Michael Ned Margolies, M.D. - US grants

DESCRIPTION: (Adapted from the applicant's abstract) A majority of antibodies raised against p-azophenylarsonate (Ars)-protein conjugates in A/J mice share a heritable cross-reactive idiotype, as they are encoded by a single combination of variable region germline gene segments termed "canonical." The dominance of the canonically-encoded structure is due to the favorable intrinsic Ars affinity of the V region germline gene combination and its ability to sustain somatic mutations conferring higher affinity leading to preferred antigen-driven selection of B cells expressing canonical V regions. The Ars systems is an important model for defining combining site structural changes occurring temporally in immune responses (affinity maturation), as functional differences among these antibodies can be related structurally by comparison to unmutated precursors. The goals of this project include: 1) To access the differentiative capacity of the germline canonical structure to generate increased affinity and to change specificity for structurally-related analogues as compared to that of a higher-affinity somatically mutated canonical antibody of known crystal structure. 2) To engineer changes in specificity and affinity ex vivo that may not be possible in vivo owing to biases inherent in the germline sequence and the somatic mutation process. A "selective" approach will be used primarily, in which libraries of mutant Fabs derived from germline (unmutated) canonical anti-Ars antibodies and high-affinity (somatically mutated) canonical anti-Ars antibodies are displayed on filamentous bacteriophage and sorted by affinity, and specificity of these mutants for Ars analogues are determined and the results interpreted in the context of the crystal structures of anti-Ars Fabs. In addition to saturation mutagenesis of different CDRs, "random" mutagenesis of entire V regions, combinatorial mutagenesis of CDRs and regions from both chains, as well as site-directed mutagenesis, will be carried out. Novel mutants will be crystallized and their structures determined. The feasibility of antibody combining site engineering to design antibodies with altered function, and in particular to increase specificity, is critical for the success of clinical immunotherapy.

The evolution of our understanding of the structure and function of the antibody combining site has advanced to the point that one can now realistically contemplate the application of antibodies as reagents or drugs of remarkable specificity. Unlike the conventional drug, which is a small organic molecule that allows a limited number of contacts with its target site, the antibody combining site is relatively capacious. This permits numerous interatomic contacts with a ligand, allowing for remarkable selectivity as well as very high affinity. The development of cell fusion methods for producing antibodies now permits the production of homogeneous antibodies of defined specificity in a reproducible manner. In the proposed program, the antibody combining site will be investigated as a major tool in cardiovascular research. On a most basic level, the combining site of antibodies specific for digoxin will be dissected with respect to its primary structure so that antibodies suitable for therapeutic use might be produced by chemical synthesis or alternatively by translation a gene message. Again utilizing digoxin specific antibody as a model, antibody fragments will be examined with respect to their pharmacologic properties in clinical studies. The resolution of molecular properties allowed by the specificity of the antibody combining site will be utilized to advantage in the dissection of the physiology, pathophysiology, and biosynthesis of renin; in the understanding of the properties and purification of adrenergic receptor antibodies; and in the elucidation of the function of prostaglandin and angiotensin receptors. The investigators in all these projects have as a common research tool the antibody combining site and utilize conjointly the facilities of a protein chemistry and cell fusion laboratory to produce and understand the reagents that they utilize.

The evolution of our understanding of the structure and function of the antibody combining site has advanced to the point that one can now realistically contemplate the application of antibodies as reagents or drugs of remarkable specificity. Unlike the conventional drug, which is a small organic molecule that allows a limited number of contacts with its target site, the antibody combining site is relatively capacious. This permits numerous interatomic contacts with a ligand, allowing for remarkable selectivity as well as very high affinity. The development of cell fusion methods for producing antibodies now permits the production of homogeneous antibodies of defined specificity in a reproducible manner. In the proposed program, the antibody combining site will be investigated as a major tool in cardiovascular research. On a most basic level, the combining site of antibodies specific for digoxin will be dissected with respect to its primary structure so that antibodies suitable for therapeutic use might be produced by chemical synthesis or alternatively by translation a gene message. Again utilizing digoxin specific antibody as a model, antibody fragments will be examined with respect to their pharmacologic properties in clinical studies. The resolution of molecular properties allowed by the specificity of the antibody combining site will be utilized to advantage in the dissection of the physiology, pathophysiology, and biosynthesis of renin; in the understanding of the properties and purification of adrenergic receptor antibodies; and in the elucidation of the function of prostaglandin and angiotensin receptors. The investigators in all these projects have as a common research tool the antibody combining site and utilize conjointly the facilities of a protein chemistry and cell fusion laboratory to produce and understand the reagents that they utilize.

Immune responses in certain inbred mouse strains are dominated by antibodies which share common variable (V) region structures (idiotypes) detected serologically by anti-idiotypic reagents. Such idiotypic determinants characterizing certain antibody specificities are useful structural and genetic markers in studies of antibody diversity and regulation. The predominant cross reactive idiotype (Id-CR) in A/J mice immunized with rho-azophenylarsonate (Ars)-protein conjugates is heritable and encoded by a single combination of germline gene segments ("canonical"). Although Jerne proposed that interactions between Id and anti-Id regulate immune responses through recognition of Id determinants, evidence in the Ars system has accumulated that Id dominance may be due to antigen-driven selection of favorable somatic mutants with higher affinity derived from preferred (more "adaptable") V region germline gene combinations. The Ars system is a model for examining regulation by defining the antibody structural changes involved in enhanced antigen affinity occurring temporally during the immune response ("affinity maturation"). Based upon the high resolution X-ray crystal structure of a somatically mutated Ars-binding Id-CR bearing antibody (36-71) and the germline tertiary structure deduced therefrom, we can now examine the detailed structural correlates of Ars binding and idiotypy, as related to somatic mutation and gene junctional variation. We will capitalize on methodologic advances in protein engineering using site-specific mutagenesis and expression, in concert with predictive computer modelling and the crystal structures to: 1) relate somatic mutation and gene junctional diversity to fine structure of the hapten binding site geometry using comparisons of the tertiary structures of germline and mutated antibodies. 2) Engineer novel mutations designed to increase affinity. 3) Examine sites of hypermutation found in vivo to assess whether or not they relate to affinity enhancement or altered idiotopes. 4) Engineer new binding specificities to Ars homologues as a measure of the differentiative capacity of a canonical V region structure. 5) Map idiotopes by mutagenesis and measurement of reactivity with monoclonal anti-Id reagents, and by determination of the crystal structures of Id-anti-Id. The use of antibody engineering to further understand and thus modify antigen-antibody complementarity is necessary to the design of antibodies for therapeutic use in targeting to drugs, toxins, hormones, and cellular receptors.