The Chemical Synthesis of Knob Domain Antibody Fragments.

Cysteine-rich knob domains found in the ultralong complementarity determining regions of a subset of bovine antibodies are capable of functioning autonomously as 3-6 kDa peptides. While they can be expressed recombinantly in cellular systems, in this paper we show that knob domains are also readily amenable to a chemical synthesis, with a co-crystal structure of a chemically synthesized knob domain in complex with an antigen showing structural equivalence to the biological product. For drug discovery, following the immunization of cattle, knob domain peptides can be synthesized directly from antibody sequence data, combining the power and diversity of the bovine immune repertoire with the ability to rapidly incorporate nonbiological modifications. We demonstrate that, through rational design with non-natural amino acids, a paratope diversity can be massively expanded, in this case improving the efficacy of an allosteric peptide. As a potential route to further improve stability, we also performed head-to-tail cyclizations, exploiting the proximity of the N and C termini to synthesize functional, fully cyclic antibody fragments. Lastly, we highlight the stability of knob domains in plasma and, through pharmacokinetic studies, use palmitoylation as a route to extend the plasma half-life of knob domains in vivo. This study presents an antibody-derived medicinal chemistry platform, with protocols for solid-phase synthesis of knob domains, together with the characterization of their molecular structures, in vitro pharmacology, and pharmacokinetics.

Isolation and characterization of antibody fragment selective for human Alzheimer's disease brain-derived tau variants.

Reagents that can selectively recognize specific toxic tau variants associated with onset and progression of Alzheimer's disease (AD) and other tauopathies can be effective diagnostic and therapeutic tools. We utilized a novel atomic force microscopy-based biopanning protocol to isolate antibody fragments (single chain variable fragments, scFvs) that selectively bind tau variants present in human AD but not cognitively normal age-matched brain tissue. We identified 6 scFvs [Alzheimer's disease tau (ADT)-1 through 6] that readily distinguished between AD and control tissue and sera samples. We utilized 3 of the scFvs (ADT-2, ADT-4, and ADT-6) to analyze longitudinal plasma samples from 50 human patients, 25 patients which converted to AD during the study and 25 that remained cognitively normal. All 3 scFvs could distinguish the AD from control samples with higher tau levels in apolipoprotein E3/3 AD cases compared to apolipoprotein E3/4. Immunohistochemical analyses of human AD brain slices indicated several but not all tau variants overlapping with phosphorylated tau staining. Several reagents also showed therapeutic potential, protecting neuronal cells against AD tau-induced toxicity.

Current research into snake antivenoms, their mechanisms of action and applications.

Snakebite is a major public health issue in the rural tropics. Antivenom is the only specific treatment currently available. We review the history, mechanism of action and current developments in snake antivenoms. In the late nineteenth century, snake antivenoms were first developed by raising hyperimmune serum in animals, such as horses, against snake venoms. Hyperimmune serum was then purified to produce whole immunoglobulin G (IgG) antivenoms. IgG was then fractionated to produce F(ab) and F(ab')2 antivenoms to reduce adverse reactions and increase efficacy. Current commercial antivenoms are polyclonal mixtures of antibodies or their fractions raised against all toxin antigens in a venom(s), irrespective of clinical importance. Over the last few decades there have been small incremental improvements in antivenoms, to make them safer and more effective. A number of recent developments in biotechnology and toxinology have contributed to this. Proteomics and transcriptomics have been applied to venom toxin composition (venomics), improving our understanding of medically important toxins. In addition, it has become possible to identify toxins that contain epitopes recognized by antivenom molecules (antivenomics). Integration of the toxinological profile of a venom and its composition to identify medically relevant toxins improved this. Furthermore, camelid, humanized and fully human monoclonal antibodies and their fractions, as well as enzyme inhibitors have been experimentally developed against venom toxins. Translation of such technology into commercial antivenoms requires overcoming the high costs, limited knowledge of venom and antivenom pharmacology, and lack of reliable animal models. Addressing such should be the focus of antivenom research.

High incidence of intact or fragmented immunoglobulin in urine of patients with multiple myeloma.

In this prospective study we determined the incidence of intact/fragmented immunoglobulin and Bence Jones protein in urine immunofixation using Sebia reagents and HydrasysTM 2 apparatus and compared the results to concentrations of serum free light chains (FLC) assessed using Siemens BNTM II nephelometer and the immunoassay Freelite (Binding Site) in 289 patients with multiple myeloma at diagnosis. It was found that in one third of IgG, IgA and IgD myeloma patients, intact/fragmented immunoglobulin can be detected in urine and is connected with impaired renal function and reduced survival. Urine immunofixation detects monoclonal protein (FLC and intact/fragmented immunoglobulin) in 66-79% of IgG and IgA myeloma patients while serum FLC immunoassay detect it in 82-94% of IgG and IgA myeloma patients. However, the latter method is inadequate for detection of intact/fragmented immunoglobulin in urine. Serum FLC immunoassay and urine immunofixation are complementary methods in diagnosing and monitoring monoclonal protein in patients with myeloma.

IgG antibodies to cyclic citrullinated peptides exhibit profiles specific in terms of IgG subclasses, Fc-glycans and a fab-Peptide sequence.

The Fc-glycan profile of IgG1 anti-citrullinated peptide antibodies (ACPA) in rheumatoid arthritis (RA) patients has recently been reported to be different from non-ACPA IgG1, a phenomenon which likely plays a role in RA pathogenesis. Herein we investigate the Fc-glycosylation pattern of all ACPA-IgG isotypes and simultaneously investigate in detail the IgG protein-chain sequence repertoire. IgG from serum or plasma (S/P, n = 14) and synovial fluid (SF, n = 4) from 18 ACPA-positive RA-patients was enriched using Protein G columns followed by ACPA-purification on cyclic citrullinated peptide-2 (CCP2)-coupled columns. Paired ACPA (anti-CCP2 eluted IgG) and IgG flow through (FT) fractions were analyzed by LC-MS/MS-proteomics. IgG peptides, isotypes and corresponding Fc-glycopeptides were quantified and interrogated using uni- and multivariate statistics. The Fc-glycans from the IgG4 peptide EEQFNSTYR was validated using protein A column purification. Relative to FT-IgG4, the ACPA-IgG4 Fc-glycan-profile contained lower amounts (p = 0.002) of the agalacto and asialylated core-fucosylated biantennary form (FA2) and higher content (p = 0.001) of sialylated glycans. Novel differences in the Fc-glycan-profile of ACPA-IgG1 compared to FT-IgG1 were observed in the distribution of bisected forms (n = 5, p = 0.0001, decrease) and mono-antennnary forms (n = 3, p = 0.02, increase). Our study also confirmed higher abundance of FA2 (p = 0.002) and lower abundance of afucosylated forms (n = 4, p = 0.001) in ACPA-IgG1 relative to FT-IgG1 as well as lower content of IgG2 (p = 0.0000001) and elevated content of IgG4 (p = 0.004) in ACPA compared to FT. One λ-variable peptide sequence was significantly increased in ACPA (p = 0.0001). In conclusion, the Fc-glycan profile of both ACPA-IgG1 and ACPA-IgG4 are distinct. Given that IgG1 and IgG4 have different Fc-receptor and complement binding affinities, this phenomenon likely affects ACPA effector- and immune-regulatory functions in an IgG isotype-specific manner. These findings further highlight the importance of antibody characterization in relation to functional in vivo and in vitro studies.

An extended range generic immunoassay for total human therapeutic antibodies in preclinical pharmacokinetic studies.

Bioanalytical support of discovery programs for human monoclonal antibody therapies involves quantitation by immunoassay. Historically, preclinical samples have been analyzed by the traditional Enzyme-Linked Immuno-Sorbent Assay (ELISA). We investigated transferring our generic ELISA for quantitating human IgG constructs in preclinical serum samples to an automated microfluidics immunoassay platform based on nanoscale streptavidin bead columns. Transfer of our immunoassay to the automated platform resulted in not only the anticipated reduction in analysts' time required for manual manipulation (ELISA) but also a substantial increase in the dynamic range of the immunoassay. The generic nature and wide dynamic range of this automated microcolumn immunoassay permit bioanalytical support of novel therapeutic candidates without the need to develop new, specific assay reagents and minimize the chances that sample reassays will be required due to out of range concentration results. Improved process efficiencies and enhanced workflow during the analysis of preclinical PK samples that enable high throughput assessment of a human monoclonal antibody lead in early discovery programs.

A comparison of capture antibody fragments in cardiac troponin I immunoassay.

OBJECTIVES: To compare cardiac troponin I (cTnI) values measured from 32 normal plasma specimens with a two-site cTnI research assay exploiting different molecular forms of a capture antibody. DESIGN AND METHODS: The current research assay consists of two capture antibodies immobilized on streptavidin-well surface and one detection antibody attached to highly fluorescent europium(III)-chelate-doped nanoparticles. Four different molecular forms of one of the capture antibodies (intact monoclonal (Mab), F(ab')2 fragment, Fab fragment and chimeric Fab fragment (cFab)) were tested. The developed immunoassays were evaluated in terms of their analytical sensitivities and assay kinetics. Furthermore, cTnI concentrations were measured from 32 heparin plasma samples from apparently healthy donors (mean age 32; range 24-60 years). RESULTS: The differences in the measured cTnI concentrations (corrected for the buffer-based zero calibrator) between the Mab and the three fragmented forms were highly significant (P<0.0001). Replacing the intact Mab with the antibody fragments also reduced the required antibody amount from 100 ng to 66 ng (F(ab')2) and 16.5 ng (Fab and cFab). Furthermore, the limit of detection was improved when Fab fragments were employed (Mab: 0.90 ng/L, Fab: 0.69 ng/L and cFab: 0.41 ng/L). The apparent normal range median (minimum/maximum) of the 32 healthy subjects was reduced from 7.28 ng/L (2.64/116 ng/L) with Mab to 1.80 ng/L (0.746/10.6 ng/L) for the cFab. CONCLUSIONS: Eliminating the Fc-part from one of the two capture antibodies in an immunofluorometric cTnI assay substantially reduced the measured cTnI concentrations, simultaneously improving the assay sensitivity and reducing the reagent consumption.

In vivo detection of amyloid-ß deposits using heavy chain antibody fragments in a transgenic mouse model for Alzheimer's disease.

This study investigated the in vivo properties of two heavy chain antibody fragments (V(H)H), ni3A and pa2H, to differentially detect vascular or parenchymal amyloid-ß deposits characteristic for Alzheimer's disease and cerebral amyloid angiopathy. Blood clearance and biodistribution including brain uptake were assessed by bolus injection of radiolabeled V(H)H in APP/PS1 mice or wildtype littermates. In addition, in vivo specificity for Aß was examined in more detail with fluorescently labeled V(H)H by circumventing the blood-brain barrier via direct application or intracarotid co-injection with mannitol. All V(H)H showed rapid renal clearance (10-20 min). Twenty-four hours post-injection (99m)Tc-pa2H resulted in a small yet significant higher cerebral uptake in the APP/PS1 animals. No difference in brain uptake were observed for (99m)Tc-ni3A or DTPA((111)In)-pa2H, which lacked additional peptide tags to investigate further clinical applicability. In vivo specificity for Aß was confirmed for both fluorescently labeled V(H)H, where pa2H remained readily detectable for 24 hours or more after injection. Furthermore, both V(H)H showed affinity for parenchymal and vascular deposits, this in contrast to human tissue, where ni3A specifically targeted only vascular Aß. Despite a brain uptake that is as yet too low for in vivo imaging, this study provides evidence that V(H)H detect Aß deposits in vivo, with high selectivity and favorable in vivo characteristics, making them promising tools for further development as diagnostic agents for the distinctive detection of different Aß deposits.

Antivenoms for snakebite: design, function, and controversies.

BACKGROUND: Animal-derived antivenoms have been used to treat snake envenomation for more than 100 years. Major technological advantages in the past 30 years have produced antivenoms that are highly purified and chemically modified to reduce the risk of acute hypersensitivity reactions. Like all pharmaceutical manufacture, commercial-scale antivenom production requires making trade-offs between cost, purity, pharmacokinetic profile, and production yield. SCOPE: This article reviews the current state of the art for antivenom production and development. Particular attention is paid to controversies and trade-offs used to achieve a balance between improved safety and pharmacokinetic performance.

Anti-CA19-9 diabody as a PET imaging probe for pancreas cancer.

BACKGROUND: Intact antibodies are poor imaging agents due to a long serum half-life (10-20 d) preventing adequate contrast between the tumor and surrounding blood. Smaller engineered antibody fragments overcome this problem by exhibiting shorter serum half-lives (4-20 h).The diabody (55 kDa) is the smallest antibody fragment, which retains the bivalency of the intact antibody. Our goal was to develop and characterize the anti-CA19-9 diabody fragment and determine its ability to provide antigen specific imaging of pancreas cancer. METHODS: The diabody DNA construct was created by isolation of the variable region genes of the intact anti-CA19-9 antibody. Diabody expression was carried out in NS0 cells and purified using HPLC from supernatant. Specific antigen binding was confirmed with flow cytometry and immunofluorescence. Radiolabeled diabody was injected into mice harboring an antigen positive xenograft (BxPC3 or Capan-2) and a negative xenograft (MiaPaca-2). MicroCT and MicroPET were performed at successive time intervals after injection. Radioactivity was measured in blood and tumor to provide objective confirmation of the microPET images. RESULTS: Immunofluorescence and flow cytometry showed specific binding of the anti-CA19-9 diabody. Pancreas xenograft imaging of BxPC3/MiaPaca-2 and Capan-2/MiaPaca-2 models with the anti-CA19-9 diabody demonstrated an average tumor:blood ratio of 5.0 and 2.0, respectively, and an average positive:negative tumor ratio of 11 and 6, respectively. With respect to the tumor:blood ratio, these data indicate five times and two times more radioactivity in the tumor than in the blood yielding adequate contrast between tumor tissue and background (i.e., blood) to create the representative microPET images. CONCLUSIONS: We successfully engineered a functional diabody against CA19-9, a tumor antigen present on the vast majority of pancreas cancers. Additionally, we demonstrate high contrast antigen specific microPET imaging of pancreas cancer in xenograft models.

Absolute quantification of a therapeutic domain antibody using ultra-performance liquid chromatography-mass spectrometry and immunoassay.

BACKGROUND: Domain antibodies (dAbs; ∼10-15 kDa) are made up of the variable heavy chain or the variable light chain of the antibody structure, and retain binding capability. dAbs have proved difficult to detect in plasma using immunoassay without specific antibodies raised against the dAb. RESULTS: A sensitive and selective UPLC-MS/MS method for the absolute quantification of a dAb in monkey plasma was developed (range: 1 to 500 ng/ml) without the need for a specific capture antibody. This method was used to analyze pharmacokinetic studies early on in drug development. Furthermore, an immunoassay was developed and the pharmacokinetic samples were reanalyzed. CONCLUSION: The two assays show good correlation (r(2) = 0.92), giving confidence in using either method for quantification of the dAb.

Nanobodies®: proficient tools in diagnostics.

With the advent of new antibody engineering technologies, conventional antibodies have been minimized into smaller antibody formats. Small size is an important advantage for current and future diagnostic development. Nanobodies® (Ablynx) are among the smallest known antigen-binding antibody fragments, and are derived from the heavy-chain only antibodies that occur naturally in the serum of Camelidae. Endowed by natural evolution, these Nanobodies inherently exhibit unique biophysical, biochemical and pharmacological characteristics. In addition to their excellent potential as molecules in drug development, Nanobodies possess very attractive functional properties that aid in their development for diagnostic tools. Here we present several examples of currently available applications of Nanobodies to the field of immunosensor for cancer, immunoaffinity chromatography, in vivo and intracellular imaging.

Isolation and biochemical characterization of plasma monoclonal free light chains in amyloidosis and multiple myeloma: a pilot study of intact and truncated forms of light chains and their charge properties.

BACKGROUND: The presence of monoclonal immunoglobulin free light chains (FLC) in the serum is commonly associated with the gammopathies, including multiple myeloma, systemic light chain amyloidosis and non-amyloid light chain deposition disease. Although a sensitive nephelometry-based assay is used for quantification of serum FLC and patient follow-up, this method does not provide information regarding the biochemical properties of these proteins. The present study focused on the development of the procedure for isolation and biochemical characterization of monoclonal FLC in small plasma specimens from patients with these disorders. METHODS: Methods used in this study were ultrafiltration, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), protein elution from gel and support membranes, dialysis, lyophilization, isoelectric focusing (IEF) and Western blotting. RESULTS: The isolation, concentration and partial purification of FLC was based on micro-preparative SDS-PAGE employing analytical scale gels. For the determination of the isoelectric point of FLC, the developed protocol included consecutive IEF, electrotransfer of IEF-separated proteins onto and elution from support membranes, and their analysis by SDS-PAGE-based Western blotting. The procedures, which require only 20-50 microL of starting plasma, allow biochemical characterization of the monomeric, dimeric and truncated forms of FLC, including their charge properties. CONCLUSIONS: The developed procedure may be applied to reveal distinguishing chemical features of FLC in serum, which could be important in predicting the pathologic form of disease, and in yielding information to better understand the mechanism(s) involved in the deposition of light chains in tissues.

Generation of high-affinity chicken single-chain Fv antibody fragments for measurement of the Pseudonitzschia pungens toxin domoic acid.

Antibody-based assay systems are now accepted by regulatory authorities for detection of the toxins produced by phytoplankton that accumulate in shellfish tissues. However, the generation of suitable antibodies for sensitive assay development remains a major challenge. We have examined the potential of using the chicken immune system to generate high-affinity, high-specificity recombinant antibody fragments against phytotoxins. Following immunization of the chicken with domoic acid-bovine serum albumin, a single-chain antibody variable region (scFv) gene library was generated from single V(H) and V(L) genes isolated from the immune cells in the spleen and bone marrow. scFvs reacting with domoic acid were isolated by phage display and affinity matured by light chain shuffling, resulting in an approximate 10-fold increase in sensitivity. The isolated scFvs were effectively expressed in Escherichia coli and readily purified by affinity chromatography. They were then used to develop a convenient and sensitive indirect competitive enzyme-linked immunosorbent assay for domoic acid, with a 50% effective dose of 156 ng/ml, which could be used reliably with shellfish extracts. This study demonstrates that chickens provide a valuable model system for the simplified, rapid generation of high-affinity recombinant antibody fragments with specificity for small toxin molecules.

Determination of concentration and binding affinity of antibody fragments by use of surface plasmon resonance.

An assay method using a surface plasmon resonance (SPR) biosensor has been developed that allows quantitative measurement of the specific antibody concentration in crude materials. By injecting non-labeled antibody samples onto a biosensor surface on which antigen was immobilized at high densities, the concentration of active antibodies can be accurately measured. To clarify applicability of this method to pharmacokinetic studies, the concentration of active antibodies in mouse plasma was measured for 4 h after injection of antibodies in mice. Although this period of measurement might be insufficient for determining the pharmacokinetics of blood pool clearance, this method has some advantages over conventional methods in measurement of single-chain antibody fragment (scFv) concentrations. Using the SPR biosensor, scFv and antibodies without epitope tag peptides were easily detected in real time, requiring as little as 20 mul of blood sample. Moreover, from the apparent dissociation rate in the dissociation phase of the sensorgrams, we could identify whether the antibody fragments existed as bivalent or monovalent in animal blood. We also evaluated the antigen binding activity of the scFvs against human CD47 and found scFvs had slightly weak affinity to their antigen (K(D), about 10 nM) compared with F(ab')2 and Fab' fragments (K(D), about 3-4 nM). This assay method promises to be a convenient tool for quality control, screening, and simple pharmacokinetic analysis of antibody fragments and other recombinant proteins not having epitope tags.

Prolonged in vivo residence times of llama single-domain antibody fragments in pigs by binding to porcine immunoglobulins.

The therapeutic parenteral application of llama single-domain antibody fragments (VHHs) is hampered by their small size, resulting in a fast elimination from the body. Here we describe a method to increase the serum half-life of VHHs in pigs by fusion to another VHH binding to porcine immunoglobulin G (pIgG). We isolated 19 pIgG-binding VHHs from an immunized llama using phage display. Six VHHs were genetically fused to model VHH K 609 that binds to Escherichia coli F4 fimbriae. All six yeast-produced genetic fusions of two VHH domains (VHH2s) were functional in ELISA and bound to pIgG with high affinity (1-33 nM). Four pIgG-binding VHH2s were administered to pigs and showed a 100-fold extended in vivo residence times as compared to a control VHH2 that does not bind to pIgG. This could provide the basis for therapeutic application of VHHs in pigs.

Kinetics of whole serum and prepurified IgG digestion by pepsin for F(ab')2 manufacture.

An alternative route for the production of polyclonal F(ab')(2) fragments that might be adopted for the facile preparation of antivenoms is assessed in this work. The method involves the digestion of whole serum by free pepsin, which results in reduction of the number of processing steps commonly in use, because it avoids the initial purification of IgG's prior to their proteolytic cleavage by the enzyme. Digestion kinetics of whole serum and caprylic acid prepurified IgG using free pepsin were monitored with SDS-PAGE followed by densitometric analysis and antigen binding activity assay of the digested samples. It was observed that with equal units of pepsin activity, caprylic acid prepurified IgG was digested more rapidly than whole serum but that the overall retention of antigen binding activity was significantly greater in the latter case. The estimated first-order digestion rate parameters were 11.8 and 4.42 microM min(-)(1) for pure IgG and whole serum, respectively. The K(m) value obtained for whole serum digestion was 33 microM and that for pure IgG digestion was 43.5 microM. Calibration with undigested whole serum and pure IgG samples of known concentrations was performed using SDS-PAGE followed by image analysis. A linear relationship was observed between the protein concentration and the respective band intensity within the range of concentrations investigated (0.63-31.2 microM IgG concentration). This technique proved to be relatively rapid, reproducible, and more precise than size-exclusion chromatography as a result of its F(ab')(2)/IgG resolving power. Staining and destaining protocols were reproduced in terms of staining and destaining times, volumes added, and compositions. Furthermore, all digestion experiments were performed in duplicate sets to monitor the extent of variation of the digestion kinetic parameters measured by this method. The results obtained from this technique confirm and quantify previous observations that pepsin digestion of whole serum is slower and easier to control than digestion of pure IgG and results in higher recovery of antigenic binding activity.

A protocol for 'enhanced pepsin digestion': a step by step method for obtaining pure antibody fragments in high yield from serum.

The digestion of ovine antiserum under acidic conditions (pH 3.5) by pepsin is highly effective at reducing all unwanted serum components to low molecular weight (< or =13 kDa) fragments while leaving the approximately 100-kDa F(ab')(2) intact. The pH is then raised to 6 to stop further digestion and the reaction mixture centrifuged or filtered to remove any insoluble contaminants. Next, unwanted low molecular weight fragments are removed by diafiltration with a 30-kDa nominal molecular weight cut-off membrane leaving an F(ab')(2) solution contaminated only with some pepsin and a small amount of the aggregated low molecular weight fragments. Material of this purity is suitable for many applications but, since all the contaminants are highly acidic, they can be easily removed by passage down an anion-exchange column to yield F(ab')(2) that is essentially free from pepsin and aggregates with a typical purity of over 96% and yields of 16-19 g/l serum. When an antivenom was processed, approximately 78% of the original serum's toxin neutralising capacity was recovered. This simple, high yield protocol for processing serum to highly purified F(ab')(2) avoids the need for an initial or any subsequent salt precipitation step and can be utilised for either bench or large scale production. If required, a mild reducing agent may be used finally to create Fab fragments.

Efficacy of a novel PEGylated humanized anti-TNF fragment (CDP870) in patients with rheumatoid arthritis: a phase II double-blinded, randomized, dose-escalating trial.

OBJECTIVE: Biological products that neutralize tumour necrosis factor alpha (TNF-alpha) are beneficial in rheumatoid arthritis (RA). We studied the effects of CDP870, a novel anti-TNF-alpha antibody fragment modified to obtain a prolonged plasma half-life ( approximately 14 days). METHODS: Thirty-six patients were randomized in a double-blind, ascending-dose group study to a single intravenous infusion of placebo (n = 12) or 1, 5 or 20 mg/kg CDP870 (each n = 8). The patients were predominantly female (30/36), had a mean age of 56 yr and a mean duration of RA of 13 years. They had received a mean of five DMARDs or experimental therapies (with 1 month washout before the study started) and had active disease. Continuation of NSAIDs and up to 7.5 mg prednisolone daily was allowed. Following the blinded dosing period, 32 patients received a single open-label infusion of either 5 or 20 mg/kg CDP870. RESULTS: In the blinded dosing period, 6/12 placebo patients withdrew from the study (for deteriorating RA < or =4 weeks after dosing). Two of 24 CDP870-treated patients withdrew, both in the 1 mg/kg group (for deteriorating RA or lost to follow up >4 weeks after dosing). The proportion of patients with ACR20 improvement for the per-protocol population with the last observation carried forward was 16.7, 50, 87.5 and 62.5% after 0, 1, 5 and 20 mg/kg CDP870 respectively (combined treatment effect, P = 0.012, primary analysis) at 4 weeks and 16.7, 25, 75 and 75% (P = 0.032) at 8 weeks. The proportion of patients with ACR50 improvement for the per-protocol population with the last observation carried forward was 0, 12.5, 12.5 and 50% after 0, 1, 5 and 20 mg/kg CDP870 respectively (combined treatment effect, P = 0.079) at 4 weeks and 0, 12.5, 12.5 and 50% (P = 0.079) at 8 weeks. Following the open-label dose of CDP870, similar beneficial effects were achieved. CONCLUSION: CDP870 is effective, was very well tolerated in this small study, and has an extended duration of action following one or more intravenous doses.

Human single-chain Fv (scFv) antibody specific to human IL-6 with the inhibitory activity on IL-6-signaling.

Human anti-IL-6 antibody may be useful for the immunotherapy of various inflammatory diseases, such as rheumatoid arthritis. Since IL-6 is a growth factor for B cell hybridoma, it is not easy to isolate murine B cell hybridomas producing the anti-IL-6 antibody with the IL-6-signaling inhibitory activity. In this study, the antibody library (Vgamma-Vkappa, Vgamma-Vlambda, Vmu-Vkappa or micro-Vlambda ligated into the pCANTAB 5E phagemid vector) was prepared from peripheral blood mononuclear cells of 20 healthy subjects. The phage display library was panned with an IL-6-coated plastic plate, and the binding specificity was confirmed by ELISA and BIAcore. From the antibody library (Vgamma-Vlambda), five IL-6-specific phage clones were isolated. The effects of the soluble scFvs purified from these phage clones were tested on the growth of the IL-6-dependent human cell line, KT-3. Two of these clones significantly inhibited the growth of KT-3, and three showed no inhibition.