V Michael Holers - US grants

We are seeking funding to identify and characterize the ligand binding sites on human Complement Receptor 2 (CR2) for C3d,g and the Epstein-Barr virus (EBV) using a peptide based pproach. In preliminary experiments utilizing a recombinant DNA based approach, we have localized these sites to a sub-domain of the receptor. We are extending these recombinant studies to further localize critical domains and amino acids by site-directed mutagenesis, homolog-scanning mutagenesis to move mouse CR2 sequence onto human CR2, and construction of soluble recombinant forms of CR2. We propose herein and are seeking funding to synthesize CR2 derived peptides which will be tested or their ability to inhibit C3d,g and EBV binding. Following initial identification of relevant peptides, we will attempt to synthesize derivatives with enhanced activity. Through this coordinated approach to receptor-ligand interaction analysis, it is anticipated that an extensive understanding of these sites will be obtained. This understanding will allow further experimental and perhaps better clinical approaches to immunotherapy and anti-viral therapy mediated through CR2.

We are seeking funding to study mouse homologues of the human Regulators of Complement Activation (RCA) gene family. These homologues include mouse Complement Receptors 1 (MCR1) and 2 (MCR2) in addition to Crry/p65, a novel mouse intrinsic complement regulatory protein. Membrane RCA proteins are proposed to play fundamental roles in the physiology of the normal immune response in addition to autoimmune diseases, reproductive biology, hyperacute transplantation rejection, and tumor cell resistance to cytolysis. In order to ultimately study the in vivo roles of these proteins and experimentally manipulate their activities, we propose to establish a mouse model system. However, the structure and biologic roles of mouse homologues of this family, including human CR1, CR2, and the intrinsic complement regulatory proteins Decay-accelerating Factor (DAF) and Membrane Cofactor Protein (MCP), have not been well understood. We have identified and characterized three mouse homologues (MCR1, MCR2, and Crry/p65) using both genetic and immunochemical criteria. We now propose to study in detail structural and functional characteristics of these proteins and compare them to the human family. In addition, we will create new monoclonal and polyclonal antibodies with the ability to detect the proteins in vivo and to block specific activities in vitro and ultimately in vivo. We also propose to create soluble forms of these proteins which block receptor activities and/or exhibit complement regulatory activity. Finally, we will determine the structure, biosynthetic characteristics and cell specific expression of Crry/p65, which we have determined to be a novel complement regulatory protein functionally analogous in vitro to human MCP and likely DAF.

Description (Adapted from Investigator's Abstract): A continuation of structural investigations on expressed domains from Complement Receptor CR2 is proposed. Complement receptor type 2 (CR2, CD21) normally serves as a receptor for complement C3 activation fragments and the immuno-modulatory protein CD23. Expression by B lymphocytes is required for the development of a normal humoral immune response to foreign antigens. This is due to the ability of CR2 to functionally link the binding of C3 fragments iC3b and C3d,g with antigen to the signal transducing capability of the B lymphocyte protein CD19. In addition the Epstein-Bar virus (EBV) utilizes CR2 as its primary means of infecting cells. The investigators along with other groups have previously studied structure-function relationships that govern the interaction of CR2 with the above ligands using strategies involving mutagenesis, and antibody binding. They have found that a domain composed of two 60-70 amino acid segments called N-terminal short consensus repeats (SCRs) contain the only two binding sites for C3 fragments and the EBV protein gp350/220, and contain one of the two sites for CD23. A model for this domain of CR2 and the location of the CD3 binding sites has been created, but the actual three dimensional structure is not known. The investigators propose to apply an NMR method for determining the three dimensional structure of the CR2 domain. They propose to further characterize ligand binding sites using mutagenesis and inhibitor techniques. They also propose to determine how this domain interacts with other regions of CR2. More specifically, their three specific aims are: 1)to determine the structure at high resolution of the human CR2 SCR 1-2 domain. 2)to define the ligand binding sites in the human CR2 SCR 1-2 domain for C3 activation fragments iC3b and C3d,g in addition to binding sites for EBV gp350/220 and CD23. 3) to establish the 3-dimensional solution phase structure of a longer CR2 region containing the SCR 1-2 domain and determine whether the orientation of this domain is influenced by its carboxy-terminal flanking SCRs.

DESCRIPTION (Adapted from the Applicant's Abstract): Human Complement Receptor type 2 (CR2/CR21) is a B lymphocyte receptor for at least three distinct ligands: the iC3b and C3d fragments of complement C3, gp350/220 of the Epstein-Barr virus and CD23 (FceRII). By interacting with these ligands, CR2 plays an important role in modulating the immune response. The ligand binding extracellular structure of CR2 consists of a linear array of 60-70 amino acid-containing modules designated short consensus repeats (SCRs). Proteins containing SCRs comprise a genetically linked family called the regulators of complement activation (RCA). Each RCA protein is involved in binding complement C3 and/or C4 and serving as a receptor or as a complement regulatory protein. Previous studies have shown that the binding site for each of these CR2 ligands is contained either completely (iC3b, C3d and gp350/220) or partially (CD23) within the amino terminal two of sixteen SCRs (SCR 1-2 domain ligand binding sites are related but not identical. Presented herein is a preliminary model of the iC3b binding site that we have recently completed by overlaying apparent iC3b ligand contact residues from CR2 onto the NMR derived coordinates of a double SCR domain from the structurally-related RCA family member, factor H. This analysis shows that the two ligand binding sites on the two SCRs are highly solvent exposed, and that a relative twist is required between the two domains in order to create a hypothetical single surface "patch" on CR2 with which iC3b would bind. Because CR2 interacts within a relatively small region with at least three distinct biologically relevant proteins, we believe that CR2 can serve as an excellent model for SCR-ligand interactions and that a comprehensive and multi-disciplinary approach should be pursued to characterize these binding sites. To that end, we propose three specific aims: 1) Determine by two- dimensional 1H NMR analysis and crystallography the three-dimensional structure of the CR2 SCR 1-2 domain, 2) further characterize and compare the binding sites for each ligand (iC3b, C3d, gp350/220 and CD23) using CR2 derived peptides and mutated forms of recombinant CR2, and 3) identify structurally informative reagents (mAbs and single chain Fv (scFv) antibody fragments from phage display libraries) that selectively inhibit binding of each ligand.

This is an investigator-initiated collaborative Phase II treatment study in which we will examine the hypothesis that treatment of patients with systemic lupus erythematosus (SLE) and active lupus nephritis with a blocking anti-human complement C5 monoclonal antibody will lead to objective improvement in renal disease parameters. The anti-C5 monoclonal antibody will lead to objective improvement in renal disease parameters. The anti-G5 monoclonal antibody will be provided by Alexion Pharmaceuticals. Several lines of investigation have supported the concept that C5 plays a central role in renal injury in antibody- mediated diseases such as SLE. While short term studies using a similar inhibitor have shown efficacy in patients with inflammatory complications of coronary artery bypass surgery, the proposed study represents the first application of this therapeutic strategy, chronic inhibition of complement C5 activation, to patients with autoimmune diseases. Patients enrolled in this double blinded, placebo controlled Phase II study will be those who have active but clinically stable nephritis and, thus, do not require immediate introduction of high dose cyclophosphamide or other cytotoxic drug therapy. Two patient groups, treated and untreated (vehicle control only as a placebo), will be studied. The primary outcome variable will be proteinuria. Secondary outcomes will include other measures of renal disease activity, other measures of lupus activity and measure of complement activation. Three Specific Aims will be pursued. Specific Aim #1. Determine the changes in renal disease activity that accompany short term treatment with an anti-C5 monoclonal antibody in patients with active lupus nephritis. Specific Aim #2. Identify changes in levels of complement activation fragments that accompany treatment with anti-c5 monoclonal antibody in patients with active lupus nephritis. Specific Aim #3. Assess these patients treated with an inhibitory anti-C5 monoclonal antibody for evidence of toxicity. This study is integrated into other components and goals of the Denver Autoimmunity Center itself in several ways. First, it utilizes a population of patients drawn from several sources in the Denver Autoimmunity Center. Second, it meets the goal of extending the use of complement inhibitors from animal models, which are being extensively studied here in the laboratory of the P.I. and others, into clinical trials in patients. Third, the analysis of the role of complement inhibitors as compared to cytokine inhibitors is a major component of the Basic Science Project #2 headed by Dr. William P. Arend.

DESCRIPTION: (Taken from the applicant?s abstract) The primary objectives of this training program will remain two-fold: to train rheumatology fellows in research techniques with the goal of becoming successful academic physician-investigators, and to train Ph.D. postdoctoral fellows in basic research related to rheumatologic diseases. The primary rationale for the first trainee group is that academic rheumatologists with a strong foundation in research remain in very short supply but are critically needed in this period of great increases in scientific understanding and substantial improvement in research technologies. A further rationale for this particular program is that the scientific and training opportunities in Denver in basic and translational immunology provide an environment for young rheumatology fellows to both perform research as well as learn about topics in the depth that is required to develop independent research careers. In addition, a newly established Ph.D. in Clinical Sciences Program will provide formal training in clinical investigation. Postdoctoral fellows with Ph.D. degrees who have interests in rheumatic and autoimmune diseases will also gain from this program by training in research methods and areas that are important to the study of human diseases. The training program will be under the direction of Dr. V. Michael Holers, Professor of Medicine and Immunology, Smyth Professor of Rheumatology and new Head of the Division of Rheumatology as of September 1, 2000. There will be two Assistant Program Directors: Dr. William P. Arend, Professor of Medicine and Immunology, Scoville Professor of Rheumatology and current Program Director, and Dr. Brian L. Kotzin, Professor of Medicine and Immunology, Claman Professor of Medicine, Program Director of a newly established NIH funded Autoimmunity Center of Excellence, and Head of the Division of Allergy and Clinical Immunology. These three individuals will be assisted by seven additional project supervisors and 22 Consultants from the Departments of Medicine, Immunology, Pediatrics, Biochemistry, Preventive Medicine (Epidemiology), Pharmacology, Cell Biology and Pharmacology as well as members of the Ph.D. in Clinical Sciences training program. These investigators are located at the University of Colorado Health Sciences Center, National Jewish Medical and Research Center, Barbara Davis Center for Childhood Diabetes, Veterans Administration Medical Center, Denver Health Medical Center, and Children?s Hospital.

DESCRIPTION (provided by applicant): This is a competitive renewal to continue the American College of Rheumatology (ACR) Basic Research Conference for the years 2002-2006. The Basic Research Conference, which is organized under the direction of the ACR Committee on Research (COR), consists of a one and one-half day scientific conference focusing on the basic science aspects of a major topic relevant to rheumatic diseases. It immediately precedes the Annual Meeting of the ACR. The purpose of the Basic Research Conference is to bring increasing numbers of leading basic scientists into contact with the physicians, scientists, and arthritis health professional members of the ACR, to impart current knowledge to the members of the ACR, to facilitate transfer of technology to patient care, and to increase the numbers of basic investigators attending the annual meeting. As outlined in the body of this proposal, we believe those goals are being achieved with the format of this conference. Each year the ACR Annual Meeting is located in a large city in the USA with adequate convention center capacity and hotel space to accommodate attendees of both the Basic Research Conference and the much larger affiliated meeting of ACR