Improving antibody affinity by mimicking somatic hypermutation in vitro.

In vivo affinity maturation of antibodies involves mutation of hot spots in the DNA encoding the variable regions. We have used this information to develop a strategy to improve antibody affinity in vitro using phage display technology. In our experiment with the antimesothelin scFv, SS(scFv), we identified DNA sequences in the variable regions that are naturally prone to hypermutations, selected a few hot spots encoding nonconserved amino acids, and introduced random mutations to make libraries with a size requirement between 10(3) and 10(4) independent clones. Panning of the hot spot libraries yielded several mutants with a 15- to 55-fold increase in affinity compared with a single clone with a fourfold increased affinity from a library in which mutagenesis was done outside the hot spots. The strategy should be generally applicable for the rapid isolation of higher-affinity mutants of Fvs, Fabs, and other recombinant antibodies from antibody phage libraries that are small in size.     

Error-prone DNA repair activity during somatic hypermutation in shark B lymphocytes

Sharks are representatives of the earliest vertebrates that possess an immune system utilizing V(D)J recombination to generate Ag receptors. Their Ab repertoire diversity is based in part on a somatic hypermutation process that introduces adjacent nucleotide substitutions of 2-5 bp. We have isolated mutant nonfunctional Ig rearrangements and intronic flank sequences to characterize the nonselected, intrinsic properties of this phenomenon; changes unique to shark were observed. Duplications and deletions were associated with N additions, suggesting participation of a DNA polymerase with some degree of template independence during the repair of DNA breaks initiated by activation-induced cytidine deaminase. Other mutations were consistent with some in vitro activities of mammalian translesion DNA polymerase η: tandem base substitutions, strand slippage, and small insertions/deletions. The nature of substitution patterns shows that DNA lesions at shark Ig genes recruit DNA repair factors with a species-specific repertoire of activities. We speculate that the tandem mutations are introduced by direct sequential misinsertions and that, in shark B cells, the mispairs tend to be extended rather than proofread. Despite extensive changes undergone by some mutants, the physical range of mutational activity remained restricted to VDJ and within the first 2-kb portion of the 6.8-kb J-C intron, perhaps a self-regulating aspect of activation-induced cytidine deaminase action that is conserved in evolution.     

A regulatory role for NBS1 in strand-specific mutagenesis during somatic hypermutation

Activation-induced cytidine deaminase (AID) is believed to initiate somatic hypermutation (SHM) by deamination of deoxycytidines to deoxyuridines within the immunoglobulin variable regions genes. The deaminated bases can subsequently be replicated over, processed by base excision repair or mismatch repair, leading to introduction of different types of point mutations (G/C transitions, G/C transversions and A/T mutations). It is evident that the base excision repair pathway is largely dependent on uracil-DNA glycosylase (UNG) through its uracil excision activity. It is not known, however, which endonuclease acts in the step immediately downstream of UNG, i.e. that cleaves at the abasic sites generated by the latter. Two candidates have been proposed, an apurinic/apyrimidinic endonuclease (APE) and the Mre11-Rad50-NBS1 complex. The latter is intriguing as this might explain how the mutagenic pathway is primed during SHM. We have investigated the latter possibility by studying the in vivo SHM pattern in B cells from ataxia-telangiectasia-like disorder (Mre11 deficient) and Nijmegen breakage syndrome (NBS1 deficient) patients. Our results show that, although the pattern of mutations in the variable heavy chain (V(H)) genes was altered in NBS1 deficient patients, with a significantly increased number of G (but not C) transversions occurring in the SHM and/or AID targeting hotspots, the general pattern of mutations in the V(H) genes in Mre11 deficient patients was only slightly altered, with an increased frequency of A to C transversions. The Mre11-Rad50-NBS1 complex is thus unlikely to be the major nuclease involved in cleavage of the abasic sites during SHM, whereas NBS1 might have a specific role in regulating the strand-biased repair during phase Ib mutagenesis.     

A role for Mlh3 in somatic hypermutation

Somatic hypermutation (SHM) and class switch recombination (CSR) allow B cells to make high affinity antibodies of various isotypes. Both processes are initiated by activation-induced cytidine deaminase (AID) to generate dG:dU mismatches in the immunoglobulin genes that are resolved differently in SHM and CSR to introduce point mutations and recombination, respectively. The MutL homolog MLH3 has been implicated in meiosis and DNA mismatch repair (MMR). Since it interacts with MLH1, which plays a role in SHM and CSR, we examined these processes in Mlh3-deficient mice. Although deficiencies in other MMR proteins result in defects in SHM, Mlh3(-/-) mice exhibited an increased frequency of mutations in their immunoglobulin variable regions, compared to wild type littermates. Alterations of mutation spectra were observed in the Jh4 flanking region in Mlh3(-/-) mice. Nevertheless, Mlh3(-/-) mice were able to switch to IgG3 or IgG1 with similar frequencies to control mice. This is the first instance where a loss of a DNA repair protein has a positive impact on the rate of SHM, suggesting that Mlh3 normally inhibits the accumulation of mutations in SHM.     

Evolution of Ig DNA sequence to target specific base positions within codons for somatic hypermutation

Ig variable (V) region genes are subjected to a somatic hypermutation process as B lymphocytes participate in immune reactions to protein Ags. Although little is known regarding the mechanism of mutagenesis, a consistent hierarchy of trinucleotide target preferences is evident. Analysis of trinucleotide regional distributions predicted and we now empirically confirm the surprising finding that the framework 2 region of kappa V region genes is highly mutable despite its importance to the structural integrity and function of the Ab molecule. Interestingly, much of this mutability appears to be focused on the third codon position where synonymous substitutions are most likely to occur. We also observed a trend for high predicted mutability for codon positions 1 and 2 in complementarity-determining regions. Consequently, amino acid replacements should occur at a higher rate in complementarity-determining regions than in framework regions due to the distribution and subsequent targeting of microsequences by the mutation mechanism. Our results reveal a subtle tier of V region gene evolution in which DNA sequence has been molded to direct mutations to specific base positions within codons in a manner that minimizes damage and maximizes the benefits of the somatic hypermutation process.     

Specificity change of antibody to (4-hydroxy-3-nitrophenyl)acetyl haptens by somatic hypermutation

Change in the specificity of anti-(4-hydroxy-3-nitrophenyl)acetyl (NP) antibodies (Abs) with time after immunization was studied. The early anti-NP Abs was specific to the ionized (phenolate) form of NP. The specificity changed with time and the late Abs became able to bind to the protonated (phenolic) form as well as the phenolate form of NP. The nucleotide sequences of mRNA coding for variable regions of heavy and light chains suggested that somatic hypermutation contributed to this change of the specificity.     

The Fanconi anemia core complex is dispensable during somatic hypermutation and class switch recombination

To generate high affinity antibodies during an immune response, B cells undergo somatic hypermutation (SHM) of their immunoglobulin genes. Error-prone translesion synthesis (TLS) DNA polymerases have been reported to be responsible for all mutations at template A/T and at least a fraction of G/C transversions. In contrast to A/T mutations which depend on PCNA ubiquitination, it remains unclear how G/C transversions are regulated during SHM. Several lines of evidence indicate a mechanistic link between the Fanconi Anemia (FA) pathway and TLS. To investigate the contribution of the FA pathway in SHM we analyzed FancG-deficient B cells. B cells deficient for FancG, an essential member of the FA core complex, were hypersensitive to treatment with cross-linking agents. However, the frequencies and nucleotide exchange spectra of SHM remained comparable between wild-type and FancG-deficient B cells. These data indicate that the FA pathway is not involved in regulating the outcome of SHM in mammals. In addition, the FA pathway appears dispensable for class switch recombination.     

Signals that initiate somatic hypermutation of B cells in vitro

Somatic hypermutation is initiated as B lymphocytes proliferate in germinal centers. The signals that switch on the mutation process are unknown. We have derived an in vitro system to define signals that will initiate mutation in normal, naive splenic B cells. We find that three signals are required to allow detection of somatic mutation in vitro; these are anti-Ig, anti-CD40, and anti-CD38. If any one of these is omitted, mutation remains off. We show that CD40 is obligatory in vivo, as CD40 knockout mice exhibit no Ag-driven mutation. In contrast, CD38 is not, as CD38 knockout mice mutate normally. We believe that, in vitro, CD38, in combination with other stimuli, drives extensive cell division, allowing the detection of mutated sequences. However, in germinal centers in vivo, proliferative activity is instigated by a different molecule. This is the first demonstration of the initiation of hypermutation in vitro with normal splenic B cells using defined stimuli.     

Deficiency in Msh2 affects the efficiency and local sequence specificity of immunoglobulin class-switch recombination: parallels with somatic hypermutation.

During maturation of the immune response, IgM+ B cells switch to expression of one of the downstream isotypes (IgG, A or E). This class switching occurs by region-specific recombination within the IgH locus through an unknown mechanism. A lack of switch recombination in mice deficient in components of the DNA-dependent protein kinase (DNA-PK)-Ku complex has pointed to a role for non-homologous end joining. Here we characterize a switching defect in mice lacking a protein involved in DNA mismatch recognition. Mice deficient in Msh2 give diminished IgG (but not IgM) responses following challenge with both T cell-dependent and T cell-independent antigens. This appears to reflect a B cell-intrinsic defect since B cells from Msh2-deficient mice also exhibit impaired switching (but not blasting or proliferation) on in vitro culture with lipopolysaccharide. Furthermore, those switches that do occur in Msh2-deficient B cells reveal a shift in the distribution of recombination sites used: the breakpoints are more likely to occur in consensus motifs. These results, which intriguingly parallel the effects of Msh2 deficiency on hypermutation, suggest a role for Msh2 in the mechanics of class-switch recombination.     

On the molecular mechanism of somatic hypermutation of rearranged immunoglobulin genes

Somatic hypermutation (SHM) diversifies the genes that encode immunoglobulin variable regions in antigen-activated germinal centre B lymphocytes. Available evidence strongly suggests that DNA deamination potentiates phase I SHM and subsequently triggers phase II SHM. A concise review of this evidence is followed by a detailed critique of two possible models which suggest that polymerase-eta potentiates phase II SHM via either its DNA-dependent or its RNA-dependent DNA synthetic activity. Quantitative analysis, in the context of extant data that define the features of SHM, favours the RNA-dependent mechanism.     

In vivo and in vitro studies of immunoglobulin gene somatic hypermutation

Following antigen encounter, two distinct processes modify immunoglobulin genes. The variable region is diversified by somatic hypermutation while the constant region may be changed by class-switch recombination. Although both genetic events can occur concurrently within germinal centre B cells, there are examples of each occurring independently of the other. Here we compare the contributions of class-switch recombination and somatic hypermutation to the diversification of the serum immunoglobulin repertoire and review evidence that suggests that, despite clear differences, the two processes may share some aspects of their mechanism in common.     

Normal somatic hypermutation of Ig genes in the absence of 8-hydroxyguanine-DNA glycosylase

The hypermutation cascade in Ig V genes can be initiated by deamination of cytosine in DNA to uracil by activation-induced cytosine deaminase and its removal by uracil-DNA glycosylase. To determine whether damage to guanine also contributes to hypermutation, we examined the glycosylase that removes oxidized guanine from DNA, 8-hydroxyguanine-DNA glycosylase (OGG1). OGG1 has been reported to be overexpressed in human B cells from germinal centers, where mutation occurs, and could be involved in initiating Ab diversity by removing modified guanines. In this study, mice deficient in Ogg1 were immunized, and V genes from the H and kappa L chain loci were sequenced. Both the frequency of mutation and the spectra of nucleotide substitutions were similar in ogg1(-/-) and Ogg1(+/+) clones. More importantly, there was no significant increase in G:C to T:A transversions in the ogg1(-/-) clones, which would be expected if 8-hydroxyguanine remained in the DNA. Furthermore, Ogg1 was not up-regulated in murine B cells from germinal centers. These findings show that hypermutation is unaffected in the absence of Ogg1 activity and indicate that 8-hydroxyguanine lesions most likely do not cause V gene mutations.     

Epstein-Barr virus and the somatic hypermutation of immunoglobulin genes in Burkitt's lymphoma cells

It has been suggested that Epstein-Barr virus (EBV) might suppress antibody maturation either by facilitating bypass of the germinal center reaction or by inhibiting hypermutation directly. However, by infecting the Burkitt's lymphoma (BL) cell line Ramos, which hypermutates constitutively and can be considered a transformed analogue of a germinal center B cell, with EBV as well as by transfecting it with selected EBV latency genes, we demonstrate that expression of EBV gene products does not lead to an inhibition of hypermutation. Moreover, we have identified two natural EBV-positive BL cell lines (ELI-BL and BL16) that hypermutate constitutively. Thus, contrary to expectations, EBV gene products do not appear to affect somatic hypermutation.     

Characterization of Ig gene somatic hypermutation in the absence of activation-induced cytidine deaminase

Somatic hypermutation (SHM) of Ig genes depends upon the deamination of C nucleotides in WRCY (W = A/T, R = A/G, Y = C/T) motifs by activation-induced cytidine deaminase (AICDA). Despite this, a large number of mutations occur in WA motifs that can be accounted for by the activity of polymerase eta (POL eta). To determine whether there are AICDA-independent mutations and to characterize the relationship between AICDA- and POL eta-mediated mutations, 1470 H chain and 1313 kappa- and lambda-chain rearrangements from three AICDA(-/-) patients were analyzed. The Ig mutation frequency of all V(H) genes from AICDA(-/-) patients was 40-fold less than that of normal donors, whereas the mutation frequency of mutated V(H) sequences from AICDA(-/-) patients was 6.8-fold less than that of normal donors. AICDA(-/-) B cells lack mutations in WRCY/RGYW motifs as well as replacement mutations and mutational targeting in complementarity-determining regions. A significantly reduced mutation frequency in WA motifs compared with normal donors and an increased percentage of transitions, which may relate to reduced uracil DNA-glycosylase activity, suggest a role for AICDA in regulating POL eta and uracil DNA-glycosylase activity. Similar results were observed in V(L) rearrangements. The residual mutations were predominantly G:C substitutions, indicating that AICDA-independent cytidine deamination was a likely, yet inefficient, mechanism for mutating Ig genes.