title: bisFabs: Tools for rapidly screening hybridoma IgGs for their activities as bispecific antibodies.
abstract: 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 full-length 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.
authors.affiliation: a Department of Protein Chemistry , Genentech , South San Francisco , CA , USA.
authors.forename: Sanket
authors.initials: S
authors.lastname: Patke
authors.affiliation: b Department of Translational Oncology , Genentech , South San Francisco , CA , USA.
authors.forename: Ji
authors.initials: J
authors.lastname: Li
authors.affiliation: b Department of Translational Oncology , Genentech , South San Francisco , CA , USA.
authors.forename: Peiyin
authors.initials: P
authors.lastname: Wang
authors.affiliation: b Department of Translational Oncology , Genentech , South San Francisco , CA , USA.
authors.forename: Dion
authors.initials: D
authors.lastname: Slaga
authors.affiliation: b Department of Translational Oncology , Genentech , South San Francisco , CA , USA.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Johnston
authors.affiliation: b Department of Translational Oncology , Genentech , South San Francisco , CA , USA.
authors.forename: Sunil
authors.initials: S
authors.lastname: Bhakta
authors.affiliation: b Department of Translational Oncology , Genentech , South San Francisco , CA , USA.
authors.forename: Siler
authors.initials: S
authors.lastname: Panowski
authors.affiliation: b Department of Translational Oncology , Genentech , South San Francisco , CA , USA.
authors.forename: Liping L
authors.initials: LL
authors.lastname: Sun
authors.affiliation: b Department of Translational Oncology , Genentech , South San Francisco , CA , USA.
authors.forename: Teemu
authors.initials: T
authors.lastname: Junttila
authors.affiliation: a Department of Protein Chemistry , Genentech , South San Francisco , CA , USA.
authors.forename: Justin M
authors.initials: JM
authors.lastname: Scheer
authors.affiliation: a Department of Protein Chemistry , Genentech , South San Francisco , CA , USA.
authors.forename: Diego A
authors.initials: DA
authors.lastname: Ellerman
mesh_terms: D000818:Animals; D018033:Antibodies, Bispecific / Q000096:biosynthesis* / Q000276:immunology / Q000302:isolation & purification; D006801:Humans; D006825:Hybridomas; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis* / Q000302:isolation & purification; D007074:Immunoglobulin G / Q000276:immunology / Q000302:isolation & purification*; D051379:Mice; D015202:Protein Engineering / Q000379:methods*
chemical_list: D018033:Antibodies, Bispecific; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G
keywords: Antibody formats; bispecific antibody; bispecific screening; crosslinked Fabs