F(ab′)2 molecules made from Escherichia coli produced Fab′ with hinge sequences conferring increased serum survival in an animal model

Humphreys, David P.; Vetterlein, O; Chapman, Andrew P.; King, David J.; Antoniw, Pari; Suitters, Amanda; Reeks, Dominic G.; Parton, Ted; King, Leslie A.; Smith, Bryan John; Lang, Volker; Stephens, Paul E.

doi:10.1016/s0022-1759(98)00061-1

Cited by 33 publications

(14 citation statements)

References 22 publications

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“…Humanized Fab’ was expressed in, and prepared from E. coli , 46,70 and further modified as detailed in Supplementary Table 3. IgG molecules were prepared from CHO SXE 69 cell culture medium by affinity chromatography on MabSelect™ SuRe™ (GE Healthcare, UK), followed by size-exclusion chromatography (SEC) on HiLoad XK 50/60 Superdex 200 PG (GE Healthcare, UK).…”

Section: Methodsmentioning

confidence: 99%

Generation and characterization of a high affinity anti-human FcRn antibody, rozanolixizumab, and the effects of different molecular formats on the reduction of plasma IgG concentration

Smith

Kießling

Lledó-García

et al. 2018

mAbs

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Rozanolixizumab (UCB7665), a humanized high-affinity anti-human neonatal Fc receptor (FcRn) monoclonal antibody (IgG4P), has been developed to reduce pathogenic IgG in autoimmune and alloimmune diseases. We document the antibody isolation and compare rozanolixizumab with the same variable region expressed in various mono-, bi- and trivalent formats. We report activity data for rozanolixizumab and the different molecular formats in human cells, FcRn-transgenic mice, and cynomolgus monkeys. Rozanolixizumab, considered the most effective molecular format, dose-dependently and selectively reduced plasma IgG concentrations in an FcRn-transgenic mouse model (no effect on albumin). Intravenous (IV) rozanolixizumab dosing in cynomolgus monkeys demonstrated non-linear pharmacokinetics indicative of target-mediated drug disposition; single IV rozanolixizumab doses (30 mg/kg) in cynomolgus monkeys reduced plasma IgG concentration by 69% by Day 7 post-administration. Daily IV administration of rozanolixizumab (initial 30 mg/kg loading dose; 5 mg/kg daily thereafter) reduced plasma IgG concentrations in all cynomolgus monkeys, with low concentrations maintained throughout the treatment period (42 days). In a 13-week toxicology study in cynomolgus monkeys, supra-pharmacological subcutaneous and IV doses of rozanolixizumab (≤ 150 mg/kg every 3 days) were well tolerated, inducing sustained (but reversible) reductions in IgG concentrations by up to 85%, with no adverse events observed. We have demonstrated accelerated natural catabolism of IgG through inhibition of IgG:FcRn interactions in mice and cynomolgus monkeys. Inhibition of FcRn with rozanolixizumab may provide a novel therapeutic approach to reduce pathogenic IgG in human autoimmune disease. Rozanolixizumab is being investigated in patients with immune thrombocytopenia (NCT02718716) and myasthenia gravis (NCT03052751).

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Section: Methodsmentioning

confidence: 99%

Generation and characterization of a high affinity anti-human FcRn antibody, rozanolixizumab, and the effects of different molecular formats on the reduction of plasma IgG concentration

Smith

Kießling

Lledó-García

et al. 2018

mAbs

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“…Fab' from the humanized antibody 40.4 was expressed in the periplasm of E. coli 8 and purified using expanded-bed chromatography with the cation exchange material Streamline SP (Amersham-Pharmacia Biotech, Uppsala, Sweden) followed by affinity chromatography on protein G-Sepharose as described 16 . PEG-maleimide (5 kDa) was prepared as described 12 , and PEG-maleimide 25 kDa and branched PEG 2 -maleimide (40 kDa) were obtained from Shearwater Polymers (Huntsville, AL).…”

Section: Experimental Protocolmentioning

confidence: 99%

Therapeutic antibody fragments with prolonged in vivo half-lives

et al. 1999

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Antibody fragments can be isolated rapidly using techniques such as phage display and can be expressed to high levels in microbial systems. However, to date such antibody fragments have been of limited use for many therapeutic applications because they are rapidly cleared from the body. We present a strategy for the site-specific chemical modification of antibody fragments with polyethylene glycol, which results in the production of antibody fragments with long in vivo half-lives and full retention of antigen-binding properties. This technology should allow more rapid and economical production of therapeutic antibodies for chronic disease therapy.

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“…FabЈ yields were typically 80 -250 mg L Ϫ1 . Fermentation cell pastes were resuspended in 0.5 harvest volume of 100 mM Tris-HCl/10 mM EDTA, pH7.4, and agitated at 250 rpm and 30°C for 16 h. Periplasmic extracts were clarified by centrifugation at 25,000g for 30 min and passed through a 0.2-m filter (Millipore) before purification on Protein G-Sepharose (GammaBind Plus, Pharmacia Biotech) as described previously (27).…”

Section: Construction and Selection Of Pcr-mutated Sp-ap Encoding Plamentioning

confidence: 99%

High-Level Periplasmic Expression in Escherichia coli Using a Eukaryotic Signal Peptide: Importance of Codon Usage at the 5′ End of the Coding Sequence

Humphreys

Sehdev

Chapman

et al. 2000

Protein Expression and Purification

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F(ab′)2 molecules made from Escherichia coli produced Fab′ with hinge sequences conferring increased serum survival in an animal model

Cited by 33 publications

References 22 publications

Generation and characterization of a high affinity anti-human FcRn antibody, rozanolixizumab, and the effects of different molecular formats on the reduction of plasma IgG concentration

Generation and characterization of a high affinity anti-human FcRn antibody, rozanolixizumab, and the effects of different molecular formats on the reduction of plasma IgG concentration

Therapeutic antibody fragments with prolonged in vivo half-lives

High-Level Periplasmic Expression in Escherichia coli Using a Eukaryotic Signal Peptide: Importance of Codon Usage at the 5′ End of the Coding Sequence

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