Dr. Sudhir Paul

My interests for the next few years are to:

1. Elucidate the immunological selection pressures governing the evolution of catalytic activity in antibodies (Abs);

2. Study the functional role of Ab catalysis in autoimmune disease and immunologicalhomeostasis;

3. Develop techniques to elicit catalytic Ab responses capable of imparting protection against cancer and microbes;

4. Study the interactions of the neuropeptide VIP with unconventional binding agents like lipids and intracellular proteins, with the aims of defining the physiological functions of these interactions, and of obtaining therapeutic effects using VIP.

The foundation for my interest in Ab catalysis lies in the ability of a subset of Abs to cleave polypeptides, DNA and other substrates. The catalytic activity can be seen as an example of several newly discovered variable domain activities that are structurally and functionally distinct from the classical antigen binding site. [In association with H. Kohler and J. Marchalonis, I have proposed the term Superantibodies as a conceptual framework from which the functions of these activities in innate and adaptive immunity can be studied, and from which rational design of anti-tumor and anti-viral activities can be undertaken]. Synthesis of the antigen-specific catalytic Abs seems to be associated preferentially with autoimmune disease (as opposed to infection with exogenous agents). The catalysts have been isolated as autoantibodies from patients with lupus, thyroiditis and airways disease; anti-idiotypic Abs to anti-enzyme Abs, Bence Jones proteins (monoclonal L chains) from multiple myeloma patients, and the L chains of Abs raised by immunization with a polypeptide antigen.

The V domains of Abs are unique in that multiplegenes encoding these proteins exist in the heritable genetic repertoire, and somatic diversification mechanisms operating under the guidance of various cellular and selection pressures permit affinity maturation of molecules specialized to recognize various ligands. This occurs over a period of a few weeks. Enzymes, in comparison are assumed to have evolved their biological functions over millions of years. A focus of the studies in my lab is the delineation of the contributions of the germline and somatic processes in evolution of the catalytic function in Abs. A primitive, non-specific catalytic function appears to be encoded by a germline V gene(s). Mutations introduced into the V domain over the course of B cell clonal selection appear to impartspecificity to catalytic Abs for recognition of individual antigens. It is interesting to contemplate whether somatic optimization for the catalytic turnover rate can occur over the course of clonal selection. Recent data suggests that highly mutated Abs can express catalytic activity. The activity in patients with autoimmune disease derives in part from improved rate constants and in part from improved autoantigen ground state binding. Teleological arguments for somatically-driven improvement of the catalytic function can be found in the possibilities that antigen digestion by the Abs might be factor in antigen processing for T cell presentation, maintenance of immunological tolerance might involve clearance of self antigens by polyreactivecatalytic Abs, and acquisition of the catalytic function by antigen-specific Abs might enhance their biological efficacy compared to stoichiometrically-binding antibodies. Cellular mechanisms for the improved catalytic function can be conceived -- for example, the improved catalysis by the B cell receptor may lead to rapid product release from the cell surface, thus saving the cells from receptor desensitization and the consequent clonal ''silencing''.

I am studying the hypothesis that autoantigen-specific catalytic autoantibodies might be the cause of tissue dysfunction in certain autoimmune diseases and in multiple myeloma. Model catalytic Abs to VIP have been shown to induce inflammatory responses in the airways, and the biological efficacy of acatalytic Ab has been observed to be greater than of its catalytically deficient mutant. Certain novel transition state analogs are presently being developed as inhibitors of the catalytic Abs to permit evaluation of the importance of the catalytic function in causing tissue damage. The design of these analogs is based on our previous mechanistic studies of model catalytic antibodies, i.e., the involvement of nucleophilic serine residues reactive and the ability to recognize flanking residues in the peptidesubstrate.

I feel that it is possible to capitalize on the above considerations to achieve the long-sought goal of applying the catalytic function towards protection against cancer and viral infections. This goal is predicated on the notion thatthecatalytic function is a superior mechanism for obtaining the protection compared to the reversible, stoichiometric binding capability of conventional Abs, because cleavage of the antigen may lead to its permanent inactivation and because a single catalytic Ab molecule will inactivate multiple antigen molecules. Both passive and active immunotherapy regimens are within the purview of this strategy. Hybridization of available, nonspecific catalytic VL domains with a tumor-specific VH domain, for example,is a feasible means to prepare catalysts capable of cleaving tumor associated antigens. Certain strains of mice prone to autoimmune disease synthesize catalytic Abs at increased levels. The desired catalysts can be isolated from these mice by applying methods designed to select Abs containing the germline encoded serine protease-like site specialized for recognition of the immunogen. A suitably designed transition state analog can be employed as the immunogen to force recruitment of the germlinecatalytic site and to enhance the probability of somatic improvement of the catalytic function. Some of these plans have been initiated in regard to developing catalytic Abs to the HIV protein gp120, and the tumor associated antigen, the receptor for epidermal growth factor. Expanded studies will be undertaken as soon funding for pending and planned NIH grant applications on these subjects comes through. Recognizing the possibility of industrial interest in these goals, a new company, CatImmun, has beenstarted to commercialize the catalytic Ab technology.

VIP was the substrate in the work leading to the discovery of catalytic Abs. My continued interest in this pleitropic neuropeptide stems from observations that: (a) VIP binds phospholipids, penetrates the hydrophobic core of lipid bilayers, acquires marked helicity upon binding phospholipids, and displays increased biological activity as a consequence of these phenomena; and (b) VIP can penetrate living cells, bind calmodulin with high affinity,and can modulate the activity of calmodulin-dependent enzymes. VIP is widely distributed in the central and peripheral nervous systems. Decreased levels of the peptide have been implicated in asthma, ulcerative colitis and impotence. We believe lipidic formulations of VIP to be promising in the therapy of certain diseases. In collaboration with Dr. Rubinstein of the the Univ of Illinois and Dr. Said of the State Univ of New York, I would like to continue exploring the contribution of the lipophilicity of VIP, its binding to calmodulin and its internalization by target cells, in the biological effects of VIP. Because of the possible therapeutic implications, Hyperpeptide, a startup company, has initiated fund-raising efforts to permit further research and development.