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C5a is a potent anaphylatoxin that modulates inflammation through the C5aR1 and C5aR2 receptors. The molecular interactions between C5a–C5aR1 receptor are well defined, whereas C5a–C5aR2 receptor interactions are poorly understood. Here, we describe the generation of a human antibody, MEDI7814, that neutralizes C5a and C5adesArg binding to the C5aR1 and C5aR2 receptors, without affecting complement–mediated bacterial cell killing. Unlike other anti–C5a mAbs described, this antibody has been shown to inhibit the effects of C5a by blocking C5a binding to both C5aR1 and C5aR2 receptors. The crystal structure of the antibody in complex with human C5a reveals a discontinuous epitope of 22 amino acids. This is the first time the epitope for an antibody that blocks C5aR1 and C5aR2 receptors has been described, and this work provides a basis for molecular studies aimed at further understanding the C5a–C5aR2 receptor interaction. MEDI7814 has therapeutic potential for the treatment of acute inflammatory conditions in which both C5a receptors may mediate inflammation, such as sepsis or renal ischemia–reperfusion injury.

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.

Ribosome display is a cell-free technology which enables in vitro selection and evolution of antibodies from very large diversified DNA libraries. It operates through the following key steps: (1) generation of PCR library; (2) formation of stable antibody-ribosome-mRNA (ARM) complexes as the selection particles, (3) selection of ligand-binding ARM complexes on an immobilized ligand and (4) recovery of the selected genetic information as DNA by RT-PCR. Since PCR-based random or/and site-directed mutagenesis can be easily used to introduce mutations into the selected DNA pool in each cycle, ribosome display offers an efficient "protein evolution" tool for antibody optimization. Both prokaryotic and eukaryotic cell-free systems have been explored for ribosome display of different proteins. In this chapter, we describe the use of the eukaryotic rabbit reticulocyte ribosome display method to isolate variants of V(H) antibody fragments with improved affinities.

Ribosome display is a cell-free technology which enables in vitro selection and evolution of antibodies from very large diversified DNA libraries. It operates through the following key steps: (1) generation of PCR library; (2) formation of stable antibody-ribosome-mRNA (ARM) complexes as the selection particles, (3) selection of ligand-binding ARM complexes on an immobilized ligand and (4) recovery of the selected genetic information as DNA by RT-PCR. Since PCR-based random or/and site-directed mutagenesis can be easily used to introduce mutations into the selected DNA pool in each cycle, ribosome display offers an efficient "protein evolution" tool for antibody optimization. Both prokaryotic and eukaryotic cell-free systems have been explored for ribosome display of different proteins. In this chapter, we describe the use of the eukaryotic rabbit reticulocyte ribosome display method to isolate variants of V(H) antibody fragments with improved affinities.

Ribosome display is a cell-free display technology for in vitro selection and optimisation of proteins from large diversified libraries. It operates through the formation of stable protein-ribosome-mRNA (PRM) complexes and selection of ligand-binding proteins, followed by DNA recovery from the selected genetic information. Both prokaryotic and eukaryotic ribosome display systems have been developed. In this chapter, we describe the eukaryotic rabbit reticulocyte method in which a distinct in situ single-primer RT-PCR procedure is used to recover DNA from the selected PRM complexes without the need for prior disruption of the ribosome.

Ribosome display is a cell-free display technology for in vitro selection and optimisation of proteins from large diversified libraries. It operates through the formation of stable protein-ribosome-mRNA (PRM) complexes and selection of ligand-binding proteins, followed by DNA recovery from the selected genetic information. Both prokaryotic and eukaryotic ribosome display systems have been developed. In this chapter, we describe the eukaryotic rabbit reticulocyte method in which a distinct in situ single-primer RT-PCR procedure is used to recover DNA from the selected PRM complexes without the need for prior disruption of the ribosome.

Apoptosis through the TRAIL receptor pathway can be induced via agonistic IgG to either TRAIL-R1 or TRAIL-R2. Here we describe the use of phage display to isolate a substantive panel of fully human anti-TRAIL receptor single chain Fv fragments (scFvs); 234 and 269 different scFvs specific for TRAIL-R1 and TRAIL-R2 respectively. In addition, 134 different scFvs that were cross-reactive for both receptors were isolated. To facilitate screening of all 637 scFvs for potential agonistic activity in vitro, a novel high-throughput surrogate apoptosis assay was developed. Ten TRAIL-R1 specific scFv and 6 TRAIL-R2 specific scFv were shown to inhibit growth of tumor cells in vitro in the absence of any cross-linking agents. These scFv were all highly specific for either TRAIL-R1 or TRAIL-R2, potently inhibited tumor cell proliferation, and were antagonists of TRAIL binding. Moreover, further characterization of TRAIL-R1 agonistic scFv demonstrated significant anti-tumor activity when expressed and purified as a monomeric Fab fragment. Thus, scFv and Fab fragments, in addition to whole IgG, can be agonistic and induce tumor cell death through specific binding to either TRAIL-R1 or TRAIL-R2. These potent agonistic scFv were all isolated directly from the starting phage antibody library and demonstrated significant tumor cell killing properties without any requirement for affinity maturation. Some of these selected scFv have been converted to IgG format and are being studied extensively in clinical trials to investigate their potential utility as human monoclonal antibody therapeutics for the treatment of human cancer.

A large 1.29 x 10(11) antibody fragment library, based upon variable (V) genes isolated from human B-cells from 160 donors has been constructed and its performance measured against a panel of 28 different clinically relevant antigens. Over 5000 different target-specific antibodies were isolated to the 28 antigens with 3340 identified as modulating the biological function (e.g. antagonism, agonism) of the target antigen. This represents an average of approximately 120 different functionally active antibodies per target. Analysis of a sample of >800 antibodies from the unselected library indicates V gene usage is representative of the human immune system with no strong bias towards any particular V(H)-V(L) pairing. Germline diversity is broad with 45/49 functional V(H) germlines and 28/30 V(lambda) and 30/35 V(kappa) light-chain germlines represented in the sample. The number of functional V(H) germlines and V(kappa) light-chain germlines present is increased to 48/49 and 31/35, respectively, when selected V gene usage is included in the analysis. However, following selection on the antigen panel, V(H)1-V(lambda)1 germline family pairings are preferentially enriched and represent a remarkable 25% of the antigen-specific selected repertoire.

A comparison has been performed, using phage display or ribosome display, of stringent selections on antibody populations derived from three rounds of phage display selection. Stringent selections were performed by reducing concentrations of the antigen, bovine insulin, down to 1 nM. Higher affinity antibodies were isolated using ribosome display in a process that introduces random mutations across the clone population. Whereas the highest affinity antibody produced by phage display, D3, has a Kd of 5.8 nM as a scFv fragment, ribosome display generated higher affinity variants of this antibody with Kd values of 189 pM and 152 pM, without or with the use of error prone mutagenesis, respectively. The affinities were further increased for each antibody on conversion of the scFv fragments to whole IgG format, to a Kd of less than 21 pM for the highest affinity variant of D3. Mutation of VH D101 of antibody D3 to glycine or valine, removing the salt bridge between K94 and D101 at the base of VHCDR3, was responsible for the enhanced affinity observed. In addition to the variants of D3, other unrelated antibodies of comparable or higher affinity for insulin, were isolated by ribosome display, but not phage display, indicating that ribosome display can enrich for different populations of antibodies. Affinity maturation of phage antibody populations using ribosome display is a valuable method of rapidly generating diverse, high affinity antibodies to antigen and should be readily applicable to the isolation of antibodies for the detection and assay of biomarkers.

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.

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 EC50 values (maximum percentage of lysis in parentheses) were 0.680 μg/mL (63.2%), 0.546 μg/mL (72.4%), and 0.391 μg/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.

Phage display is a powerful technique for the rapid selection and isolation of antibodies to any given target antigen. We have applied this technology to isolate over 100 different human antibodies that bind to antigens expressed in situ on the human adipocyte cell surface. This is a diverse panel of antibodies, as indicated by the V-region sequences. The binding profile of each anti-adipocyte antibody has been characterised using phage antibody immunocytochemistry against a panel of normal human tissues. Although there was some variation in the intensity of the adipocyte staining, each antibody consistently recognised adipocytes, where present, irrespective of the tissue source. In addition, all of the antibodies recognised at least one other cell type other than the adipocyte cell surface. In total, over 50 different tissue-binding profiles were recorded, with the most frequently recognised tissues identified as capillaries or smooth muscle. Extensive tissue binding profiles were generated for some antibodies using a panel of 37 different human tissues. This identified anti-adipocyte antibodies with unexpected profiles, such as FAT.13, which binds only to adipocytes and capillaries in the entire tissue panel. We believe this is the most extensive survey ever undertaken of the human adipocyte cell surface. Moreover, similar methodology could be used to derive complete tissue-binding profiles of antibodies against cell-surface antigens of any cell type. Indeed, by screening antibodies on both normal and diseased tissues, it may be possible to identify antigenic associations between different cell types and the pathologies of many diseases.