Bispecific antibodies with natural architecture produced by co-culture of bacteria expressing two distinct half-antibodies

Spiess, Christoph; Merchant, Mark; Huang, Arthur; Zheng, Zhong; Yang, Nai‐Ying; Peng, Jing; Ellerman, Diego; Shatz, Whitney; Reilly, Dorothea; Yansura, Daniel G.; Scheer, Justin M.

doi:10.1038/nbt.2621

Cited by 171 publications

(136 citation statements)

References 30 publications

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“…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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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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show abstract

“…[1][2][3][4][5][6][7][8][9][10][11][12] Bispecific molecules described thus far can be divided into five classes based on their molecular format: 1) BsAb with IgG1-like structure, which are monovalent for each antigen; 2) BsAb prepared by appending binding domains to the IgG, which are bivalent for each antigen; 3) BsAb prepared using antibody fragments, which are monovalent for each antigen and often lack the Fc region; 4) bispecific fusion proteins, which are formed of an antibody fragment genetically linked to a protein to add additional functionality or specificity; and 5) bispecific conjugates, which are prepared by chemical conjugation of antibody fragments. While some such bispecific molecules have demonstrated manufacturing robustness, in vivo drug-like properties, suitable pharmacokinetics (PK) and efficacy have been achieved, platforms that yield multifunctional bispecific antibodies with different spatial configurations and flexibility suitable for a variety of target and disease applications are still needed.…”

Section: Introductionmentioning

confidence: 99%

Insertion of scFv into the hinge domain of full-length IgG1 monoclonal antibody results in tetravalent bispecific molecule with robust properties

Bezabeh¹,

Fleming²,

Fazenbaker³

et al. 2016

mAbs

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By simultaneous binding two disease mediators, bispecific antibodies offer the opportunity to broaden the utility of antibody-based therapies. Herein, we describe the design and characterization of Bs4Ab, an innovative and generic bispecific tetravalent antibody platform. The Bs4Ab format comprises a full-length IgG1 monoclonal antibody with a scFv inserted into the hinge domain. The Bs4Ab design demonstrates robust manufacturability as evidenced by MEDI3902, which is currently in clinical development. To further demonstrate the applicability of the Bs4Ab technology, we describe the molecular engineering, biochemical, biophysical, and in vivo characterization of a bispecific tetravalent Bs4Ab that, by simultaneously binding vascular endothelial growth factor and angiopoietin-2, inhibits their function. We also demonstrate that the Bs4Ab platform allows Fc-engineering similar to that achieved with IgG1 antibodies, such as mutations to extend half-life or modulate effector functions.

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“…Production and characterization of full-length bispecific antibodies was done as described elsewhere. 9,10,11 To explore potential differences between the 2 formats in the context of different binding geometries and antigen densities, we used 10 different antibodies directed against 5 different antigens: human epidermal growth factor receptor 2 (HER2); B-cell antigens CD20, CD79, and antigen A; and melanoma antigen B. A description of the isotypes, enzymes used and yields obtained for those 10 clones is shown in Table S1.…”

Section: Validation Of the Bisfab Format As Surrogate Of Full-length mentioning

confidence: 99%

bisFabs: Tools for rapidly screening hybridoma IgGs for their activities as bispecific antibodies

Patke¹,

Li²,

Wang³

et al. 2017

mAbs

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Bispecific antibodies are a growing class of therapeutic molecules. Many of the current bispecific formats require DNA engineering to convert the parental monoclonal antibodies into the final bispecific molecules. We describe here a method to generate bispecific molecules from hybridoma IgGs in 3-4 d using chemical conjugation of antigen-binding fragments (Fabs) (bisFabs). Proteolytic digestion conditions for each IgG isotype were analyzed to optimize the yield and quality of the final conjugates. The resulting bisFabs showed no significant amounts of homodimers or aggregates. The predictive value of murine bisFabs was tested by comparing the T-cell redirected cytotoxic activity of a panel of antibodies in either the bisFab or full-length IgG formats. A variety of antigens with different structures and expression levels was used to extend the comparison to a wide range of binding geometries and antigen densities. The activity observed for different murine bisFabs correlated with those observed for the fulllength IgG format across multiple different antigen targets, supporting the use of bisFabs as a screening tool. Our method may also be used for the screening of bispecific antibodies with other mechanisms of action, allowing for a more rapid selection of lead therapeutic candidates.

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Bispecific antibodies with natural architecture produced by co-culture of bacteria expressing two distinct half-antibodies

Cited by 171 publications

References 30 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

Insertion of scFv into the hinge domain of full-length IgG1 monoclonal antibody results in tetravalent bispecific molecule with robust properties

bisFabs: Tools for rapidly screening hybridoma IgGs for their activities as bispecific antibodies

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