The use of CrossMAb technology for the generation of bi- and multispecific antibodies

Klein, Christian; Schaefer, W.; Regula, Jörg T.

doi:10.1080/19420862.2016.1197457

Cited by 150 publications

(121 citation statements)

References 65 publications

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“…Fit parameters N and K D are listed in Table 2, and SPR sensorgrams are shown in Figure S2. 32 That framework had been briefly described in a review and described in a publicly available patent application. 32,33 While our work was under review for publication, a complete characterization of DuoMAbs was published.…”

Section: Discussionmentioning

confidence: 99%

Design and characterization of novel dual Fc antibody with enhanced avidity for Fc receptors

et al. 2019

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Monoclonal antibodies (mAbs) have become an important class of therapeutics, particularly in the realm of anticancer immunotherapy. While the two antigen‐binding fragments (Fabs) of an mAb allow for high‐avidity binding to molecular targets, the crystallizable fragment (Fc) engages immune effector elements. mAbs of the IgG class are used for the treatment of autoimmune diseases and can elicit antitumor immune functions not only by several mechanisms including direct antigen engagement via their Fab arms but also by Fab binding to tumors combined with Fc engagement of complement component C1q and Fcγ receptors. Additionally, IgG binding to the neonatal Fc receptor (FcRn) allows for endosomal recycling and prolonged serum half‐life. To augment the effector functions or half‐life of an IgG1 mAb, we constructed a novel “2Fc” mAb containing two Fc domains in addition to the normal two Fab domains. Structural and functional characterization of this 2Fc mAb demonstrated that it exists in a tetrahedral‐like geometry and retains binding capacity via the Fab domains. Furthermore, duplication of the Fc region significantly enhanced avidity for Fc receptors FcγRI, FcγRIIIa, and FcRn, which manifested as a decrease in complex dissociation rate that was more pronounced at higher densities of receptor. At intermediate receptor density, the dissociation rate for Fc receptors was decreased 6‐ to 130‐fold, resulting in apparent affinity increases of 7‐ to 42‐fold. Stoichiometric analysis confirmed that each 2Fc mAb may simultaneously bind two molecules of FcγRI or four molecules of FcRn, which is double the stoichiometry of a wild‐type mAb. In summary, duplication of the IgG Fc region allows for increased avidity to Fc receptors that could translate into clinically relevant enhancement of effector functions or pharmacokinetics.

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Section: Discussionmentioning

confidence: 99%

Design and characterization of novel dual Fc antibody with enhanced avidity for Fc receptors

et al. 2019

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“…261 This CrossMAb approach reliably generates the desired HC-LC pairings without the use of potentially destabilizing mutations and has inspired a whole family of multispecific Ig frameworks. 262 In cases where it is possible to generate functional Fvs against 2 distinct antigens using a common LC or common HC, the LC and HC problems can be avoided, and these chain-pairing strategies become unnecessary.…”

Section: Asymmetric Igg-like Frameworkmentioning

confidence: 99%

Considerations for the Design of Antibody-Based Therapeutics

Goulet

Atkins

2020

Journal of Pharmaceutical Sciences

199

154

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Keywords: antibody(s) antibody drug(s) antibody drug conjugate(s) (ADC) physicochemical properties pharmacokinetics monoclonal antibody(s) immunotherapy IgG antibody(s) drug design developability a b s t r a c t Antibody-based proteins have become an important class of biologic therapeutics, due in large part to the stability, specificity, and adaptability of the antibody framework. Indeed, antibodies not only have the inherent ability to bind both antigens and endogenous immune receptors but also have proven extremely amenable to protein engineering. Thus, several derivatives of the monoclonal antibody format, including bispecific antibodies, antibody-drug conjugates, and antibody fragments, have demonstrated efficacy for treating human disease, particularly in the fields of immunology and oncology. Reviewed here are considerations for the design of antibody-based therapeutics, including immunological context, therapeutic mechanisms, and engineering strategies. First, characteristics of antibodies are introduced, with emphasis on structural domains, functionally important receptors, isotypic and allotypic differences, and modifications such as glycosylation. Then, aspects of therapeutic antibody design are discussed, including identification of antigen-specific variable regions, choice of expression system, use of multispecific formats, and design of antibody derivatives based on fragmentation, oligomerization, or conjugation to other functional moieties. Finally, strategies to enhance antibody function through protein engineering are reviewed while highlighting the impact of fundamental biophysical properties on protein developability.

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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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The use of CrossMAb technology for the generation of bi- and multispecific antibodies

Cited by 150 publications

References 65 publications

Design and characterization of novel dual Fc antibody with enhanced avidity for Fc receptors

Design and characterization of novel dual Fc antibody with enhanced avidity for Fc receptors

Considerations for the Design of Antibody-Based Therapeutics

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

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