7xl0

Publication Abstract from PubMed

The camelid single domain antibody, referred to VHH or Nanobody, is considered a versatile tool for various biotechnological and clinical applications because of its favorable biophysical properties. To take advantage of these characteristics and for its application in biotechnology and therapy, research on VHH engineering is currently vigorously conducted. To humanize a camelid VHH, we performed complementarity determining region (CDR) grafting using a humanized VHH currently in clinical trials, and investigated the effects of these changes on the biophysical properties of the resulting VHH. The chimeric VHH exhibited a significant decrease in affinity and thermal stability and a large conformational change in the CDR3. To elucidate the molecular basis for these changes, we performed mutational analyses on the framework regions revealing the contribution of individual residues within the framework region. It is demonstrated that the mutations resulted in the loss of affinity and lower thermal stability, revealing the significance of bulky residues in the vicinity of the CDR3, and the importance of intramolecular interactions between the CDR3 and the framework-2 region. Subsequently, we performed back-mutational analyses on the chimeric VHH. Back-mutations resulted in an increase of the thermal stability and affinity. These data suggested that back-mutations restored the intramolecular interactions, and proper positioning and/or dynamics of the CDR3, resulting in the gain of thermal stability and affinity. These observations revealed the molecular contribution of the framework region on VHHs and further designability of the framework region of VHHs without modifying the CDRs.

Molecular basis for thermal stability and affinity in a VHH: Contribution of the framework region and its influence in the conformation of the CDR3.,Kinoshita S, Nakakido M, Mori C, Kuroda D, Caaveiro JMM, Tsumoto K Protein Sci. 2022 Nov;31(11):e4450. doi: 10.1002/pro.4450. PMID:36153698^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.