Improving biophysical properties of a bispecific antibody scaffold to aid developability

Kreudenstein, Thomas Spreter von; Escobar-Carbrera, Eric; Lario, Paula I.; D’Angelo, I.; Brault, Karine; Kelly, John F.; Durocher, Yves; Baardsnes, Jason; Woods, R. Jeremy; Xie, Michael; Girod, Pierre‐Alain; Suits, M.D.L.; Boulanger, Martin J.; Poon, D; Ng, Gordon; Dixit, Surjit B.

doi:10.4161/mabs.25632

Cited by 104 publications

(98 citation statements)

References 48 publications

(63 reference statements)

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“…For example, any format isolating a variable fragment (Fv) by replacing its adjacent constant fragment (Fc) with a peptide linking heavy or light chain Fv domains is associated with decreased Fv stability. 2,8 Introduction of disulfide bridges or improved interface interactions stabilize Fv dimerization in specific cases; however, such strategies remain associated with loss of antigen affinity or increased aggregation propensity. [9][10][11][12] Asymmetric bispecific antibodies including Fc domains give rise to improperly paired side-products even when optimized.…”

Section: Introductionmentioning

confidence: 99%

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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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Section: Introductionmentioning

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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“…A number of alternative approaches for the same purpose have been developed recently. 9,10,11,12,13,14 Upon application of one of these methods alone, however, there still remains a mixture of 4 compounds except for cases where the chain association issue is circumvented by a common LC approach. 15 The LC mispairing problem is more difficult to address because a total of 4 possible pairings of heavy and light chains have to be considered.…”

Section: Introductionmentioning

confidence: 99%

Heavy and light chain pairing of bivalent quadroma and knobs-into-holes antibodies analyzed by UHR-ESI-QTOF mass spectrometry

Schaefer

Völger

Lorenz

et al. 2015

mAbs

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(2016) Heavy and light chain pairing of bivalent quadroma and knobs-into-holes antibodies analyzed by UHR-ESI-QTOF mass spectrometry, mAbs, 8:1, 49-55, DOI: 10.1080/19420862.2015 To link to this article: https://doi.org/10. 1080/19420862.2015 Keywords: bispecific antibody, CrossMab, chain pairing, ESI-QTOF mass spectrometry, knobs-into-holes, quadromaAbbreviations: Ang2, angiopoietin-2; bsAbs, bispecific antibodies; ESI, electrospray ionization; GuA HCl, guanidine hydrochloride; HEK, human embryonic kidney; HC, heavy chain; IgG1, immunoglobulin G1; ISCID, ion source collision induced dissociation; KiH, knobs-into-holes; LC, light chain; MS, mass spectrometry; TCEP, tris(2-carboxyethyl)phosphine; UHR, ultrahigh-resolution; VEGF-A, vascular endothelial growth factor A; QTOF, quadrupole time-of-flightThe quadroma antibody represents the first attempt to produce a bispecific heterodimeric IgG antibody by somatic fusion of 2 hybridoma cells each expressing monoclonal antibodies with distinctive specificities. However, because of random heavy and light chain pairing, the desired functional bispecific antibody represents only a small fraction of the protein produced. Subsequently, the knobs-into-holes (KiH) approach was developed to enforce correct heavy chain heterodimerization. Assuming equimolar expression of 4 unmodified chains comprising 2 heavy and 2 light chains, the statistical distribution of all paired combinations can be calculated. With equimolar expression as the goal, we transfected HEK cells with 1:1:1:1 plasmid ratios and analyzed the protein A affinity-purified antibodies from the quadroma and KiH approaches qualitatively and quantitatively with regard to the estimated relative amounts of the products using electrospray quadrupole time-of-flight mass spectrometry. Our results show that all expected species are formed, and that, within the methodological limits, the species distribution in the mixtures corresponds approximately to the statistical distribution.

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“…Antibody asymmetry is facilitated through engineering of the CH 3 domain (13)(14)(15)(16) or the hinge region of the antibody (17,18), promoting heterodimerization of the constant domains. Some of the constant domain mutations required to enable IgG heterodimerization compromise stability and may affect binding to Fc receptors as well (48). Solving these issues requires extensive antibody engineering (48).…”

Section: Discussionmentioning

confidence: 99%

Bispecific antibody generated with sortase and click chemistry has broad antiinfluenza virus activity

Wagner

Kwakkenbos

Claassen

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Significance Bispecific antibodies expand the function of conventional antibodies. However, therapeutic application of bispecifics is hampered by the reduced physiochemical stability of such molecules. We present a format for bispecific antibodies, fusing two full-sized antibodies via their C termini. This format does not require mutations in the antibody constant domains beyond installation of a five-residue tag, ensuring that the native antibody structure is fully retained in the bispecific product. We have validated the approach by linking two anti-influenza A antibodies, each active against a different subgroup of the virus. The bispecific antibody dimer retains the activity and the stability of the two original antibodies.

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Improving biophysical properties of a bispecific antibody scaffold to aid developability

Cited by 104 publications

References 48 publications

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

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

Heavy and light chain pairing of bivalent quadroma and knobs-into-holes antibodies analyzed by UHR-ESI-QTOF mass spectrometry

Bispecific antibody generated with sortase and click chemistry has broad antiinfluenza virus activity

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