Engineering bispecific antibodies with defined chain pairing

Krah, Simon; Sellmann, Carolin; Rhiel, Laura; Schröter, Christian; Dickgießer, Stephan; Beck, Jan; Zielonka, Stefan; Hock, Björn; Kolmar, Harald; Becker, Stefan

doi:10.1016/j.nbt.2016.12.010

Cited by 43 publications

(43 citation statements)

References 53 publications

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“…B-Ab formats are diverse including bispecific immunoglobulin G (IgGs), appended IgGs, and bispecific fragments such as bispecific T-cell engagers (BiTEs; Spiess, Zhai, & Carter, 2015). In general, a heterodimer and multichain bispecific IgG has two different light chains and two different heavy chains that fold and must correctly pair via a diversity of engineered interchain interactions (e.g., orthogonal interface, domain crossover, charged mutations, sterically complementary mutations; Gunasekaran et al, 2010;Klein, Schaefer, & Regula, 2016;Krah et al, 2017;Lewis et al, 2014;Merchant et al, 1998).…”

Section: Introductionmentioning

confidence: 99%

Culture temperature modulates half antibody and aggregate formation in a Chinese hamster ovary cell line expressing a bispecific antibody

Gómez

Wieczorek

et al. 2018

Biotech & Bioengineering

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Therapeutic bispecific antibodies are formed by assembly of multichain polypeptides. In general, a bispecific antibody has two different light chains and two different heavy chains that fold and correctly pair via engineered interchain interactions. Because of some incorrect assembly, product-related impurities can be prevalent (e.g., half molecules, mispaired light chains, homodimers), requiring its removal during subsequent purification. In this study, we investigated the modulation of impurity levels in a stable Chinese hamster ovary cell line X expressing a bispecific antibody A formed by two light chains (LC1 and LC2) and two heavy chains (HC1 and HC2) that assembled intracellularly into a heterodimer (LC1-HC1 + LC2-HC2) via engineered charged residues. Cell line X exhibited the best volumetric productivity, growth, and viability in culture compared with other clones but also showed higher levels of half antibody species (>10%); therefore, to minimize process yield loss, better understanding, and control of impurity formation was pursued. We found this cell line decreased half antibody levels from 16% to 1% when temperature changed from 36°C to 32.5°C or 31.5°C. However, lower temperature also increased high-molecular-weight (HMW) species from 4% to 12%. To determine the impurity species composition, we characterized enriched fractions with half antibody or HMW. Intact mass spectrometry analysis revealed half antibody was LC2-HC2, whereas HMW was a mixture with ~50% as LC1-HC1 homodimer. Results suggested LC2-HC2 was easily folded and could be secreted as half antibody, especially at 36°C. On the contrary, LC1-HC1 was more susceptible to misfold or aggregate, a phenomenon more acute for cell line X at lower culture temperature because of 60% increased LC1 and HC1 messenger RNA levels. Although temperature modulation was cell line X-specific, the propensity of LC2-HC2 to form half antibodies and LC1-HC1 to aggregate appeared in other cell lines also expressing bispecific antibody A, suggesting an amino-acid sequence-dependent mechanism. In summary, impurity formation in cell line X was temperature-dependent and was influenced by different molecule characteristics between the LC1-HC1 and LC2-HC2 parts. Ultimately, we selected a biphasic cell culture process with a growth phase followed by a lower temperature phase to improve product quality and purification yield.

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Section: Introductionmentioning

confidence: 99%

Culture temperature modulates half antibody and aggregate formation in a Chinese hamster ovary cell line expressing a bispecific antibody

Gómez

Wieczorek

et al. 2018

Biotech & Bioengineering

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“…Los estudios preclínicos realizados por estos investigadores exhibieron una relación entre la supervivencia en ratones y la inducción de la respuesta inmune humoral mediada por anticuerpos [9, Sin embargo, ya no está siendo producido por Fresenius Biotech GmbH logísticas. En junio de autorización de mercado en la Unión Europea a petición del titular de la autorización comercialización, Neovii Biotech GmbH [4,9].…”

Section: Catumaxomab (Removab®)unclassified

“…En consecuencia, surgieron los anticuerpos monoclonales biespecíficos (bsAbs). Comprenden dos entidades de enlace individual físicamente conectadas, que permiten la unión simultánea a dos epítopos distintos en uno o dos antígenos diferentes [4].…”

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Anticuerpos monoclonales biespecíficos: desarrollo, producción y uso como terapia a anticancerígena

Carvajal¹,

Carrillo²,

Mata³

et al. 2019

Rev Med UCR

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Los anticuerpos monoclonales son glicoproteínas especializadas del sistema inmune. Se producen a partir de células B y presentan la capacidad de reconocer un antígeno específico. A partir de la teoría de generación de hibridomas desarrollada por Köhler y Milstein en 1975, se convirtieron en herramientas esenciales para los ámbitos clínico y biotecnológico, y a través de los años han sido útiles en el diagnóstico y el tratamiento de enfermedades infecciosas, inmunológicas y neoplásicas. Dichos anticuerpos a menudo se unen únicamente a un objetivo específico, lo que limita su potencial terapéutico en enfermedades causadas por múltiples mediadores y mecanismos. En consecuencia, surgieron los anticuerpos monoclonales biespecíficos, los cuales, junto con los avances de los mecanismos fisiopatológicos del cáncer, se han convertido en una eficaz y atractiva alternativa terapéutica. En Costa Rica, existe una considerable inversión en la compra de anticuerpos monoclonales por parte del sistema de Salud Pública. Se espera que, una vez que se aprueben los estudios clínicos y se comercialicen más anticuerpos biespecíficos, estos puedan llegar a ser ofertados a sus pacientes.

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“…Da sich hier jedoch vier unterschiedliche Ketten korrekt zusammenschließen müssen -man spricht vom chain association issue -ist die Produktion und Reinigung solcher Moleküle schwierig. 19,20) Zur Heterodimerisierung schwerer Ketten finden sich in der Literatur mehrere Methoden wie knobs into holes oder SEEDbodies. 21,22) Die korrekte Assoziation von leichten an die entsprechenden schweren Ketten erwies sich jedoch als Problem.…”

Section: Yeast Surface Displayunclassified