Contribution of heavy chain junctional amino acid diversity to antibody affinity among p-azophenylarsonate-specific antibodies.

Abstract We showed previously that heavy chain gene junctional amino acid differences among unmutated p-azophenylarsonate (Ars) Abs that share a unique gene segment combination encoding these V regions, termed "canonical," alter affinity. To determine the contribution of junctional amino acid differences to binding, we introduced, by site-directed mutagenesis, various amino acids at position 100 and/or 107 (sequential numbering) into the unmutated Ab 36-65. Among 22 mutant Abs, 15 preserved or showed increased Ars binding (1-to 12.9-fold increase) relative to Ab 36-65, while 7 Abs exhibited lower affinity (&amp;lt; or = 0.5-fold). As much as a 150-fold difference in Ars binding was observed between 2 Abs with different sets of junctions (Asn100/Tyr107 and Val100/Lys107). Thus, amino acid replacements at D gene junctions can produce changes in affinity greater than those for any V region somatic mutation observed thus far in vivo among anti-Ars Abs and, potentially, can result in preferential selection of Abs containing certain junctions during affinity maturation. We combined five different junctional residue pairs with mutations at H chain positions 58 and 59 that are known to be recurrent in vivo and are associated with increased Ars affinity. The mutant Abs all showed increased affinity, indicating that despite variation in D gene junctions of Ars-binding canonical Abs, the combined mutations are additive for enhancement of Ars affinity. These additive effects reflect the "adaptability" of the canonical gene segment combination in sustaining somatic mutations leading to affinity maturation.</jats:p

A functional analysis of the antigenicity of streptokinase using monoclonal antibody mapping and recombinant streptokinase fragments.

Abstract Streptokinase (SK), a bacterial product of pathogenic Streptococcus species, is now widely used as an effective therapy for the treatment of heart attacks. Because naturally occurring antibody to SK is ubiquitous, serious allergic reactions to SK therapy are common. To begin to identify regions of the molecule that are important for the antigenicity of SK we performed studies using a panel of 51 hybridomas producing anti-SK antibodies, recombinant SK fragments, and assays of SK activity. Antibodies generated from mice hyperimmunized with wild-type SK were shown to fall into six distinct complementation groups by competitive binding studies. Recombinant SK fragments were used to determine the peptide regions recognized by these complementation groups. Correlation of the effects of the mAb on SK function, with knowledge of their SK fragment-binding pattern, suggested regions of the SK molecule that are important for the construction and the catalytic function of the SK-plasminogen activator complex.</jats:p

Structural characterization of H chain-associated idiotopes of anti-p-azophenylarsonate monoclonal antibodies.

Abstract The majority of antibodies directed against p-azophenylarsonate (Ars) protein conjugates elicited during secondary immune responses of A/J mice bear a heritable cross-reactive Id (CRIa or IdCR) which corresponds to the utilization of a unique combination of variable region gene segments that can differ by somatic mutations. One such monoclonal anti-Ars antibody, 44-10, bears IdCR as defined by rabbit antisera but does not react with two anti-idiotypic mAb, 5Ci and AD8, which react with all primary (unmutated) IdCR+ antibodies and some secondary response IdCR+ antibodies. We therefore determined the complete sequence of antibody 44-10, which differs from the germline encoded (unmutated) IdCR+ antibody 36-65 at four positions in the H chain V region (VH): position 55 in the second complementarity determining region, 100 and 107 (D-gene junctions) and 110 (in JH2). The 44-10 L chain is unmutated. Sequence analyses of five other secondary immune response anti-Ars IdCR+ antibodies chosen on the basis of sharing one or more of the amino acid substitutions found in 44-10, were correlated with idiotypic expression of this set of antibodies. The results suggest that the mutation at VH position 55 (Asn----Lys) is responsible for loss of the 5Ci idiotope. To substantiate this hypothesis, oligonucleotide-directed mutagenesis of the germline encoded (unmutated) IdCR+ antibody was used to produce two mutants, one with VH Lys 55 and the other containing residues at positions 100, 107 and 110 identical to those found in 44-10. Id binding studies on these mutants confirm that 5Ci idiotope loss is due to conformational changes resulting from a mutation at VH position 55. This mutation also results in loss of the AD8 idiotope in the structural context of antibody 44-10.</jats:p

The amino acid residues at the VH-D-JH junctions affect the affinity of anti-p-azophenylarsonate antibodies.

Abstract Murine A/J anti-p-azophenylarsonate (Ars) antibodies sharing a predominant idiotype are encoded by a single combination of germ-line V region gene segments. The dominance of this idiotype among secondary immune response anti-Ars antibodies has been explained by the Ag-driven selection of favorable somatic mutants of this gene segment combination, associated with an intrinsic Ars-affinity of the germ-line V region higher than that of other possible combinations. To determine the effect of junctional diversity upon affinity for Ag, independently of somatic mutation, we determined the V region sequences and affinity for Ars of five primary response antibodies. These antibodies share identical unmutated V regions but differ only at the D gene junctions. Among the five antibodies, Ars-affinity differed up to 10-fold depending upon the identity of the amino acid residues at the VH-D and the D-JH junctions. The combination of junctional residues observed in two primary response antibodies with relatively low Ars-affinity has not been observed among secondary response antibodies. Thus the identity of junctional residues resulting from gene rearrangement prior to antigen stimulation must be taken into account in hypotheses which account for idiotype dominance by selection on the basis of affinity.</jats:p