BACKGROUND:E-selectin is an endothelial cell specific adhesion molecule that is believed to play an important role in the early stages of leukocyte extravasation.OBJECTIVES:Here we describe the construction and evaluation of an engineered human monoclonal antibody that blocks E-selectin function.RESULTS:SPLAT-1 is an engineered human monoclonal antibody that has a very similar affinity for E-selectin as its murine parent antibody. In vitro SPLAT-1 blocks the binding of human leukocytes to E-selectin and does not mediate antibody-dependent cellular cytotoxicity (ADCC) or complement-mediated lysis of endothelial cells. In vivo, SPLAT-1 inhibits the recruitment of leukocytes to cytokine-inflamed human skin grafted on to SCID mice and has a long circulating half-life in primates. It does not appear to provoke an immune response in primates even on repeat administration.CONCLUSIONS:SPLAT-1 has the characteristics of a antibody suitable for human therapy studies.

We have found that amino acid residues necessary for C1q and Fc gamma R binding of human IgG1 are located in the N-terminal region of the CH2 domain, residues 231-238, using a matched set of engineered antibodies based on the anti-HLA-DR antibody L243. Changing the leucine 235 in the CH2 region of IgG3 and IgG4 to glutamic acid was already known to abolish Fc gamma RI binding. We have confirmed this for IgG1 and also found a concomitant abolition of human complement lysis with retention of Fc gamma RIII-mediated function. Changing the glycine at 237 to alanine of IgG1 also abolished Fc gamma RI binding and reduced human complement lysis and Fc gamma RIII-mediated function. Exchanging the whole region 233-236 with the sequence found in human IgG2, abolished Fc gamma RI binding and human complement lysis and reduced Fc gamma RIII-mediated function of IgG1. In contrast, a change in the previously described C1q-binding motif, from lysine at 320 to alanine, had no effect on IgG1-mediated complement lysis.

A murine antibody to human tumour necrosis factor-alpha (TNF-alpha) (CB0010) was complementarity-determining region (CDR)-grafted using human IgG4 heavy and kappa light chain constant regions. In cynomolgus monkeys, the grafted antibody (CDP571) was eliminated with a half-life of 40-90 hr, two to three times longer than CB0010, and immunogenicity was reduced by > 90%. Responses to the constant regions were almost entirely eliminated and responses to the CDR loop (anti-idiotype) were lowered. CDP571 was given to 24 human volunteers in doses from 0.1 to 10.0 mg/kg. It was well tolerated, with a half-life of approximately 13 days. Anti-CDP571 antibodies were low or undetectable at higher doses. At lower doses, anti-CDP571 peaked at 2 weeks and then declined. The response was primarily IgM (in contrast to the cynomolgus monkey, where by 5 weeks IgG predominated) and was against a conformational epitope comprising heavy and light chain CDR loops. No antibodies were detected against the gamma 4/kappa domains or frameworks. The response had little or no effect on CDP571 binding to TNF-alpha or on plasma clearance.

OBJECTIVE--To investigate the role of tumour necrosis factor alpha (TNF alpha) in the development of antigen induced arthritis (AIA) in rabbits. METHODS--Monoclonal antibodies to rabbit TNF alpha were developed in rats and were used to detect TNF alpha in synovial fluid by enzyme linked immunosorbent assay and to localise it in tissue sections of synovium and cartilage from rabbits up to 21 days after induction of AIA. An antibody which neutralised TNF alpha activity in vitro was injected into rabbits to block TNF alpha action in vivo in AIA. Joint swelling, leucocyte infiltration into synovium and proteoglycan loss from cartilage were measured and compared with a control group, which were injected with sterile saline. RESULTS--Monoclonal antibodies to purified rabbit TNF alpha were prepared in rats and two were selected which were able to neutralise rabbit TNF alpha in a cytotoxicity bioassay. TNF alpha was detected in significant concentrations (21.7 (SE 0.5) pg/ml) in the arthritic joint fluid of rabbits with AIA only at one day after induction and it was then also sparsely localised in cells of the synovium, but from day 3 onwards it was localised more strongly in the deep zone of articular cartilage. Injection of anti-TNF monoclonal antibody R6 over three days into rabbits with AIA reduced joint swelling and leucocyte infiltration into joint fluid and decreased the expression of CD11b and CD18 on cells in the joint fluid. However, there was no significant reduction in the loss of proteoglycan from articular cartilage, although the joint fluid at three days contained a lower glycosaminoglycan content. The antibody R6 gave most effect at a dose of 0.6 mg/kg and there was no increase in its effectiveness at a fivefold greater dose (3.0 mg/kg). Treatment over 10 days gave a more complete suppression of joint swelling, but did not result in any less proteoglycan loss from cartilage. Treatment for five days with a 16 day follow up gave a significant reduction in swelling for several days beyond the treatment, but the swelling then slowly returned, until by day 21 there was no significant difference in joint swelling and there was also no recovery of cartilage proteoglycan content. A rabbit anti-rat immunoglobulin response was detected at 21 days, which may have limited the long term effectiveness of the antibody. CONCLUSIONS--In AIA in rabbits, TNF alpha was only detected in synovial fluid at one day after induction and there was only limited cellular localisation of TNF alpha in synovium and cartilage from three days. However, neutralising TNF alpha with a monoclonal antibody was effective in suppressing inflammatory changes in the joint during the acute onset of AIA, but it had little effect on the loss of proteoglycan from cartilage. The results suggest that blocking inflammation and synovitis with anti-TNF alpha may be more easily achieved than preventing damage to articular cartilage.

A one-step protocol for the purification of recombinant human tumour necrosis factor-alpha (TNF alpha) has been developed based on the use of antibody affinity chromatography. The method allows for the preparation of large amounts of the protein (>15 mg). The overall recovery of the purified material from Esherichia coli lysate after buffer exchange into 0.8% mannitol is 48%, with no apparent loss of bioactivity. This method has been utilized for the preparation of 3-fluoro-tyrosine labelled human TNF alpha. Data indicate that the protein produced in minimal media is a heterotrimer consisting of two 17 kDa monomers and one proteolytically cleaved 14 kDa unit. Preliminary F-19 n.m.r. spectroscopy indicated that the 3-fluoro-tyrosine labelled protein is suitable for further study using this technique.

Yttrium binding ligands DOTA, caDTPA and CT-DTPA were each conjugated to monoclonal antibody B72.3, labelled with 90Y and injected into mice in order to assess the in vivo inertness of the antibody-linked 90Y-ligand complexes. Levels of 90Y in femur shafts of the DOTA-B72.3 mice were low, being ~ 7 and 44%, respectively, of levels in the femur shafts of the caDTPA-B72.3 and CT-DTPA-B72.3 treated mice. This finding demonstrates the greater inertness and by implication the greater suitability for immunotherapy of the DOTA-90Y complex.

Bovine whey proteins and caseins were separated by hydrophobic interaction chromatography with the new Pharmacia fast protein liquid chromatography column, phenyl-Superose. Total casein was separated using a decreasing gradient of 0.8 to 0.05 M sodium phosphate and a constant 3.75 M urea concentration at pH 6.0. The order of elution of caseins was β < γ, αs2 < ϰ < αs1, and β-casein was always eluted first. Whey proteins were separated with a decreasing salt gradient of 1.5 to 0 M ammonium sulphate in 0.05 M sodium phosphate at ph 7.0. The order of elution was β-lactoglobulin < bovine serum albumin < immunoglobulin < α-lactalbumin. The elution order of proteins from the column did not correlate with the calculated average hydrophobicities but the method was considered to be a measure of the “effective” hydrophobicity of proteins and therefore of more use for attempting to relate hydrophobicity to functional properties of proteins. The method shows significant advantages over conventional techniques allowing rapid optimization of elution conditions and reducing run times from 24 h or more to less than 2 h.