Edgar Haber - US grants

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.

Idiotypic determinants characterizing certain antibody specificities have proven valuable structural and genetic markers in studies of antibody diversity and regulation. The major crossreacting idiotype in strain A/J mice immunized with para-azophenylarsonate is heritable and encoded by germline genes. We have demonstrated (in collaboration with M. Gefter) that hybridoma proteins bearing this predominant idiotype are serologically and structurally microheterogeneous but are all derived from a single VH germline gene. In addition, a set of arsonate-nonbinding hybridoma proteins bearing this same predominant idiotype have been produced by immunization with anti-idiotype. Structural studies have demonstrated that these anti-idiotype antibodies are closely related to the arsonate-binding, idiotype-bearing antibodies and are derived from the same V[unreadable]H[unreadable] and V[unreadable]L[unreadable] germline genes. The loss of antigen binding in these molecules has been correlated with somatic mutation involving either the V[unreadable]H[unreadable] gene and/or J[unreadable]H[unreadable] gene segments. In addition, among arsonate-binding hybridomas it is possible to identify a set that have lost idiotype by virtue of somatic mutation in the V[unreadable]H[unreadable] gene or by utilization of different D-gene segments than are ordinarily utilized. The Fab fragment from an arsonate-binding, idiotype-bearing hybridoma has been crystallized, which makes it likely that the three-dimensional structure responsible for this idiotype will be known. In addition to the predominant idiotype, a second idiotype family (Id[unreadable]36-60[unreadable]) among A/J anti-azophenylarsonate antibodies, which are structurally and serologically distinct from the predominant idiotype, have been characterized. The complete variable region protein sequences of Id[unreadable]36-60[unreadable] hybridomas for both the A/J and BALB/c strains have been determined. The entire Id[unreadable]36-60[unreadable] family arises by somatic mutation from single germline V[unreadable]H[unreadable] genes which are closely related in each strain. In addition, the complete light chain variable region sequences of hybridomas from the two strains bearing Id[unreadable]36-60[unreadable] have been determined. These studies, in combination with chain recombination studies, indicate that the protein encoded directly by the germline gene in the BALB/c strain is associated with low affinity for the antigen, indicating that somatic mutation in the system is necessary to enhance arsonate affinity. (AB)

A diverse group of investigators including cardiac physiologists, cardiac surgeons, radiologists, pathologists, biochemists, and immunologists propose to attack the problem of ischemic heart disease both from a fundamental and an applied direction. Studies range from the mechanism of ischemic injury of tissue at a cellular or molecular level to the diagnosis and control of ischemic injury in man.

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.