Enhancement and destruction of antibody function by somatic mutation: unequal occurrence is controlled by V gene combinatorial associations.

We examined the positive and negative effects of somatic mutation on antibody function using saturation mutagenesis in vitro to mimic the potential of the in vivo process to diversify antibodies. Identical mutations were introduced into the second complementarity determining region of two anti-phosphocholine antibodies, T15 and D16, which share the same germline VH gene sequence. T15 predominates in primary responses and does not undergo affinity maturation. D16 is representative of antibodies that co-dominate in memory responses and do undergo affinity maturation. We previously reported that > 50% of T15 mutants had decreased antigen binding capacity. To test if this high frequency of binding loss was unique to T15 or a consequence of random point mutations applicable to other combining sites, we analyzed the same mutations in D16. We show that D16 suffers a similar loss of function, indicating an equally high potential for B-cell wastage. However, only D16 displayed the capacity for somatic mutation to improve antigen binding, which should enhance its persistence in memory responses. Mutation of residues contacting the haptenic group, as determined by molecular modeling, did not improve binding. Instead, productive mutations occurred in residues that either contacted carrier protein or were distant from the antigen binding site, possibly increasing binding site flexibility through long-range effects. Targeting such residues for mutation should aid in the rational design of improved antibodies.     

In vitro affinity maturation of a natural human antibody overcomes a barrier to in vivo affinity maturation

Antibodies isolated from human donors are increasingly being developed for anti-infective therapeutics. These antibodies undergo affinity maturation in vivo, minimizing the need for engineering of therapeutic leads for affinity. However, the affinities required for some therapeutic applications may be higher than the affinities of the leads obtained, requiring further affinity maturation in vitro. To improve the neutralization potency of natural human antibody MSL-109 targeting human cytomegalovirus (CMV), we affinity matured the antibody against the gH/gL glycoprotein complex. A phage display library where most of the six complementary-determining regions (CDRs) were allowed to vary in only one amino acid residue at a time was used to scan for mutations that improve binding affinity. A T55R mutation and multiple mutations in position 53 of the heavy chain were identified that, when present individually or in combination, resulted in higher apparent affinities to gH/gL and improved CMV neutralization potency of Fab fragments expressed in bacterial cells. Three of these mutations in position 53 introduced glycosylation sites in heavy chain CDR 2 (CDR H2) that impaired binding of antibodies expressed in mammalian cells. One high affinity (K_D < 10 pM) variant was identified that combined the D53N and T55R mutations while avoiding glycosylation of CDR H2. However, all the amino acid substitutions identified by phage display that improved binding affinity without introducing glycosylation sites required between two and four simultaneous nucleotide mutations to avoid glycosylation. These results indicate that the natural human antibody MSL-109 is close to a local affinity optimum. We show that affinity maturation by phage display can be used to identify and bypass barriers to in vivo affinity maturation of antibodies imposed by glycosylation and codon usage. These constraints may be relatively prevalent in human antibodies due to the codon usage and the amino acid sequence encoded by the natural human repertoire.     

In vitro affinity maturation of a natural human antibody overcomes a barrier to in vivo affinity maturation

Antibodies isolated from human donors are increasingly being developed for anti-infective therapeutics. These antibodies undergo affinity maturation in vivo, minimizing the need for engineering of therapeutic leads for affinity. However, the affinities required for some therapeutic applications may be higher than the affinities of the leads obtained, requiring further affinity maturation in vitro. To improve the neutralization potency of natural human antibody MSL-109 targeting human cytomegalovirus (CMV), we affinity matured the antibody against the gH/gL glycoprotein complex. A phage display library where most of the six complementary-determining regions (CDRs) were allowed to vary in only one amino acid residue at a time was used to scan for mutations that improve binding affinity. A T55R mutation and multiple mutations in position 53 of the heavy chain were identified that, when present individually or in combination, resulted in higher apparent affinities to gH/gL and improved CMV neutralization potency of Fab fragments expressed in bacterial cells. Three of these mutations in position 53 introduced glycosylation sites in heavy chain CDR 2 (CDR H2) that impaired binding of antibodies expressed in mammalian cells. One high affinity (K_D < 10 pM) variant was identified that combined the D53N and T55R mutations while avoiding glycosylation of CDR H2. However, all the amino acid substitutions identified by phage display that improved binding affinity without introducing glycosylation sites required between two and four simultaneous nucleotide mutations to avoid glycosylation. These results indicate that the natural human antibody MSL-109 is close to a local affinity optimum. We show that affinity maturation by phage display can be used to identify and bypass barriers to in vivo affinity maturation of antibodies imposed by glycosylation and codon usage. These constraints may be relatively prevalent in human antibodies due to the codon usage and the amino acid sequence encoded by the natural human repertoire.     

Key mutations stabilize antigen‐binding conformation during affinity maturation of a broadly neutralizing influenza antibody lineage

Affinity maturation, the process in which somatic hypermutation and positive selection generate antibodies with increasing affinity for an antigen, is pivotal in acquired humoral immunity. We have studied the mechanism of affinity gain in a human B‐cell lineage in which two main maturation pathways, diverging from a common ancestor, lead to three mature antibodies that neutralize a broad range of H1 influenza viruses. Previous work showed that increased affinity in the mature antibodies derives primarily from stabilization of the CDR H3 loop in the antigen‐binding conformation. We have now used molecular dynamics simulations and existing crystal structures to identify potentially key maturation mutations, and we have characterized their effects on the CDR H3 loop and on antigen binding using further simulations and experimental affinity measurements, respectively. In the two maturation pathways, different contacts between light and heavy chains stabilize the CDR H3 loop. As few as two single‐site mutations in each pathway can confer substantial loop stability, but none of them confers experimentally detectable stability on its own. Our results support models of the germinal center reaction in which two or more mutations can occur without concomitant selection and show how divergent pathways have yielded functionally equivalent antibodies. Proteins 2014; 83:771–780. © 2014 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.     

Hypermutation at A/T sites during G.U mismatch repair in vitro by human B-cell lysates

Somatic hypermutation in the variable regions of immunoglobulin genes is required to produce high affinity antibody molecules. Somatic hypermutation results by processing G.U mismatches generated when activation-induced cytidine deaminase (AID) deaminates C to U. Mutations at C/G sites are targeted mainly at deamination sites, whereas mutations at A/T sites entail error-prone DNA gap repair. We used B-cell lysates to analyze salient features of somatic hypermutation with in vitro mutational assays. Tonsil and hypermutating Ramos B-cells convert C-->U in accord with AID motif specificities, whereas HeLa cells do not. Using tonsil cell lysates to repair a G.U mismatch, A/T and G/C targeted mutations occur about equally, whereas Ramos cell lysates make fewer mutations at A/T sites (approximately 24%) compared with G/C sites (approximately 76%). In contrast, mutations in HeLa cell lysates occur almost exclusively at G/C sites (> 95%). By recapitulating two basic features of B-cell-specific somatic hypermutation, G/C mutations targeted to AID hot spot motifs and elevated A/T mutations dependent on error-prone processing of G.U mispairs, these cell free assays provide a practical method to reconstitute error-prone mismatch repair using purified B-cell proteins.     

Memory in the B-cell compartment: antibody affinity maturation

In the humoral arm of the immune system, the memory response is not only more quickly elicited and of greater magnitude than the primary response, but it is also different in quality. In the recall response to antigen, the antibodies produced are of higher affinity and of different isotype (typically immunoglobulin G rather than immunoglobulin M). This maturation rests on the antigen dependence of B-cell maturation and is effected by programmed genetic modifications of the immunoglobulin gene loci. Here we consider how the B-cell response to antigen depends on the affinity of the antigen receptor interaction. We also compare and draw parallels between the two processes, which underpin the generation of secondary-response antibodies: V gene somatic hypermutation and immunoglobulin heavy-chain class switching.     

Protein evolution by hypermutation and selection in the B cell line DT40

Genome-wide mutations and selection within a population are the basis of natural evolution. A similar process occurs during antibody affinity maturation when immunoglobulin genes are hypermutated and only those B cells which express antibodies of improved antigen-binding specificity are expanded. Protein evolution might be simulated in cell culture, if transgene-specific hypermutation can be combined with the selection of cells carrying beneficial mutations. Here, we describe the optimization of a GFP transgene in the B cell line DT40 by hypermutation and iterative fluorescence activated cell sorting. Artificial evolution in DT40 offers unique advantages and may be easily adapted to other transgenes, if the selection for desirable mutations is feasible.     

Altered spectra of hypermutation in DNA repair-deficient mice

Affinity maturation of the humoral immune response is based on the ability of immunoglobulin variable genes to undergo a process of rapid and extensive somatic mutation followed by antigenic selection for antibodies with higher affinity. While the behaviour of this somatic hypermutation phenomenon has been well characterized over the last 20 years, the molecular mechanism responsible for inserting mutations has remained shrouded. To better understand this mechanism, we studied the interplay between hypermutation and other DNA associated activities such as DNA repair. There was no effect on the frequency and pattern of hypermutation in mice deficient for nucleotide excision repair, base excision repair and ataxia-telangiectasia mutated gene repair of double strand breaks. However, variable genes from mice lacking some components of mismatch repair had an increased frequency of tandem mutations and had more mutations of G and C nucleotides. These results suggest that the DNA polymerase(s) involved in the hypermutation pathway produces a unique spectra of mutations, which is then altered by mismatch repair and antigenic selection. We, also describe the differential pattern of expression of some nuclear DNA polymerases in hypermutating versus non-hypermutating B lymphocytes. The rapidly dividing germinal centre B cells expressed DNA polymerases alpha, beta, delta, epsilon and zeta, whereas the resting non-germinal centre cells did not express polymerases alpha or epsilon at detectable levels, although they did express polymerases beta, delta and zeta. The lack of expression of polymerase epsilon in the non-germinal centre cells suggests that this enzyme has a critical role in chromosomal replication but does not participate in DNA repair in these cells.     

The mutation patterns in B-cell immunoglobulin receptors reflect the influence of selection acting at multiple time-scales

During the several-week course of an immune response, B cells undergo a process of clonal expansion, somatic hypermutation of the immunoglobulin (Ig) genes and affinity-dependent selection. Over a lifetime, each B cell may participate in multiple rounds of affinity maturation as part of different immune responses. These two time-scales for selection are apparent in the structure of B-cell lineage trees, which often contain a ‘trunk’ consisting of mutations that are shared across all members of a clone, and several branches that form a ‘canopy’ consisting of mutations that are shared by a subset of clone members. The influence of affinity maturation on the B-cell population can be inferred by analysing the pattern of somatic mutations in the Ig. While global analysis of mutation patterns has shown evidence of strong selection pressures shaping the B-cell population, the effect of different time-scales of selection and diversification has not yet been studied. Analysis of B cells from blood samples of three healthy individuals identifies a range of clone sizes with lineage trees that can contain long trunks and canopies indicating the significant diversity introduced by the affinity maturation process. We here show that observed mutation patterns in the framework regions (FWRs) are determined by an almost purely purifying selection on both short and long time-scales. By contrast, complementarity determining regions (CDRs) are affected by a combination of purifying and antigen-driven positive selection on the short term, which leads to a net positive selection in the long term. In both the FWRs and CDRs, long-term selection is strongly dependent on the heavy chain variable gene family.     

Selection of phage antibodies by binding affinity. Mimicking affinity maturation.

We describe a process, based on display of antibodies on the surface of filamentous bacteriophage, for selecting antibodies either by their affinity for antigen or by their kinetics of dissociation (off-rate) from antigen. For affinity selection, phage are mixed with small amounts of soluble biotinylated antigen (less than 1 microgram) such that the antigen is in excess over phage but with the concentration of antigen lower than the dissociation constant (Kd) of the antibody. Those phage bound to antigen are then selected using streptavidin-coated paramagnetic beads. The process can distinguish between antibodies with closely related affinities. For off-rate selection, antibodies are preloaded with biotinylated antigen and diluted into excess unlabelled antigen for variable times prior to capture on streptavidin-coated paramagnetic beads. To mimic the affinity maturation process of the immune system, we introduced random mutations into the antibody genes in vitro using an error-prone polymerase, and used affinity selection to isolate mutants with improved affinity. Starting with a small library (40,000 clones) of mutants (average 1.7 base changes per VH gene) of the mouse antibody B1.8, and using several rounds of affinity selection, we isolated a mutant with a fourfold improved affinity to the hapten 4-hydroxy-5-iodo-3-nitrophenacetyl-(NIP)-caproic acid (mutant Kd = 9.4(+/- 0.3) nM compared with B1.8 Kd = 41.9(+/- 1.6) nm). The relative increase in affinity of the mutant is comparable to the increase seen in the anti-4-hydroxy-3-nitrophenylacetyl/NIP-caproic acid murine secondary immune response.     

Parameters and determinants of responses to selection in antibody libraries

The sequences of antibodies from a given repertoire are highly diverse at few sites located on the surface of a genome-encoded larger scaffold. The scaffold is often considered to play a lesser role than highly diverse, non-genome-encoded sites in controlling binding affinity and specificity. To gauge the impact of the scaffold, we carried out quantitative phage display experiments where we compare the response to selection for binding to four different targets of three different antibody libraries based on distinct scaffolds but harboring the same diversity at randomized sites. We first show that the response to selection of an antibody library may be captured by two measurable parameters. Second, we provide evidence that one of these parameters is determined by the degree of affinity maturation of the scaffold, affinity maturation being the process by which antibodies accumulate somatic mutations to evolve towards higher affinities during the natural immune response. In all cases, we find that libraries of antibodies built around maturated scaffolds have a lower response to selection to other arbitrary targets than libraries built around germline-based scaffolds. We thus propose that germline-encoded scaffolds have a higher selective potential than maturated ones as a consequence of a selection for this potential over the long-term evolution of germline antibody genes. Our results are a first step towards quantifying the evolutionary potential of biomolecules.     

In vitro antibody evolution targeting germline hot spots to increase activity of an anti-CD22 immunotoxin

Recombinant immunotoxin BL22, containing the Fv portion of an anti-CD22 antibody, produced complete remissions in most patients with drug-resistant hairy cell leukemia but had less activity in leukemias with low CD22 expression. Complementarity-determining region (CDR) mutagenesis is used to increase antibody affinity but can be difficult to perform successfully. We previously showed that antibodies with increased affinity and immunotoxins with increased activity could be obtained by directing mutations at specific DNA residues called hot spots. Because hot spots can arise either by somatic mutation or be present in the germline, we examined which type of hot spot is preferred for increasing antibody affinity. Initially, a second generation antibody phage-display library targeting a germline hot spot (Ser(30)-Asn(31)) within CDR1 of the antibody light chain was mutated. Substitution of serine 30 or asparagine 31 with arginine produced mutant immunotoxins with an affinity (0.8 nM) increased 7-fold over BL22 (5.8 nM) and 3-fold over the first generation mutant HA22 (2.3 nM). More importantly, a 10-fold increase in activity over BL22 and a 2-3-fold increase over HA22 were observed in various B lymphoma cell lines including WSU-CLL that contains only 5500 CD22 sites per cell. For comparison, two phage-display libraries targeting non-germline hot spots in heavy chain CDR1 and CDR3 were generated but did not produce Fv with increased affinity. Our results demonstrate that germline hot spots but not non-germline hot spots are effective for in vitro antibody affinity maturation.     

Demonstration of an in vivo generated sub-picomolar affinity fully human monoclonal antibody to interleukin-8

The high specificity and affinity of monoclonal antibodies make them attractive as therapeutic agents. In general, the affinities of antibodies reported to be high affinity are in the high picomolar to low nanomolar range and have been affinity matured in vitro. It has been proposed that there is an in vivo affinity ceiling at 100 pM and that B cells producing antibodies with affinities for antigen above the estimated ceiling would have no selective advantage in antigen-induced affinity maturation during normal immune responses. Using a transgenic mouse producing fully human antibodies, we have routinely generated antibodies with sub-nanomolar affinities, have frequently rescued antibodies with less than 10 pM affinity, and now describe the existence of an in vivo generated anti-hIL-8 antibody with a sub-picomolar equilibrium dissociation constant. This confirms the prediction that antibodies with affinities beyond the proposed affinity ceiling can be generated in vivo. We also describe the technical challenges of determining such high affinities. To further understand the importance of affinity for therapy, we have constructed a mathematical model to predict the relationship between the affinity of an antibody and its in vivo potency using IL-8 as a model antigen.     

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.     

The NK model of rugged fitness landscapes and its application to maturation of the immune response

Adaptive evolution is, to a large extent, a complex combinatorial optimization process. Such processes can be characterized as "uphill walks on rugged fitness landscapes". Concrete examples of fitness landscapes include the distribution of any specific functional property such as the capacity to catalyze a specific reaction, or bind a specific ligand, in "protein space". In particular, the property might be the affinity of all possible antibody molecules for a specific antigenic determinant. That affinity landscape presumably plays a critical role in maturation of the immune response. In this process, hypermutation and clonal selection act to select antibody V region mutant variants with successively higher affinity for the immunizing antigen. The actual statistical structure of affinity landscapes, although knowable, is currently unknown. Here, we analyze a class of mathematical models we call NK models. We show that these models capture significant features of the maturation of the immune response, which is currently thought to share features with general protein evolution. The NK models have the important property that, as the parameter K increases, the "ruggedness" of the NK landscape varies from a single peaked "Fujiyama" landscape to a multi-peaked "badlands" landscape. Walks to local optima on such landscapes become shorter as K increases. This fact allows us to choose a value of K that corresponds to the experimentally observed number of mutational "steps", 6-8, taken as an antibody sequence matures. If the mature antibody is taken to correspond to a local optimum in the model, tuning the model requires that K be about 40, implying that the functional contribution of each amino acid in the V region is affected by about 40 others. Given this value of K, the model then predicts several features of "antibody space" that are in qualitative agreement with experiment: (1) The fraction of fitter variants of an initial "roughed in" germ line antibody amplified by clonal selection is about 1-2%. (2) Mutations at some sites of the mature antibody hardly affect antibody function at all, but mutations at other sites dramatically decrease function. (3) The same "roughed in" antibody sequence can "walk" to many mature antibody sequences. (4) Many adaptive walks can end on the same local optimum. (5) Comparison of different mature sequences derived from the same initial V region shows evolutionary hot spots and parallel mutations. All these predictions are open to detailed testing by obtaining monoclonal antibodies early in the immune response and carrying out in vitro mutagenesis and adaptive hill climbing with respect to affinity for the immunizing antigen.     

Removal of N-linked glycosylation sites in the V1 region of simian immunodeficiency virus gp120 results in redirection of B-cell responses to V3

One mechanism of immune evasion utilized by human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) envelope glycoproteins is the presence of a dense carbohydrate shield. Accumulating evidence from in vitro and in vivo experiments suggests that alterations in N-linked glycosylation of SIV gp120 can enhance host humoral immune responses that may be involved in immune control. The present study was designed to determine the ability of glycosylation mutant viruses to redirect antibody responses to shielded envelope epitopes. The influence of glycosylation on the maturation and specificity of antibody responses elicited by glycosylation mutant viruses containing mutations of specific N-linked sites in and near the V1 and V2 regions of SIVmac239 gp120 was determined. Results from these studies demonstrated a remarkably similar maturation of antibody responses to native, fully glycosylated envelope proteins. However, analyses of antibodies to defined envelope domains revealed that mutation of glycosylation sites in V1 resulted in increased antibody recognition to epitopes in V1. In addition, we demonstrated for the first time that mutation of glycosylation sites in V1 resulted in a redirection of antibody responses to the V3 loop. Taken together, these results demonstrate that N-linked glycosylation is a determinant of SIV envelope B-cell immunogenicity in addition to in vitro antigenicity. In addition, our results demonstrate that the absence of N-linked carbohydrates at specific sites can influence the exposure of epitopes quite distant in the linear sequence.     

Multi‐constraint computational design suggests that native sequences of germline antibody H3 loops are nearly optimal for conformational flexibility

The limited size of the germline antibody repertoire has to recognize a far larger number of potential antigens. The ability of a single antibody to bind multiple ligands due to conformational flexibility in the antigen‐binding site can significantly enlarge the repertoire. Among the six complementarity determining regions (CDRs) that generally comprise the binding site, the CDR H3 loop is particularly variable. Computational protein design studies showed that predicted low energy sequences compatible with a given backbone structure often have considerable similarity to the corresponding native sequences of naturally occurring proteins, indicating that native protein sequences are close to optimal for their structures. Here, we take a step forward to determine whether conformational flexibility, believed to play a key functional role in germline antibodies, is also central in shaping their native sequence. In particular, we use a multi‐constraint computational design strategy, along with the Rosetta scoring function, to propose that the native sequences of CDR H3 loops from germline antibodies are nearly optimal for conformational flexibility. Moreover, we find that antibody maturation may lead to sequences with a higher degree of optimization for a single conformation, while disfavoring sequences that are intrinsically flexible. In addition, this computational strategy allows us to predict mutations in the CDR H3 loop to stabilize the antigen‐bound conformation, a computational mimic of affinity maturation, that may increase antigen binding affinity by preorganizing the antigen binding loop. In vivo affinity maturation data are consistent with our predictions. The method described here can be useful to design antibodies with higher selectivity and affinity by reducing conformational diversity. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

Mimicking Somatic Hypermutation: Affinity Maturation of Antibodies Displayed on Bacteriophage Using a Bacterial Mutator Strain

Human antibodies can now be isolated from antibody repertoires displayed on the surface of filamentous bacteriophage in a process that mimics the primary immune response. Here we have attempted to mimic the secondary response, the natural process of affinity maturation of antibodies occurring in germinal centres, by multiple cycles of random mutation and selection. Phage displaying a human antibody fragment recognising the hapten 2-phenyl-5-oxazolone were grown in a mutator strain of bacteria (Escherichia coli: mutD5) to generate a large repertoire of antibodies that should include the majority of possible single nucleotide point mutations. The repertoire of phage antibody mutants was then selected by binding to hapten. By multiple rounds of growth in the mutator strain, and increasingly stringent selection, we succeeded in isolating mutants with improved binding affinities; furthermore, the distribution of mutations and nucleotide substitution preferences strongly resembled those of somatic hypermutation. We then constructed a genealogical tree from the sequences of mutants taken at different rounds, and identified four sequentially acquired mutations that together improve the binding affinity of the antibody by a factor of 100-fold (fromK_d320 nM to 3.2 nM).     

From affinity selection to kinetic selection in Germinal Centre modelling

Affinity maturation is an evolutionary process by which the affinity of antibodies (Abs) against specific antigens (Ags) increases through rounds of B-cell proliferation, somatic hypermutation, and positive selection in germinal centres (GC). The positive selection of B cells depends on affinity, but the underlying mechanisms of affinity discrimination and affinity-based selection are not well understood. It has been suggested that selection in GC depends on both rapid binding of B-cell receptors (BcRs) to Ags which is kinetically favourable and tight binding of BcRs to Ags, which is thermodynamically favourable; however, it has not been shown whether a selection bias for kinetic properties is present in the GC. To investigate the GC selection bias towards rapid and tight binding, we developed an agent-based model of GC and compared the evolution of founder B cells with initially identical low affinities but with different association/dissociation rates for Ag presented by follicular dendritic cells in three Ag collection mechanisms. We compared an Ag collection mechanism based on association/dissociation rates of B-cell interaction with presented Ag, which includes a probabilistic rupture of bonds between the B-cell and Ag (Scenario-1) with a reference scenario based on an affinity-based Ag collection mechanism (Scenario-0). Simulations showed that the mechanism of Ag collection affects the GC dynamics and the GC outputs concerning fast/slow (un)binding of B cells to FDC-presented Ags. In particular, clones with lower dissociation rates outcompete clones with higher association rates in Scenario-1, while remaining B cells from clones with higher association rates reach higher affinities. Accordingly, plasma cell and memory B cell populations were biased towards B-cell clones with lower dissociation rates. Without such probabilistic ruptures during the Ag extraction process (Scenario-2), the selective advantage for clones with very low dissociation rates diminished, and the affinity maturation level of all clones decreased to the reference level.