Hypermutation in antibody affinity maturation.

By studying the role of mismatch repair in hypermutation at the immunoglobulin loci, the field of antibody hypermutation has been integrated into the larger area of DNA repair. Trans-acting factors - Ku70, Ku80 and possibly SWAP-70 - have been identified for the temporally related but not mechanistically related immunoglobulin heavy-chain class-switch.     

Interferon-gamma potentiates antibody affinity in mice with a genetically controlled defect in affinity maturation.

Interferon-gamma (IFN-gamma), the tetrapeptide tuftsin and the synthetic nonapeptide from interleukin-1 beta (IL-1 beta) (amino acids 163-171) have previously been shown to act on macrophages and/or T cells and to enhance antibody titres to T cell-dependent antigens. The ability of these immunomodulatory agents to potentiate antibody affinity in addition to antibody titre has been studied in a line of mice that fail to demonstrate normal maturation of antibody affinity (low N/M mice). The results presented here confirm that each of the agents potentiate antibody levels following simultaneous injection with a T cell-dependent antigen but demonstrate that only IFN-gamma is able to enhance antibody affinity in these mice. The observation that IFN-gamma can enhance both antibody affinity and antibody levels suggests that it could be an important adjuvant for vaccine use.     

Trypanosome infection of mice depresses antibody affinity and delays affinity maturation

Infection with either a pathogenic species of trypanosome (Trypanosoma brucei brucei) or a non-pathogenic trypanosome (Trypanosoma musculi) had differing effects on the response of mice to a soluble protein antigen (human serum albumin, HSA) injected in either Freund's incomplete adjuvant or in saline. T. brucei suppressed the response to HSA to a level undetectable by ammonium sulphate globulin precipitation, irrespective of the mode of immunization, whereas T. musculi did not suppress the amount of antibody produced in response to either form of antigen presentation. The affinity of the antibody produced in response to antigen in adjuvant was unaffected, but antibody affinity was significantly reduced in infected animals in which the antigen was given in saline. This depression of antibody affinity was related to the period of infection and arose as a result of a delay in the normal maturation of affinity. Furthermore, the depression was only observed when infection preceded the exposure to antigen. Possible mechanisms which may lead to a depression of affinity without a corresponding effect upon antibody levels are discussed in context of current knowledge of immunosuppression in trypanosome infections.     

Studies on the control of antibody synthesis. VI. Effect of antigen dose and time after immunization on antibody affinity and heterogeneity in the mouse

The effect of antigen dose and time after immunization on the affinity of serum antihapten antibody was studied in the mouse by the Farr technique and by equilibrium dialysis. A progressive increase in affinity was seen with time after immunization at all antigen dose levels. The rate of increase in affinity was faster with lower doses of antigen. However, the increase in affinity continued for a longer time in animals immunization with larger doses of antigen. Consequently, at 3 months after immunization animals injected with a larger dose of antigen had higher average affinity antibody than did animals immunized with low doses of antigen. Low affinity antibody was produced in significant amounts at all immunizing dose levels and was present throughout the course of the immune response. Certain technical problems in interpretation of equilibrium dialysis data are discussed.     

Affinity maturation in trout: clonal dominance of high affinity antibodies late in the immune response.

The ability of an animal to develop a highly specific antibody response through affinity maturation has been considered an integral part of the adaptive immune response. However, much of the literature dealing with teleostean antibody responses suggests that little or no affinity maturation may occur within these taxa. As trout antibodies are similar to multimeric mammalian IgM, it has been reasoned that affinity maturational shifts in intrinsic affinity might be similarly small. Such small increases in affinity can, however, lead to potentially great avidity changes for multimeric antibodies. We therefore employed a partition-based immunoassay that permits the dissection of a single antiserum into discrete, affinity-based antibody subpopulations. Such partitioning assays provide for enhanced sensitivity and resolution of these affinity subpopulations over that which can be obtained by fluorescence quenching or equilibrium dialysis. Through the use of the partition-based immunoassay, we were able to detect a consistent increase in affinity within trout anti-TNP antisera. Furthermore, it was determined that trout are capable of generating new, higher affinity antibodies relatively late in the antibody response, which then come into dominance. Such evidence suggests that either somatic mutation does occur or that a unique form of affinity-based regulation of antibody expression is employed.     

Studies on the control of antibody synthesis. XII. Genetic influences on antibody affinity.

Genetic controls on the concentration and affinity of the anti-dinitrophenyl antibodies produced by different inbred strains of mice in response to immunization with dinitrophenylated bovine gamma globulin in Freund's complete adjuvant were demonstrated. Based upon data on F1 hybrids and backcrosses, it can be concluded that there are genetic controls on antibody affinity which are not linked to the major histocompatibility complex. In addition, the genetic controls on affinity which were studied here appear to be inherited independently of genetic controls on antibody concentration.     

噬菌体展示技术在抗体亲和力成熟中的应用进展

抗体因其高度的特异性在诊断和靶向治疗上一直备受青睐,相关行业发展也是突飞猛进。噬菌体展示技术作为一种抗体库构建技术,不仅可应用于特异性抗体的筛选,也可应用于对已获得的低亲和力抗体进行亲和成熟的研究。该方法主要利用VH和VL的随机重组,能在一定程度上模拟体内抗体亲和力成熟的过程,使噬菌体展示技术在提升抗体亲和力方面拥有许多优势,以下对噬菌体展示技术在亲和力成熟方面的应用进行综述。     

Antibody response and antibody affinity maturation in cats with experimental proliferative immune complex glomerulonephritis.

An experimental model of proliferative glomerulonephritis (GN) in the cat, which closely resembles human proliferative forms of GN, has been used to study the role of antibody and antibody affinity in the development of immune complex-mediated renal disease. The serum IgG and IgM antibody response to antigen, average antibody affinity (avidity) and affinity heterogeneity of the IgG and IgM populations was assessed at varying times after commencement of chronic immunization with the antigen, human serum albumin (HSA), by enzyme immunoassay. Cats could be classified according to whether they were "low", "intermediate" or "high" IgG responders, by quantification of serum IgG values. Cats with the lowest serum IgG values failed to develop glomerulonephritis. However, there was no relationship between actual IgG values and the severity of the induced disease. In contrast to IgG, there was no division of cats into low or high IgM anti-HSA responders. Again, cats with the lowest IgM values failed to develop GN, but, more interestingly, a late, marked increase in serum IgM anti-HSA occurred only in cats that developed clinical signs of GN (anterior uveitis and nephrotic syndrome). Maturation of average, functional IgG affinity (avidity) for HSA following chronic immunization was clearly demonstrated for all cats. At the end of the experiment, all cats had IgG of high affinity for HSA and the average affinity heterogeneity of the IgG populations was less than in measurements taken earlier. Values of IgG affinity at the end of the experiment were very similar both in cats which developed GN and in those which remained clinically, biochemically and pathologically normal. In contrast to IgG antibody, some cats developed IgM of increased affinity, whilst others produced antibody of reduced affinity, following chronic immunization. There was no correlation between the development of disease and the production of either low or high affinity IgM antibody. Data indicated that an alteration in IgM affinity occurred at a late stage, as serum IgM levels increased, in cats which progressed to develop GN. These findings suggested that an increase in both serum IgG and IgM anti-HSA values, in particular IgM, was associated with the development of a more severe immune complex renal disease in these cats. Although there was no evidence that differences in the average affinity of either the IgG or IgM antibody populations for HSA, were associated with the development of disease, the increase in IgM values was also accompanied by a concomitant alteration in IgM affinity for antigen.(ABSTRACT TRUNCATED AT 400 WORDS)     

Rabbit lymphoid cells. II. Anti-allotype antisera, lipopolysaccharide and other bacterial and fungal mitogens as probes for the identification of B-cell subpopulations.

Removal of adherent cells or complement-mediated killing of rabbit thymus lymphocyte antigen (BTLA) bearing rabbit T lymphocytes did not abolish the responsiveness (increased thymidine incorporation) of lymphoid cells to antibody against immunoglobulin allotype, Nocardia water-soluble mitogen (NWSM), pneumococcal polysaccharide SIII (PPSIII), S. abortus lipopolysaccharide (LPS), lipid A conjugated to bovine serum albumin and a crude preparation containing C polysaccharide from the cell wall of Diplococcus pneumoniae. Isologous and heterologous antisera, directed against different portions of the Ig receptor, differed in their capacity to enhance thymidine incorporation. The difference in mitogenicity of these antisera was discussed in terms of the accessibility of cell-bound immunoglobulin receptor sites. Spleen cells, responsive to anti-allotype (Ab4) antiserum and B-cell mitogens, NWSM and PPSIII, were characterized by velocity sedimentation. The mean volume of NWSM- and PPSIII-responsive cells was larger than that of the cells responsive to anti-allotype antiserum. Fractionation of spleen cells on glass bead columns yielded a population of non-adherent cells which were two to six times as responsive to anti-Ab4 antiserum as the original spleen cell suspension. The responsiveness of peripheral blood lymphocytes to anti-Ab4 antiserum was significantly greater than that of spleen cells. On the other hand, spleen cells were more responsive to PPSIII than were cells from the peripheral blood, the popliteal and the mesenteric lymph nodes.     

Low antigen dose favours selection of somatic mutants with hallmarks of antibody affinity maturation.

The immunization schedule is critical for the derivation of high-affinity antibodies, low antigen dose being particularly favourable for the development of a more efficient memory response. To analyse the molecular events underpinning this preference, we analysed the early maturation of the response to the hapten 2-phenyloxazolone (phOx) using low and high doses of immunogen. The phOx response is initially dominated by antibodies expressing the VkOx1-Jk5 light chain and the hallmark of the early stages of maturation is the substitution of His 34 by Asn or Gln increasing affinity 10- or eightfold, respectively, and of Tyr 36 by Phe. High-affinity antibodies express mutations at both sites. We cloned and sequenced VkOx1-Jk5 light chains from antigen-specific B cells taken 14 and 21 days after immunization with high and low antigen doses. We found that overall, the derived sequences were more mutated both at longer times and at higher dose. At day 14, His 34 was more frequently mutated at the higher than at the lower dose, while at day 21 the reverse was true. On the other hand, the His 34/Tyr 36 mutation pair was more frequent at low than high doses at both 14 and 21 days. Furthermore, at both times, the low immunization protocol yielded double mutants in cells with a lower mutation background. It appears therefore that while the higher dose may favour the acquisition of individual critical mutations, low-dose immunization favours the selection of a more focused mutational pattern, whereby advantageous mutations are associated with a low mutational background.     

DNA affinity column chromatography: application in the isolation of distinct antibody populations from SLE sera.

An improved method was developed for purification of anti-DNA antibodies by single-stranded DNA (ssDNA), or double-stranded DNA (dsDNA) affinity column chromatography. The effectiveness of this method was examined by measuring the recoveries and purifications of various monoclonal and polyclonal antibodies. Results showed that this method gave good recoveries and purifications of anti-DNA antibodies. Furthermore, by this method, it was possible to isolate antibody populations with distinct specificities from polyclonal antibodies, as shown by enzyme linked immunosorbent assay (ELISA). Therefore, this method should be useful for characterization of polyclonal antibodies in the sera of patients with systemic lupus erythematosus (SLE) as well as of autoimmune lupus mice.     

In vitro improvement of a shark IgNAR antibody by Qbeta replicase mutation and ribosome display mimics in vivo affinity maturation

We have employed a novel mutagenesis system, which utilizes an error-prone RNA dependent RNA polymerase from Qbeta bacteriophage, to create a diverse library of single domain antibody fragments based on the shark IgNAR antibody isotype. Coupling of these randomly mutated mRNA templates directly to the translating ribosome allowed in vitro selection of affinity matured variants showing enhanced binding to target, the apical membrane antigen 1 (AMA1) from Plasmodium falciparum. One mutation mapping to the IgNAR CDR1 loop was not readily additive to other changes, a result explained by structural analysis of aromatic interactions linking the CDR1, CDR3, and Ig framework regions. This combination appeared also to be counter-selected in experiments, suggesting that in vitro affinity maturation is additionally capable of discriminating against incorrectly produced protein variants. Interestingly, a further mutation was directed to a position in the IgNAR heavy loop 4 which is also specifically targeted during the in vivo shark response to antigen, providing a correlation between natural processes and laboratory-based affinity maturation systems.     

Calculation of average antibody affinity in anti-hapten sera from data obtained by competitive radioimmunoassay

A method is described for rapid and precise determination of average antibody affinity in anti-hapten sera from the following data obtained by competitive radioimmunoassay (RIA): the molar hapten (inhibitor) concentration giving 50% inhibition of tracer-antibody binding under equilibrium conditions, the molar tracer concentration in the assay and the amount of tracer bound by the antibodies in the absence of inhibitor. Thus, no additional experiments are required once a competitive RIA has been established. As an example, the average antibody affinity constants for 2′-deoxyguanosine, O⁶-ethylguanosine, and O⁶-ethyl-2′-deoxyguanosine were determined in antisera raised against the respective nucleoside-protein conjugates.     

Polysaccharide vaccines as probes of antibody repertoires in man

Summary: Antibodies specific for capsular polysaccharide epitopes mediate immunity to encapsulated bacterial pathogens, and accordingly, vac-cine development has focused upon the induction of these specificities. Efficacious vaccines, consisting of either polysaccharide alone or polysaccharide coupled to protein carriers, have been developed for a number of pathogens. Their clinical importance not with standing, these vaccines serve as model antigens to study the genetic and somatic forces molding adaptive immunity in man. In this article we review progress aimed at delineating the structure and dynamics of the human antibody repertoire to the Hoemophilus influenzoe type b poiysaccharide (Hib PS), a system which has been studied from infancy to old age. Collectively, the data reveal a repertoire which is encoded by a relatively iarge number of germline variable (V) region gene segments, but which is typically expressed within individuals as a markedly restricted, oligoclonal population. One particular V domain has attained canonical status because of its high penetrance at the population level and its predominance in individual repertoires, Although this combining site is assembled in early infancy and retains its prominence throughout life, its frequency of expression, affinity and protective function are dictated by the molecular form of the Hib PS immunogen (vaccine). The determinants of Hib PS binding affinity can include both germline and somatically-acquired V region polymorphisms. We discuss how these properties of the Hib PS repertoire could impact immunity to Htb, and we consider the implications of these findings towards understanding die evolution of immunoglobulin germline V genes.     

T-cell maturation and clonal deletion in cyclosporine-induced autoimmunity.

In some circumstances cyclosporine (CyA) induces autoimmune phenomena, and a lethal form of syngeneic graft-vs-host disease (SGVHD) can be induced in rats by the administration of CyA. Although several rat strains develop this disease, in this study, we report that of six mouse strains tested, only the DBA/2 strain developed SGVHD. A comparison of the effect of CyA on thymocytic and peripheral T-cell populations revealed that CyA-induced alterations were similar in the two species, although more marked in rats. Notably, CyA-treated syngeneic bone marrow chimeras (SBMC) had transiently increased numbers of peripheral CD4+ CD8+ T-cells, expressing a marker normally limited to thymocytes. Examination of T-cell receptor (TCR) V beta expression in CyA-treated mice revealed a normal pattern of clonal deletion in all strains (including disease-prone DBA/2 mice), suggesting that CyA may induce autoimmunity without blocking intrathymic clonal deletion.     

A study of the structural correlates of affinity maturation: antibody affinity as a function of chemical interactions, structural plasticity and stability

Mutations introduced in an antibody germline sequence as a result of somatic hypermutation could cause its derivatives to have an altered affinity for its target. Affinity maturation favors the selection of the antibodies which exhibit increased affinity. The mutations in 80 high affinity anti-thyroid peroxidase sequences derived from six germlines were analysed in terms of the physicochemical properties of the replacement residues, namely hydrophilicity, size and polarizability, and charge and polarity, in the context of its position and probable solvent accessibility. The effects of these substitutions were evaluated in terms of the resultant increased chemical interactivity potential of the affinity-matured antibodies relative to the germline. The results of the analysis would be useful in the rational design of antibodies and of other proteins for improved binding properties.     

Studies on the control of antibody synthesis. VIII. Selection for high affinity antibody production in the secondary response.

Priming with haptenic determinants can augment the antibody response to a new haptenic determinant presented on a different carrier to which the original haptens are also coupled. This augmentation may be accompanied by a slight increase in the affinity of the antibody formed. However, only if the test hapten is present on the original priming antigen is there a selection for high affinity antibody-forming cells and a marked increase in the affinity of the antihapten antibody produced upon boosting is seen. The data are consistent with the assumption that antibody affinity is primarily controlled by the selection of B lymphocytes by antigen, while additional factors may be involved in controlling the magnitude of the response.     

Studies on the control of antibody synthesis. Effect of antibody affinity upon its ability to suppress antibody formation

Suppression of anti-DNP antibody formation by passively administered anti-DNP antibody was studied quantitatively. The ability of an antiserum to suppress antibody formation was found to be related to the affinity of the antibody for the homologous antigenic determinant (ε-DNP-L-lysine), high affinity antibody being capable of causing suppression at far lower concentrations than low affinity antibody. In addition, very low concentrations of high affinity antibody were found to bring about enhancement of antibody formation. The results are discussed with respect to the significance of circulating antibody in the control of antibody synthesis.Partial suppression of antibody formation by a single injection of passive antibody slightly lowered the affinity of the antibody synthesized. The affinity of the antibody synthesized was independent of the affinity of the passive antibody used to bring about partial suppression.     

In vitro affinity maturation of human GM-CSF antibodies by targeted CDR-diversification

The mammalian immune system applies somatic hypermutation to select for antibodies with improved dissociation rates in vivo up to an intrinsic limit, previously termed as affinity ceiling. However, for certain therapeutic applications it may be desirable to further improve antibody affinities beyond that limit. In this study the selection of antibodies specific for the pro-inflammatory cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) from the HuCAL GOLD human antibody library is described. In order to increase affinity and also functional activity, in vitro affinity maturation of a pool of lead Fab candidates was carried out. CDR-L3 and parallel CDR-H2 diversification using trinucleotide consensus cassettes were followed by the combination of optimized CDR-L3 and CDR-H2 leading to a 5000-fold improved affinity finally reaching a K(D) of 400 fM. Cytokine neutralizing potential of MOR04357 was evaluated in a TF-1 proliferation assay. Along with affinity optimization a 2000-fold increase in potency was observed compared to the parental antibody. Due to species cross-reactivity MOR04357 also blocks rat GM-CSF induced proliferation of FDCP-1 cells. Receptor inhibition studies showed that MOR04357 prevents the interaction of GM-CSF with the GM-CSF receptor alpha chain. As a consequence this leads to a blockade in signal transduction as measured by abolished STAT5 phosphorylation in the presence of GM-CSF and antibody. Due to its pro-inflammatory role GM-CSF has been implicated in the pathophysiology of inflammatory diseases like rheumatoid arthritis or asthma. Based on the mode of action described herein MOR04357 shows favourable antibody features as a potential drug candidate.     

Anti-bovine antibody in human sera as a cause of nonspecificity in enzyme immunoassay.

Much nonspecific activity in enzyme immunoassay is due to reactions between anti-bovine antibodies which are commonly present in human serum samples and residual calf serum in viral antigens. Calf serum is used in cultured cells which are used to produce viral antigens. Addition of bovine proteins to serum samples to be tested for viral antibodies reduces this activity.