Mutations In Complementarity Determining Regions Research Articles

Structure-guided mutations in CDRs for enhancing the affinity of neutralizing SARS-CoV-2 nanobody

The optimization of antibodies to attain the desired levels of affinity and specificity holds great promise for the development of next generation therapeutics. This study delves into the refinement and engineering of complementarity-determining regions (CDRs) through in silico affinity maturation followed by binding validation using isothermal titration calorimetry (ITC) and pseudovirus-based neutralization assays. Specifically, it focuses on engineering CDRs targeting the epitopes of receptor-binding domain (RBD) of the spike protein of SARS-CoV-2. A structure-guided virtual library of 112 single mutations in CDRs was generated and screened against RBD to select the potential affinity-enhancing mutations. Protein-protein docking analysis identified 32 single mutants of which nine mutants were selected for molecular dynamics (MD) simulations. Subsequently, biophysical ITC studies provided insights into binding affinity, and consistent with in silico findings, six mutations that demonstrated better binding affinity than native nanobody were further tested in vitro for neutralization activity against SARS-CoV-2 pseudovirus. Leu106Thr mutant was found to be most effective in virus-neutralization with IC50 values of ∼0.03 μM, as compared to the native nanobody (IC50 ∼0.77 μM). Thus, in this study, the developed computational pipeline guided by structure-aided interface profiles and thermodynamic analysis holds promise for the streamlined development of antibody-based therapeutic interventions against emerging variants of SARS-CoV-2 and other infectious pathogens.

In Vitro Nanobody Library Construction by Using Gene Designated-Region Pan-Editing Technology.

Camelid single-domain antibody fragments (nanobodies) are an emerging force in therapeutic biopharmaceuticals and clinical diagnostic reagents in recent years. Nearly all nanobodies available to date have been obtained by animal immunization, a bottleneck restricting the large-scale application of nanobodies. In this study, we developed three kinds of gene designated-region pan-editing (GDP) technologies to introduce multiple mutations in complementarity-determining regions (CDRs) of nanobodies in vitro. Including the integration of G-quadruplex fragments in CDRs, which induces the spontaneous multiple mutations in CDRs; however, these mutant sequences are highly similar, resulting in a lack of sequences diversity in the CDRs. We also used CDR-targeting traditional gRNA-guided base-editors, which effectively diversify the CDRs. And most importantly, we developed the self-assembling gRNAs, which are generated by reprogrammed tracrRNA hijacking of endogenous mRNAs as crRNAs. Using base-editors guided by self-assembling gRNAs, we can realize the iteratively diversify the CDRs. And we believe the last GDP technology is highly promising in immunization-free nanobody library construction, and the full development of this novel nanobody discovery platform can realize the synthetic evolution of nanobodies in vitro.

IgA natural antibodies are produced following T-cell independent B-cell activation following stroke

Up to 30% of stroke patients experience cognitive decline within one year of their stroke. There are currently no FDA-approved drugs that can prevent post-stroke cognitive decline, in part due to a poor understanding of the mechanisms involved. We have previously demonstrated that a B-lymphocyte response to stroke, marked by IgA + cells, can cause delayed cognitive dysfunction in mice and that a similar adaptive immune response occurs in the brains of some human stroke patients that suffer from vascular dementia. The stimuli which trigger B-lymphocyte activation following stroke, and their target antigens, are still unknown. Therefore, to learn more about the mechanisms by which B-lymphocytes become activated following stroke we first characterized the temporal kinetics of the B-lymphocyte, T-lymphocyte, and plasma cell (PC) response to stroke in the brain by immunohistochemistry (IHC). We discovered that B-lymphocyte, T-lymphocyte, and plasma cell infiltration within the infarct progressively increases between 2 and 7 weeks after stroke. We then compared the B-lymphocyte response to stroke in WT, MHCII-/-, CD4-/-, and MyD88-/- mice to determine if B-lymphocytes mature into IgA + PCs through a T-lymphocyte and MyD88 dependent mechanism. Our data from a combination of IHC and flow cytometry indicate that following stroke, a population of IgA + PCs develops independently of CD4 + helper T-lymphocytes and MyD88 signaling. Subsequent sequencing of immunoglobulin genes of individual IgA + PCs present within the infarct identified a novel population of natural antibodies with few somatic mutations in complementarity-determining regions. These findings indicate that a population of IgA + PCs develops in the infarct following stroke by B-lymphocytes interacting with one or more thymus independent type 2 (TI-2) antigens, and that they produce IgA natural antibodies.

Somatic hypermutation of TCRα contributes to thymic positive selection in sharks

Abstract In mammals and probably all vertebrates, receptor editing of TCR alpha genes enhances immature T cell positive selection over a three-day interval in the thymic cortex. Surprisingly we found extensive somatic hypermutation (SHM) operating at the TCRα locus in the nurse shark thymus, implying that SHM contributes to receptor modifications that enhance positive selection. We analyzed mutation in TCRα families of clones with the same VJ rearrangement. Additionally, in situ hybridization showed the strongest activation-induced cytidine deaminase (AID) expression in the central thymic cortex and bordering the corticomedullary junction. The frequency of mutation at TCRα was as high as that seen at B cell receptor (BCR) loci in sharks and mammals. Complementarity determining regions (CDRs) accumulated significantly more mutations than framework regions (FWs), and significantly more mutations in CDRs resulted in amino acid replacement than in silent mutation. We saw a preference for transition mutations as well as a strong bias toward G:C substitutions within AID hotspots, especially within CDR regions. We suggest that TCRα utilizes SHM to boost positive selection and perhaps to broaden diversification of the αβ T cell repertoire in sharks, the first reported use of this process in thymic diversification in vertebrates.

Efficient affinity maturation of antibody variable domains requires co-selection of compensatory mutations to maintain thermodynamic stability

The ability of antibodies to accumulate affinity-enhancing mutations in their complementarity-determining regions (CDRs) without compromising thermodynamic stability is critical to their natural function. However, it is unclear if affinity mutations in the hypervariable CDRs generally impact antibody stability and to what extent additional compensatory mutations are required to maintain stability during affinity maturation. Here we have experimentally and computationally evaluated the functional contributions of mutations acquired by a human variable (VH) domain that was evolved using strong selections for enhanced stability and affinity for the Alzheimer’s Aβ42 peptide. Interestingly, half of the key affinity mutations in the CDRs were destabilizing. Moreover, the destabilizing effects of these mutations were compensated for by a subset of the affinity mutations that were also stabilizing. Our findings demonstrate that the accumulation of both affinity and stability mutations is necessary to maintain thermodynamic stability during extensive mutagenesis and affinity maturation in vitro, which is similar to findings for natural antibodies that are subjected to somatic hypermutation in vivo. These findings for diverse antibodies and antibody fragments specific for unrelated antigens suggest that the formation of the antigen-binding site is generally a destabilizing process and that co-enrichment for compensatory mutations is critical for maintaining thermodynamic stability.

Activation-induced cytidine deaminase (AID) is strongly expressed in the fetal bovine ileal Peyer's patch and spleen and is associated with expansion of the primary antibody repertoire in the absence of exogenous antigens.

Due to a limited range of immunoglobulin (Ig) genes, cattle and several other domestic animals rely on postrecombinatorial amplification of the primary repertoire. We report that activation-induced cytidine deaminase (AID) is strongly expressed in the fetal bovine ileal Peyer's patch and spleen but not in fetal bone marrow. The numbers of IGHV (immunoglobulin heavy chain variable) mutations correlate with AID expression. The mutational profile in the fetuses is similar to postnatal and immunized calves, with targeting of complementarity-determining region (CDR) over framework region (FR), preference of replacement over silent mutations in CDRs but not in FRs, and targeting of the AID hotspot motif RGYW/WRCY. Statistical analysis indicates negative selection on FRs and positive selection on CDRs. Our results suggest that AID-mediated somatic hypermutation and selection take place in bovine fetuses, implying a role for AID in the diversification of the primary antibody repertoire in the absence of exogenous antigens.

Aid-Mediated Hypermutation of B-Cell Receptors with Subsequent Subclone Selection Define Biological Differences Between IgM-Expressing and Isotype-Switched Follicular Lymphoma

AbstractAbstract 680Pathogenesis of follicular lymphoma (FL) progresses from acquisition of the t(14;18) with upregulation of the anti-apoptotic BCL2 protein to establishment of neoplastic follicles after entry of FL cells into germinal centers (GC), and eventual transformation to diffuse large B-cell lymphoma. The initial t(14;18) appears to occur during immunoglobulin (Ig) VDJ recombination in the bone marrow. FL cells in GC persistently express activation-induced cytidine deaminase (AID) that continuously introduces mutations in the Ig variable region (IgV). Eventually, this somatic hypermutation (SHM) process generates novel glycosylation sites within the antigen-binding regions that are subsequently preserved. The rules governing subclone evolution upon entry into the GC until acquisition of glycosylation sites are unclear.Based on an unbiased A-PCR amplification strategy, a mutation analysis of a total of 585 lymphoma-derived IgV sequences from 79 FL cases demonstrated a different mutation pattern in isotype-switched (IgG/IgA-FL; n=34) in comparison to non-switched (IgM-FL; n=45) FL. Despite lack of evidence for differences in the total numbers of mutations [IgM-FL: median 29.5 (range 1–182) vs. IgG/IgA-FL: 34 (12–121); p=0.15], IgG/IgA-FL showed accumulation of silent mutations in the VDJ region [15 (4–30) vs. 11 (0–48); p=0.010] with a lower replacement-to-silent (R/S) mutation ratio in complementarity determining regions (CDR) [1.8 (0.5–6) vs. 2.6 (0–13); p=0.024]. Most of IgG/IgA-FL showed evidence of selection against replacement mutations in CDR as determined by 3 different independent algorithms (Chang: 61%; Lossos: 32%; Hershberg: 78%), arguing for active preservation of a selected BCR with a given antigen binding site. In contrast, a significant number of IgM-FL (Fisher’s exact test: p=0.002) was characterized by an unambiguous footprint of positive selection as seen in normal B cells during affinity maturation. FL displaying evidence for BCR preservation according to these algorithms also showed a longer diversification process of the malignant clones compared to cases with active subclone selection as determined by a higher outgoing degree and path length in clonal diversification analysis (2.7±0.1 vs.3.1±0.1; p=0.01).In order to explore whether AID activity had an influence on the hypermutation pattern, AID expression was measured by quantitative PCR and immunohistochemistry in a subgroup of 16 IgG/IgA-FL and 23 IgM-FL with sufficient archived material. While AID levels did not differ overall between both groups, AID expression was positively correlated with somatic IgV mutations in IgM-FL (r=0.48; p=0.03). Surprisingly, AID expression tended to be inversely correlated with IgV mutations in IgG/IgA-FL (r= −0.5; p=0.07).Our data indicate opposing possible outcomes of the selection pressures acting in FL: In some cases, AID-induced subclones are positively selected based on advantageous variations in antigen binding sites. In this FL subgroup, AID effects are directly dependent on its expression level, and isotype switching has not yet occurred. Shorter diversification paths suggest an earlier stage in FL development. Overall, this FL subtype resembles physiological clonal selection of normal B cells driven by improved affinity for the cognate antigen. In contrast, the second FL subtype appears to be dependent on a particular BCR that is preserved against continuous AID attack. Hence, AID levels no longer correlate with the mutation rate. Most cases of this FL group have undergone isotype switching, and longer diversification paths suggest a later maturation stage.In summary, we provide evidence for a maturation model with FL evolution from an early stage with positive, possibly antigen-driven subclone selection to a later stage characterized by BCR preservation. Indeed, one informative case with two FL subpopulations expressing the same VDJ sequence, one as IgM and the other as IgG, supports this hypothesis through evidence of positive selection in the IgM compartment and BCR preservation in the isotype-switched compartment. Nevertheless, an alternative scenario with early dichotomy between both defined FL subtypes cannot be excluded with sufficient certainty by this study. The consequences of the differences between the identified biological FL subtypes with respect to overall prognosis and risk for histological transformation remain to be evaluated. Disclosures:No relevant conflicts of interest to declare.

Antigen selection in the IgE response of allergic and nonallergic individuals

Affinity maturation within germinal centers should usually lead to an accumulation of replacement mutations in complementarity-determining regions (CDRs) of Ig genes as a result of antigen selection. A number of studies have suggested, but not statistically demonstrated, that antigen selection might not guide such an accumulation of replacement mutations in allergic IgE sequences. This has been suggested to reflect the nature of allergens themselves or of the allergic response. We sought to investigate the role of antigen selection in the evolution of the IgE response by mean of analysis of Ig sequences derived from both allergic and nonallergic individuals. IgE sequences were amplified from peripheral blood of allergic and nonallergic individuals by using seminested RT-PCR. Additional IgE and IgG sequences were obtained from public databases. Analysis considered replacement mutations in the CDRs as a proportion of total mutations and compared IgE sequences with IgG sequences. The nonallergic IgE sequences were significantly less mutated than both the allergic IgE (P < .001) and IgG (P < .01) sequences. There was a low proportion of replacement mutations in the CDRs of both nonallergic and allergic IgE sequences and a significantly increased proportion of such mutations in IgG sequences (P < .001). IgE antibodies in both nonallergic and allergic individuals appear to accumulate few somatic point mutations as a consequence of antigen selection. Allergic and nonallergic IgE responses might often develop along a common pathway that is distinct from the conventional germinal center reaction through which the IgG response develops.

Variable region genes of human monoclonal autoantibodies to histones H2A and H2B from a systemic lupus erythematosus patient

An antibody phage library obtained from peripheral blood lymphocytes of a systemic lupus erythematosus (SLE) patient was used to isolate four monoclonal autoantibodies against histones H2A and H2B. Analysis of the variable region sequences revealed that the anti-histone monoclonal antibodies were not clonally related; they used V H genes from three different V H gene families (V H3, V H4, and V H5) and distant members of the Vκ group (L25, L6, A27, and O8) in conjunction with different D and J gene segments. These observations suggest that certain gene families or segments are not critical in producing anti-histone autoantibodies in SLE. Most of the utilized V H and Vκ sequences were highly mutated and the complementarity-determining regions (CDRs) varied greatly in length. The V H region of the antibody SLEhis18 had an isoelectric point of 6.1, and 29% of the mutations were changes to acidic amino acid residues. The second CDR (CDR2) of SLEhis18 V H contained one basic and three acidic residues. Acidic residues were observed in the CDR3 regions of V H, but not V L, in all isolated clones; this is unusual, as most autoantibodies are comprised predominantly of non-acidic residues. This is the first report of a systematic sequence analysis of human anti-histone monoclonal antibodies. Our results suggest that certain V genes are not important for autoreactive specificity to histones in SLE; instead, other mechanisms such as an existence of acidic residues and somatic mutations in CDRs are required for specific binding to histones, which might play a role as a stimulatory autoantigen for the activation of autoantibody-producing B-cells and the selection of high affinity antibody.

Targeting and subsequent selection of somatic hypermutations in the human V kappa repertoire.

The number and distribution of nucleotide substitutions in human VkappaJkappa genes were examined using a PCR technique that analyzed nonproductive and productive rearrangements amplified from genomic DNA of individual B cells. The results indicate that the mutational mechanism introduces replacement (R) mutations comparably throughout the length of the VkappaJkappa rearrangement, but tends to target specific triplets. Moreover, hotspots of mutational activity were identified in complementarity determining regions (CDR). A marked increase in the frequency of R mutations in CDR was noted when productive were compared to nonproductive rearrangements, indicating that these were selected into the expressed repertoire. Of note, amino acids encoded by codons adjacent to hotspots of mutation were also positively selected implying that similar regions were targeted for hypermutation and subsequent selection. In contrast to the distribution of CDR mutations, R mutations in the framework (FR) regions tended to be eliminated from productive VkappaJkappa rearrangements, implying that the somatic hypermutational machinery frequently introduced amino acid changes that were deleterious to the structural integrity of the kappa chain protein. The difference in the ratio of R to silent mutations in CDR and FR in the expressed repertoire, therefore, reflects the summation of positive selection of R mutations in the CDR and the elimination of R mutations in the FR. The data indicate that the balance between targeted mutation of VkappaJkappa rearrangements and subsequent selection and elimination governs the pattern of mutations manifest within the expressed kappa repertoire.

Lupus-specific antibodies reveal an altered pattern of somatic mutation.

The F4 idiotype is a heavy chain determinant expressed almost exclusively on IgG immunoglobulins and is highly associated with specificity for double-stranded DNA. Since high-titered F4 expression is present predominantly in sera of patients with systemic lupus erythematosus (SLE), we thought F4+ IgG antibodies might constitute a useful subset of immunoglobulins in which to investigate lupus-specific alterations in variable (V) region gene expression or in the process of somatic mutation. This molecular analysis of F4+ B cell lines generated from lupus patients demonstrates that despite the strong association of F4 reactivity with specificity for native DNA, there is no apparent VH gene restriction. Furthermore, VH gene segments encoding these antibodies are also used in protective immune responses. An examination of the process of somatic mutation in F4+ antibodies showed no abnormality in frequency of somatic mutation nor in the distribution of mutations in complementarity-determining regions or framework regions. However, there was a decrease in targeting of mutations to putative mutational hot spots. This subtle difference in mutations present in these antibodies may reflect an intrinsic defect in mutational machinery or, more likely, altered state of B cell activation that affects the mutational process and perhaps also negative selection.

Hypermutation generating the sheep immunoglobulin repertoire is an antigen-independent process

Somatic hypermutation of light chain V genes during development of B cells in sheep ileal Peyer's patches was studied i n three experimental conditions: in sterile fragments of the ileum surgically isolated from the gut during fetal life, in germ-free sheep, and in animals thymectomized during early fetal life. The somatic mutation pattern was found identical to control tissues in all three experiments. The same age-dependent amount of mutations, a higher than theoretical R/S ratio in complementarity-determining regions (CDRs), and a similar clustering of mutations in CDRs were observed. The mechanism, as estimated from the silent mutation pattern, appears to target mutations to CDRs; moreover, the major Vλ genes have a specific codon usage with a high purine content at the first two bases of the codons and a low content at the third position, which, together with a specific targeting of mutations to purines, favors replacement mutations in CDRs.

Somatic mutation in constant regions of mouse lambda 1 light chains.

To study the distribution of somatic mutation, we determined nucleotide sequences of rearranged lambda 1-chain genomic DNA from four hybridomas obtained from C57BL/6 mice that had been immunized with (4-hydroxy-3-nitrophenyl)acetyl-conjugated chicken gamma globulin. In total, 114 nucleotide substitutions were observed, with neither insertion nor deletion. Sixty-one mutations occurred in the variable-joining region genes (V lambda 1-J lambda 1) and 49 in joining-constant (J lambda 1-C lambda 1) introns. Although frequency decreased with distance from the V lambda 1-J lambda 1 coding region, somatic mutations occurred in the entire J lambda 1-C lambda 1 intron and even in the C lambda 1 region. We found four nucleotide substitutions in C lambda 1 genes, all of which were replacement mutations. Therefore, the mechanism responsible for somatic mutation is operative into the C lambda 1 exons. Nucleotide sequences of rearranged but inactive lambda 2-chain genes from two hybridomas were also examined and compared with those of lambda 1-chain genes. The clustering of replacement mutations in complementarity-determining regions in the inactive lambda 2-chain genes similar to the active lambda 1-chain genes suggested a mechanism that induces somatic mutation preferentially in this region even in the absence of antigenic selection.

Somatic hypermutagenesis in immunoglobulin genes. I. Correlation between somatic mutations and repeats. Somatic mutation properties and clonal selection

Based on the analysis of some immunoglobulin V-gene sequences, somatic mutations are assumed to occur by correction of complementary violations in heteroduplexes formed by direct or inverted repeats. Correlation between somatic mutations and repeats is investigated by a statistical weights method in 323 somatic substitutions in 14 V-genes. Assuming absence of correlation, the probability of observing data in the sample would be very low (0.00004). This result supports the idea that somatic mutations may arise from heteroduplex repair. The high frequency of these mutations in complementarity-determining regions (CDRs) of V-genes may be due to a high concentration of repeats in these regions. Analysis of somatic substitutions has revealed that stabilizing selection seems to provide conservation of framework regions (FRs) (which leads to preservation of the protein's three-dimensional structure). Positive selection may be provided by B-lymphocyte proliferation with large changes in CDRs.