Antibody engineering for the analysis of affinity maturation of an anti-hapten response.

Abstract

The influence of structural variation, previously observed in a panel of V186.2 VH/V lambda 1‐expressing anti‐NP antibodies from the secondary response, on the affinity of these antibodies was examined by site‐specific mutagenesis and recombinant antibody construction. A tryptophan––leucine exchange at position 33 in the VH segment of all but one of the high‐affinity antibodies is the most frequently observed somatic mutation and by itself leads to a 10‐fold higher affinity; all other somatic exchanges are irrelevant for affinity selection. In the single case of a high‐affinity antibody without this common exchange, high affinity is mediated by a combination of mutations (including a one‐codon deletion) in VH and the particular D‐JH rearrangement carried by this antibody. The data indicate that the pattern of somatic diversification through hypermutation is shaped by affinity selection, but that only a single point mutation is available in the VH and the VL gene of lambda 1 chain‐bearing anti‐NP antibodies which by itself leads to an increase of hapten‐binding affinity. Based on the analysis of two secondary response antibodies from which somatic mutations in VH and VL have been eliminated, it is also concluded that the recruitment of B cell clones into the pathway of hypermutation involves a mechanism which is not based upon affinity differences towards the antigen.

Generation of High-Affinity Antibody against T Cell-Dependent Antigen in the Ganp Gene-Transgenic Mouse

“…However, mutations in the V L regions were not apparently increased in the comparison of the anti-NP mAbs from Ganp Tg and C57BL/6 mice. The generation of high-affinity BCR without the exchange at position 33 is a rare event, which suggested that the combination of particular D-J H sequences and/or many SHMs do not result in high affinity (18). A recent report only showed the case of mutation, Y99G in V H 186.2, which generated similar high-affinity mAb against NP-hapten comparable to W33L (10).…”

Origin and diversification of the clonogenic cell in multiple myeloma: lessons from the immunoglobulin repertoire

“…39 It is possible that a minimum number of amino acid substitutions in CDRs might ultimately confer a great increase in affinity for antigen and favor selection; this has been convincingly shown in hybridomas producing monoclonal antibodies, where a single amino acid replacement in CDRs by site-directed mutagenesis may cause a 10-fold rise in affinity. 40 Therefore, it is not impossible to have a relatively modest R:S mutation ratio in CDRs and yet attain optimal conditions for high-affinity antigen binding. Moreover, a statistically significant low R:S ratio in FWRs in such situations might in itself constitute a surrogate measure for non-random distribution of mutations suggestive of selection by antigen.…”

Histologic transformation of follicular lymphoma to diffuse lymphoma represents tumor progression by a single malignant B cell.

“…Third, the DJ join that contains the 3-bp deletion in the tDL is otherwise identical to the FL consensus sequence for this region. Fourth, somatic mutation in Ig genes can include deletion of an entire codon (46,47) . The finding that 4 of the 28 mutations observed among the eight FL clones occurred in this 15-bp stretch where only one would have been expected if the mutations were distributed evenly suggests that the DJ join is a target for active mutation .…”