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 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.

Bispecific antibody target pair discovery by high-throughput phenotypic screening using in vitro combinatorial Fab libraries

“…High-throughput combinatorial target discovery on this scale has not been described to date, although unbiased bispecific functional screening has been reported to be necessary for identification of the optimal V-region pair. 8,9 We were able to generate sufficient numbers of bispecific antibodies at high purity for screening owing to the unique way in which our bispecific Fab-K D -Fab antibody format is formed. Following in vitro mixing of the two component Fab-X and Fab-Y halves, the resulting bispecific complex is of sufficiently high purity and thus requires no further purification or processing prior to screening.…”

“…34 Homogenous Fab conjugates have been generated by chemical conjugation of proteolytically cleaved IgGs, though reduction and oxidation in addition to multiple purification steps were required. 9 More recently, split protein reconstitution methods such as the SpyTag/SpyCatcher system have been used to covalently couple antibody fragments; however, SpyCatcher fusions can suffer from low expression 35 and multiple polishing steps are required to isolate homogeneous-ligated product. 36,37 Sortasemediated covalent antibody conjugates have been successfully screened in biological assays without further purification of the conjugated product, 38 but certain assays may require removal of the sortase prior to screening.…”

“…7 Bispecific target selection to date has been largely hypothesis-driven; either by pre-selection of a defined antigen pair or a small number of functionally related targets, based on their known biological function. Although extensive functional screening of panels of bispecific antibodies has been reported to be necessary for optimal antibody selection for a given target pair, 8,9 a systematic, large-scale screening approach has not yet been fully exploited for the co-discovery of the optimal target antigen pairs for modulation of a given cellular response. The functional activity of bispecific antibodies, particularly those targeted to cell surface receptors, is difficult to predict from that of the individual parental antibodies.…”

The renaissance of chemically generated bispecific antibodies

“…73 In the case of Fab' fragments with two cysteines, N-ethylmaleimide was used to cap one of the cysteines and conditions that preferentially re-oxidize the interchain disulfide were used to remove all other free thiols from the fragment. 74 Recently, an optimization of the Fab' crosslinking was reported, where mild re-oxidation of the Fab' fragments with dehydroascorbic acid promoted re-formation of disulfide bridges from intramolecular pairs of cysteines, leaving only one sulfhydryl group to react with the bis-maleimide crosslinker. The selective re-oxidation process was ~80% efficient, with <10% Fab-Fab dimer formation and <12% Fab without any re-oxidation as shown by SDS-PAGE, and as such a purification step before bispecific formation was needed.…”

Brief introduction of current technologies in isolation of broadly neutralizing HIV-1 antibodies

“…While successful in-vitro, recombinant gp120 has failed to generate cross neutralizing antibodies in vivo possibly due to the natural conformation of gp120 (Prevost, 2017;Wang et al, 2016;Doores et al, 2015;Doores et al, 2010;Zhou et al, 2010;Wu et al, 1997;Watkins et al, 1996;Robert-Guroff et al, 1992). Monoclonal antibodies were first generated in mice in 1975 using a hybridoma technique (Patke et al, 2017;Holzlohner and Hanack, 2017;Bhatia et al, 2016;Pogson et al, 2016;Abusneina and Gauthier, 2016;Milstein, 1999), and were subsequently accepted globally by manufacturers. Now, it is well understood that rodent and murine derived antibodies are immunogenic in humans; when binding to antigens within the human body, the complexes can be recognized as immunogenic agents.…”