The remarkable flexibility of the human antibody repertoire; isolation of over one thousand different antibodies to a single protein, BLyS.

It is well established that the humoral immune response can generate antibodies to many different antigens. The antibody diversity required to achieve this is believed to be substantial. However, the extent to which the immune repertoire can generate structural diversity against a single target antigen has never been addressed. Here, we have used phage display to demonstrate the extraordinary capacity of the human antibody repertoire. Over 1000 antibodies, all different in amino acid sequence, were generated to a single protein, B-lymphocyte stimulator (BLyS protein). This is a highly diverse panel of antibodies as exemplified by the extensive heavy and light chain germline usage: 42/49 functional heavy chain germlines and 19/33 V(lambda) and 13/35 V(kappa) light chain germlines were all represented in the panel of antibodies. Moreover, a high level of sequence diversity was observed in the V(H) CDR3 domains of these antibodies, with 568 different amino acid sequences identified. Thus we have demonstrated that specific recognition of a single antigen can be achieved from many different VDJ combinations, illustrating the remarkable problem-solving ability of the human immune repertoire. When studied in a biochemical assay, around 500 (40%) of these antibodies inhibited the binding of BLyS to its receptors on B-cell lines. The most potent antibodies inhibited BLyS binding with sub-nanomolar IC(50) values and with sub-nanomolar affinities. Such antibodies provide excellent choices as candidates for the treatment of BLyS-associated autoimmune diseases.

Antibody-induced lysis of isolated rat epididymal adipocytes and complement activation in vivo.

OBJECTIVE: To identify human monoclonal antibodies selectively binding to human adipocytes and to evaluate their ability to induce lysis of isolated rat adipocytes in vitro and to reduce rat complement levels in vivo. RESEARCH METHODS AND PROCEDURES: Using phage display technology, human monoclonal antibodies binding to human adipocyte plasma membranes were identified. Three antibodies (Fat 13, Fat 37, and Fat 41) were selected based on their additional cross-reaction with rat adipocytes and reformatted as a rat chimeric IgG2bs. The ability of these antibodies, both singly and in combination, to induce lysis of rat epididymal adipocytes in vitro and the reduction of serum complement levels in vivo in the rat was evaluated. RESULTS: All antibodies caused similar time- and dose-dependent lysis of isolated rat adipocytes. Calculated mean EC(50) values (maximum percentage of lysis in parentheses) were 0.680 microg/mL (63.2%), 0.546 microg/mL (72.4%), and 0.391 microg/mL (73.7%) for Fat 13, Fat 37, and Fat 41, respectively. Combinations were no more effective than individual antibodies in inducing lysis. Anti-adipocyte antibodies (both singly and in combination) were also similarly effective in vivo. In rats, doses of monoclonal antibody up to 10 mg/kg intraperitoneal generally caused almost complete depletion of serum complement up to 24 hours after dosing recovering to baseline values by day 5. DISCUSSION: Individual and combinations of monoclonal anti-adipocyte antibodies produced a complement-dependent and concentration-dependent activity to lyse adipocytes in vitro and in vivo as measured by a dramatic depletion in serum complement.

Generation and characterization of LymphoStat-B, a human monoclonal antibody that antagonizes the bioactivities of B lymphocyte stimulator.

OBJECTIVE: To identify and characterize a fully human antibody directed against B lymphocyte stimulator (BLyS), a tumor necrosis factor-related cytokine that plays a critical role in the regulation of B cell maturation and development. Elevated levels of BLyS have been implicated in the pathogenesis of autoimmune diseases. METHODS: A human phage display library was screened for antibodies against human BLyS. A human monoclonal antibody, LymphoStat-B, specific for human BLyS was obtained from the library screening and subsequent affinity optimization mutagenesis. The antibody was tested for inhibition of human BLyS in vitro and in an in vivo murine model. Additionally, the consequences of BLyS inhibition were tested in vivo by administration of LymphoStat-B to cynomolgus monkeys. RESULTS: LymphoStat-B bound with high affinity to human BLyS and inhibited the binding of BLyS to its 3 receptors, TACI, BCMA, and BLyS receptor 3/BAFF-R. LymphoStat-B potently inhibited BLyS-induced proliferation of B cells in vitro, and administration of LymphoStat-B to mice prevented human BLyS-induced increases in splenic B cell numbers and IgA titers. In cynomolgus monkeys, administration of LymphoStat-B resulted in decreased B cell representation in both spleen and mesenteric lymph nodes. CONCLUSION: A fully human monoclonal antibody has been isolated that binds to BLyS with high affinity and neutralizes human BLyS bioactivity in vitro and in vivo. Administration of this antibody to cynomolgus monkeys resulted in B cell depletion in spleen and lymph node. This antibody may prove therapeutically useful in the treatment of autoimmune diseases in humans.