Academic literature on the topic 'Affinity maturation ; Libraries with insertions and deletions ; Antibody'

We report a systematic combinatorial exploration of affinity enhancement of antibodies by insertions and deletions (InDels). Transposon-based introduction of InDels via the method TRIAD (transposition-based random insertion and deletion mutagenesis) was used to generate large libraries with random in-frame InDels across the entire single-chain variable fragment gene that were further recombined and screened by ribosome display. Knowledge of potential insertion points from TRIAD libraries formed the basis of exploration of length and sequence diversity of novel insertions by insertional-scanning mutagenesis (InScaM). An overall 256-fold affinity improvement of an anti–IL-13 antibody BAK1 as a result of InDel mutagenesis and combination with known point mutations validates this approach, and suggests that the results of this InDel mutagenesis and conventional exploration of point mutations can synergize to generate antibodies with higher affinity.

During a germinal center reaction, random mutations are introduced into immunoglobulin V genes to increase the affinity of antibody molecules and to further diversify the B cell repertoire. Antigen-directed selection of B cell clones that generate high affinity surface Ig results in the affinity maturation of the antibody response. The mutations of Ig genes are typically basepair substitutions, although DNA insertions and deletions have been reported to occur at a low frequency. In this study, we describe five insertion and four deletion events in otherwise somatically mutated VH gene cDNA molecules. Two of these insertions and all four deletions were obtained through the sequencing of 395 cDNA clones (∼110,000 nucleotides) from CD38+IgD− germinal center, and CD38−IgD− memory B cell populations from a single human tonsil. No germline genes that could have encoded these six cDNA clones were found after an extensive characterization of the genomic VH4 repertoire of the tonsil donor. These six insertions or deletions and three additional insertion events isolated from other sources occurred as triplets or multiples thereof, leaving the transcripts in frame. Additionally, 8 of 9 of these events occurred in the CDR1 or CDR2, following a pattern consistent with selection, and making it unlikely that these events were artifacts of the experimental system. The lack of similar instances in unmutated IgD+CD38− follicular mantle cDNA clones statistically associates these events to the somatic hypermutation process (P = 0.014). Close scrutiny of the 9 insertion/deletion events reported here, and of 25 additional insertions or deletions collected from the literature, suggest that secondary structural elements in the DNA sequences capable of producing loop intermediates may be a prerequisite in most instances. Furthermore, these events most frequently involve sequence motifs resembling known intrinsic hotspots of somatic hypermutation. These insertion/deletion events are consistent with models of somatic hypermutation involving an unstable polymerase enzyme complex lacking proofreading capabilities, and suggest a downregulation or alteration of DNA repair at the V locus during the hypermutation process.

During the B–cell response to T–cell–dependent antigens, the B cells undergo a rapid proliferative phase in the germinal centre. This is accompanied by the introduction of mutations into the immunoglobulin (Ig) variable region (V) genes. The B cells are then selected according to the affinity of the encoded immunoglobulin for antigen, resulting in affinity maturation of the response. Analysis of mutations in IgV genes has given insight into the history of individual B cells and their malignancies. In most cases, analysis of mutations confirms classifications of B–cell lineage designated by studies of cellular morphology and surface antigen expression. However, of particular interest is the subdivision of groups of malignancies by analysis of somatic hypermutation. It is now apparent that there are two subsets of chronic lymphocytic leukaemia (CLL), one with a low load of mutations and poor prognosis, and one with a heavy load of mutations with a much more favourable prognosis. In addition, in Burkitt's lymphoma, sporadic and endemic subtypes are now considered possibly to have a different pathogenesis, reflected in differences in the numbers of mutations. Hodgkin's disease, which was a mystery for many years, has now been shown to be a B–cell tumour. Although in many cases the Ig genes are crippled by somatic hypermutation, it is thought that failure to express Ig is more likely to be associated with problems of transcription. It has been proposed that the distribution of mutations in a B–cell lymphoma can be used to determine whether a lymphoma is selected. We have investigated the load and distribution of mutations in one group of lymphomas–marginal zone B–cell lymphomas of mucosa–associated lymphoid tissues (MALT–type lymphoma), which are dependent on Helicobacter pylori for disease progression, to investigate the limits of information that can be derived from such studies. Comparison of the load of mutations demonstrates that these tumours have approximately the same load of mutations as normal mucosal marginal zone B cells from the Peyer's patches and mucosal plasma cells. This is consistent with the origin of these cells from mucosal marginal zone B cells with plasma cell differentiation. To investigate selection in MALT lymphomas we compared a region of the framework region three in ten MALT lymphomas which use the V H4 family, with the same codons in groups of V H4 genes that are out of frame between V and J. The latter accumulate mutations but are not used and are not selected. A group of V H4 genes are in–frame between V and J were also included for comparison. There were no obvious differences in the distribution of mutations between the groups of genes; the same hot spots and cold spots were apparent in each. In the MALT lymphomas, selection was apparent in the framework regions only and the tendency was to conserve. We therefore feel that there is selection to conserve antibody structure and that this does not reflect selection for antigen. We do not believe that antigen selection can be deduced reliably from sequence information alone. It is possible that somatic hypermutation could be a cause of malignancy since it has been shown that the process may generate DNA strand breaks and is known to be able to generate insertions and deletions. Such events may mediate the translocation of genes—a process that is pivotal in the evolution of many lymphomas.

ABSTRACT The structural and functional significance of somatic insertions and deletions in antibody chains is unclear. Here, we demonstrate that a naturally occurring three-amino-acid insertion within the influenza virus-specific human monoclonal antibody 2D1 heavy-chain variable region reconfigures the antibody-combining site and contributes to its high potency against the 1918 and 2009 pandemic H1N1 influenza viruses. The insertion arose through a series of events, including a somatic point mutation in a predicted hot-spot motif, introduction of a new hot-spot motif, a molecular duplication due to polymerase slippage, a deletion due to misalignment, and additional somatic point mutations. Atomic resolution structures of the wild-type antibody and a variant in which the insertion was removed revealed that the three-amino-acid insertion near the base of heavy-chain complementarity-determining region (CDR) H2 resulted in a bulge in that loop. This enlarged CDR H2 loop impinges on adjacent regions, causing distortion of the CDR H1 architecture and its displacement away from the antigen-combining site. Removal of the insertion restores the canonical structure of CDR H1 and CDR H2, but binding, neutralization activity, and in vivo activity were reduced markedly because of steric conflict of CDR H1 with the hemagglutinin antigen. IMPORTANCE Antibody diversification through VDJ gene recombination, junctional variation, and somatic hypermutation has clear importance for the generation of mature, high-affinity antibodies. Between 1.3 and 6.5% of antibody variable gene sequences have been reported to contain insertions or deletions, but their structural and functional significance remains less well defined. The pandemic influenza virus hemagglutinin antibody 2D1 data suggest that somatic insertions and deletions in antibody genes contribute important structural and functional features. We predict that such features can be critical for affinity and functional maturation of the human antibody repertoire.

In Nature, antibodies are capable of recognizing a huge variety of different molecular structures on the surface of antigens. The primary factor that defines the structural diversity of the antibody antigen combining site is the length variation of the complementarity determining region (CDR) loops. Following antigen stimulation, further diversification through the process called somatic hypermutation (SHM) leads to antibodies with improved affinity and specificity. Sequence diversification by SHM is mainly achieved by introduction of point substitutions and a small percentage of insertions/deletions (indels). Although the percentage of indels in affinity matured antibodies is low, probably due to the low rate incorporation of in-frame indels throughout the course of the SHM diversification process, it is likely that the antibody fold can accommodate higher diversity of affinity-enhancing indels. By in vitro evolution, other researchers have sampled either only restricted diversity of indels or extended diversity of insertions only in specific positions chosen based on structural information and natural length variation. The aim of this thesis was to study the impact of random and high diversity indels on antibody affinity by in vitro evolution. New approaches for construction of libraries with in-frame amino acid indels were applied to enable sampling of indels of different lengths across the entire antibody variable domains. I followed two different approaches for construction of indel libraries. Firstly, a recently developed random approach allowed the construction of libraries with random insertions and deletions. Secondly, a semi-random approach was developed to build libraries with different lengths of insertions that could be widely applied in future in vitro antibody affinity maturation campaigns. Libraries constructed by either of these approaches yielded variants with insertions with improved affinity. Overall, this thesis demonstrates that insertions besides offering alternative routes to affinity maturation can also be combined with point substitutions to take advantage of additive effects on function.