Protein design of IgG/TCR chimeras for the co-expression of Fab-like moieties within bispecific antibodies

Wu, Xiufeng; Sereno, Arlene; Huang, Flora; Zhang, Kai; Batt, Micheal A; Fitchett, Jonathan R.; Dan, He; Rick, Heather L; Conner, Elaine M.; Demarest, Stephen J.

doi:10.1080/19420862.2015.1007826

Cited by 23 publications

(18 citation statements)

References 39 publications

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“… 27 This resulted in multiple CDR residues changing rotomer positions and subtle alterations in substrate specificity and catalytic efficiency of the catalytic antibody. We have not observed significant differences in antigen binding by replacing Cκ with Cλ 28 or CH1/Cκ with Cα/Cβ 17 having tested multiple Fabs with mismatched domains. It seems likely, as observed with scFvs, that subtle changes in affinity (2–4-fold) may occur when making unnatural variable and constant domain pairings, but that catastrophic changes in affinity/activity are likely not induced by modifying the constant domain subunits within Fabs.…”

Section: Discussionmentioning

confidence: 71%

“…Constructs containing the TCR Cα/Cβ domains were engineered to have optimized HC/LC association based on optimized V-gene/C-gene linker regions including an FG loop replaced with a canonical type II' β-turn. 17 Both scFv constructs were generated in the VH/VL format with a flexible (G 4 S) 4 linker. All constructs contained C-terminal histidine tags; for HC/LC heterodimers, the histags were placed at the C-terminus of the HC following the cysteine that forms the inter-chain disulfide.…”

Section: Resultsmentioning

confidence: 99%

“…We also evaluated replacing the entire CH1/Cκ subunit with the TCR Cα/Cβ subunit, which is structurally homologous and has been shown capable of pairing with VH/Vκ. 17 …”

Section: Resultsmentioning

confidence: 99%

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Comparing domain interactions within antibody Fabs with kappa and lambda light chains

Toughiri

Ruiz

et al. 2016

mAbs

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IgG antibodies are multi-domain proteins with complex inter-domain interactions. Human IgG heavy chains (HCs) associate with light chains (LCs) of the κ or λ isotype to form mature antibodies capable of binding antigen. The HC/LC interaction involves 4 domains: VH and CH1 from the HC and VL and CL from the LC. Human Fabs with κ LCs have been well characterized for their unfolding behaviors and demonstrate a significant level of cooperativity and stabilization when all 4 domains are intact. Very little is known regarding the thermodynamic properties of human Fabs with λ LCs. Here, we dissect the domain contributions to Fab stability for both κ and λ LC-containing Fabs. We find the cooperativity of unfolding between the constant domains, CH1/Cλ, and variable domains, VH/Vλ, within λ LC-containing Fabs is significantly weaker than that of κ LC-containing Fabs. The data suggests there may not be an evolutionary necessity for strong variable/constant domain cooperativity within λ LC-containing Fabs. After investigating the biophysical properties of Fabs with mismatched variable and constant domain subunits (e.g., VH/Vκ paired with CH1/Cλ or T cell receptor Cα/Cβ), the major role of the constant domains for both κ- and λ-containing Fabs may be to reduce the hydrophobic exposure at the VH/VL interface. Even though Fabs with these non-native pairings were thermodynamically less stable, they secreted well from mammalian cells as well behaved monodisperse proteins, which was in contrast to what was observed with the VH/Vκ and VH/Vλ scFvs that secreted as a mixture of monomer and aggregates.

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Section: Discussionmentioning

confidence: 71%

Section: Resultsmentioning

confidence: 99%

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Comparing domain interactions within antibody Fabs with kappa and lambda light chains

Toughiri

Ruiz

et al. 2016

mAbs

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“…For instance, N-or C-BsAbs Her2 x Her2 in which the heavy chain of one IgG HER2 is N-or C-terminal extended by a second Fv-containing unit directed against another HER2 epitope have distinct cellular activities. 21 Also, IgG-scFv and IgG-Fab built with the two previously mentioned Fv HER2 significantly differ in cellular assays. 22 Similarly, bispecific tandem scFv and tandem Fabs comprising the same Fv EGFR and Fv CD3 also show distinct outcome in cellular evaluations.…”

Section: Discussionmentioning

confidence: 99%

“…2 Thus, they generally require further engineering, extensive purification, or special production systems imposing specific limitations. 8,[13][14][15][16][17][18][19][20][21][22] Furthermore, their avidity per antigen is reduced compared to the parental antibodies.…”

Section: Introductionmentioning

confidence: 99%

CODV-Ig, a universal bispecific tetravalent and multifunctional immunoglobulin format for medical applications

Steinmetz

Vallée

Beil

et al. 2016

mAbs

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Bispecific immunoglobulins (Igs) typically contain at least two distinct variable domains (Fv) that bind to two different target proteins. They are conceived to facilitate clinical development of biotherapeutic agents for diseases where improved clinical outcome is obtained or expected by combination therapy compared to treatment by single agents. Almost all existing formats are linear in their concept and differ widely in drug-like and manufacture-related properties. To overcome their major limitations, we designed cross-over dual variable Ig-like proteins (CODV-Ig). Their design is akin to the design of circularly closed repeat architectures. Indeed, initial results showed that the traditional approach of utilizing (G4S) x linkers for biotherapeutics design does not identify functional CODV-Igs. Therefore, we applied an unprecedented molecular modeling strategy for linker design that consistently results in CODV-Igs with excellent biochemical and biophysical properties. CODV architecture results in a circular self-contained structure functioning as a self-supporting truss that maintains the parental antibody affinities for both antigens without positional effects. The format is universally suitable for therapeutic applications targeting both circulating and membrane-localized proteins. Due to the full functionality of the Fc domains, serum half-life extension as well as antibody-or complement-dependent cytotoxicity may support biological efficiency of CODV-Igs. We show that judicious choice in combination of epitopes and paratope orientations of bispecific biotherapeutics is anticipated to be critical for clinical outcome. Uniting the major advantages of alternative bispecific biotherapeutics, CODV-Igs are applicable in a wide range of disease areas for fast-track multi-parametric drug optimization.

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Bispecifics

McCluskey

Ghayur

2017

Methods and Principles in Medicinal Chemistry

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Protein design of IgG/TCR chimeras for the co-expression of Fab-like moieties within bispecific antibodies

Abstract: (2015) Protein design of IgG/TCR chimeras for the co-expression of Fab-like moieties within bispecific antibodies, mAbs, 7:2, 364-376,

Cited by 23 publications

References 39 publications

Comparing domain interactions within antibody Fabs with kappa and lambda light chains

Comparing domain interactions within antibody Fabs with kappa and lambda light chains

CODV-Ig, a universal bispecific tetravalent and multifunctional immunoglobulin format for medical applications

Bispecifics

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