A single amino acid substitution abolishes the heterogeneity of chimeric mouse/human (IgG4) antibody

Angal, Sarojani; King, David J.; Bodmer, Mark; Turner, Alison; Lawson, Alastair D. G.; Roberts, Gerald D.; Pedley, R. Barbara; Adair, John R.

doi:10.1016/0161-5890(93)90432-b

Cited by 228 publications

(164 citation statements)

References 24 publications

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“…To enable further characterization, this V region was expressed in multiple antibody formats (Fab’, Fab’PEG, IgG1, IgG4P, triFab’, FabFv, Fab’human albumin conjugate and Fab’mouse albumin fusion; Supplementary Table 3). When subcloned as a humanized IgG4P (serine-to-proline mutation at residue 241 to reduce propensity for Fab-arm exchange 31 ) isotype, this antibody is known as rozanolixizumab (Supplementary Figure 1; these sequence data have been submitted to the DDBJ/EMBL/GenBank databases under accession number MG976502 (light chain) and MG976503 (heavy chain)).…”

Section: Resultsmentioning

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: Resultsmentioning

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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“…Interactions in both the IgG4 core hinge sequence and the IgG4 CH3 domain are critical for this process to occur (31,32). Substitution of serine with proline at position 228, as found in IgG1 and IgG2 core hinge sequences (i.e., CPSCP to CPPCP) stabilized the inter-H chain disulfide linkage in NIB(28SA)-G4-S228P with the resulting loss of half-molecules after SDS-PAGE, as shown for other IgG4 mAbs with "wild-type" hinges (10,16,33,34). Exchange of the IgG4 for the IgG1 core hinge was previously shown to abrogate Fab arm-exchange in vitro and in vivo (10,32).…”

Section: Responses Of Pbmcs To Nib(28sa)-g4 Nib(28sa)-g1 and Nib(28mentioning

confidence: 97%

Antibody C Region Influences TGN1412-like Functional Activity In Vitro

Ball

Fox

Hufton

et al. 2012

The Journal of Immunology

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The unexpected outcome of the clinical trial of the superagonistic CD28 mAb TGN1412 (IgG4κ) continues to stimulate interest. We show that TGN1412 binds similarly to human and cynomolgus macaque FcγR, eliminating the possibility that differences in Fc-mediated interactions with FcγR contributed to the failure of preclinical testing in macaques to predict toxicity in humans. The influence of the Fc domain and C region structure on the in vitro functional activity of TGN1412 was investigated using F(ab′)2 and Fab fragments derived from TGN1412 recovered from the trial and recombinant TGN1412 subclass variants and mutants. Superagonistic activity, as measured by cytokine release and proliferation, was assessed by exposing PBMCs to immobilized mAbs/fragments or to aqueous mAbs/fragments in the presence of HUVEC monolayers. Removing the Fc generally curtailed or abolished PBMC activation. However, eliminating detectable FcγR-binding of the IgG4 by mutation (L235E) did not abrogate activity. Stabilizing the “wild-type” IgG4 hinge (S228P) enhanced activity without increasing FcγR binding, which could only partially be explained by inhibition of Fab arm–exchange. Subclass switching the IgG4 mAb to IgG1 decreased activity, whereas switching to IgG2 markedly increased activity. We conclude that the C region strongly influences in vitro CD28-mediated superagonistic signaling. Superagonism requires an intact Fc, as shown by the absence of activity of TGN1412 Fab and F(ab′)2 fragments, but, notably, appears to be relatively independent of FcγR-binding properties. We propose that the Fc, potentially through restricting flexibility, maintains a favorable V region conformation to allow superagonistic activity. These findings have important implications for Ab design strategies.

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“…1989: Tan et al. 1990: Canfield & Morrison, 1991: King et at.. 1992: Angal et al, 1993. The percentage of tetramers consisting of noncovalently associated half IgG molecules varies from S to 10%-of the total secreted IgG (Colcher et at.. 1989: King et al.…”

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confidence: 99%

“…1991: King et at.. 1992. Highresolution ion-exchange chromatography can effect partial separation of the noncovalently associated half IgG molecules from the tetramer with inter-heavy chain disulfide bonds (Angal et al, 1993).…”

mentioning

confidence: 99%

“…1987). Angal et al (1993) reasoned that the presence of a serine at position 241 of IgG4 rather than the proline seen in lgGl and IgG2 might be related to the presence of noncovalently associated half-molecules in preparations of IgG4. Substitution of Pro 241 for Ser 241 led to the secretion of a homogeneous mouse/human chimeric IgG4 [cB72.3(y4)] with interheavy chain disulfide bonds.…”

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confidence: 99%

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Intrachain disulfide bond in the core hinge region of human IgG4

et al. 1997

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IgG is a tetrameric protein composed of two copies each of the light and heavy chains. The four-chain structure is maintained by strong noncovalent interactions between the amino-terminal half of pairs of heavy-light chains and between the carboxyl-terminal regions of the two heavy chains. In addition, interchain disulfide bonds link each heavy-light chain and also link the paired heavy chains. An engineered human IgG4 specific for human tumor necrosis factor-cy (CDP571) is similar to human myeloma IgG4 in that it is secreted as both disulfide bonded tetramers (approximately 75% of the total amount of IgG) and as tetramers composed of nondisulfide bonded half-IgG4 (heavy chain disulfide bonded to light chain) molecules. However, when CDP571 was genetically engineered with a proline at residue 229 of the core hinge region rather than serine, CDP57 I(S229P), or with an IgGl rather than IgG4 hinge region, CDP571(yl), only trace amounts of nondisulfide bonded half-lgG tetramers were observed. Trypsin digest reversephase HPLC peptide mapping studies of CDP57 1 and CDP57 1 (y I ) with on-line electrospray ionization mass spectroscopy supplemented with Edman sequencing identified the chemical factor preventing inter-heavy chain disulfide bond formation between half-IgG molecules: the two cysteines in the IgG4 and IgGl core hinge region KPSCP and GPPCP, respectively) are capable of forming an intrachain disulfide bond. Conformational modeling studies on cyclic disulfide bonded CPSCP and CPPCP peptides yielded energy ranges for the low-energy conformations of 31-33 kcallmol and 40-42 kcal/mol, respectively. In addition, higher torsion and angle bending energies were observed for the CPPCP peptide due to backbone constraints caused by the extra proline. These modeling results suggest a reason why a larger fraction of intrachain bonds are observed in IgG4 rather than IgGl molecules: the serine in the core hinge region of IgG4 allows more hinge region flexibility than the proline of IgGl and thus may permit formation of a stable intrachain disulfide bond more readily.

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A single amino acid substitution abolishes the heterogeneity of chimeric mouse/human (IgG4) antibody

Cited by 228 publications

References 24 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

Antibody C Region Influences TGN1412-like Functional Activity In Vitro

Intrachain disulfide bond in the core hinge region of human IgG4

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