Mutations of other residues selected on the basis of alternative criteria might further increase antigen affinity by a different mechanism

Mutations of other residues selected on the basis of alternative criteria might further increase antigen affinity by a different mechanism. point mutation. The affinity-increased mutants are shown to fluctuate less in their free form and to form a more packed structure in their antigen-bound form. Introduction An increasing quantity of antibodies are now in therapeutic use1. Antibodies can be very Rabbit Polyclonal to TEAD1 easily acquired by immunization of model animals and humanization but, because their amino acid sequences are optimized by random shuffling, the producing affinities are not fully matured against the target molecules2. In terms of therapeutic use, there is a large demand for higher-affinity antibodies that enable the dose and cost to be minimized. It is thus important both to design antibodies with higher affinity and to elucidate the detail of antibodyCantigen interactions in order to establish a basis for optimizing antibodies. To improve our understanding of antibodyCantigen interactions, many biophysical studies, including crystallographic, kinetic, and thermodynamic analyses, have characterized the events that occur at the antibodyCantigen interface3C7. Based on the results of such studies, a large number of antibody mutants have been designed and tested8. Most of the studies aiming to increase the affinity of an antibody have focused on residues at the antibodyCantigen interface, known as the complementary determining region (CDR)3C7. This strategy is affordable because antibodies in nature switch their specificity and affinity by randomly mutating the amino acid sequences of the CDR. In particular, the technique of molecular development, or phage display, has proved to be successful in affinity maturation of the CDR6. Computer-assisted structure-based affinity optimization has also been successful9. Therefore, several strategies are available to increase the affinity by modifying the CDR. However, it is hard to increase the affinity solely by changing the CDR residues10 because a CDR sequence is relatively well optimized by nature itself, and other strategies to design antibodies with higher affinity are desired2. One encouraging strategy might be to regulate 5-Hydroxy Propafenone D5 Hydrochloride the antigen-binding process of an antibody by introducing a mutation in a non-CDR region. The variable domains of the heavy chain (VH) and light chain (VL) represent potential target regions for mutation because they are sequentially and spatially connected to the CDR. At present, however, there is no established rational method that can specifically identify residues appropriate for mutation in non-CDR regions to increase the affinity to antigen. Here, we focused on the process of dynamic binding between an antibody and its antigen in order to rationally design an antibody with higher affinity. We considered that many residues in non-CDR regions that do not directly interact with the antigen potentially contribute to the process of antibodyCantigen complex formation via conformational fluctuation. Elucidating the mechanism by which these residues contribute to the binding process would provide the basis of a strategy to regulate the antibodyCantigen conversation; however, the conformational fluctuations that occur in the antibodyCantigen binding process are poorly comprehended. A problem in attempting to regulate the conformational fluctuation of an antibody is how to choose the residues to be mutated. Without specific criteria, you will find nearly 200 candidate 5-Hydroxy Propafenone D5 Hydrochloride residues in non-CDR regions that might be mutated. Here, we describe a method to identify conformational fluctuations that are relevant to the antigen-binding process. Fluctuations of an antibody can be measured by relaxation dispersionan NMR method that quantitates conformational exchange rates around the millisecond timescale11, which applies to many biologically relevant fluctuations such as ligand-binding 5-Hydroxy Propafenone D5 Hydrochloride and folding12. We devised a plan to select residues for mutation on the basis of relaxation dispersion data. We used the Fv fragment (the hypervariable region comprising the VH and VL domains) of an anti-lysozyme antibody, HyHEL-1013, 14, to establish the method. The Fv fragment of HyHEL-10 is easy to overexpress in.