization of the most promising Affitin (N13) improved affinity by 60-fold (from 35 nM to 0.57 nM). The high specificity of N13 was demonstrated on cell co-cultures under static or dynamic conditions. This Affitin, monomeric or dimeric, was also stable under a wide range of temperatures and upon repeated freeze/thaw cycles. Finally, bispecific natural killer (NK) cell engagers (BiKEs) composed of an anti-cluster of differentiation 16 (CD16) variable heavy domain of heavy chain (VHH) fused to a monomer or a dimer of N13 Affitins were constructed. Using a cytotoxicity assay, we showed the specific lysis of hMSLN-expressing cells in the presence of BiKE and NK cells, supporting the potential therapeutic application of these affinity agents.