In spite of a similar general architecture between bisFab and full-length antibodies, bisFabs likely have higher flexibility round the linker than Fabs in an IgG. for a more rapid selection of lead restorative candidates. CHDI-390576 strong class=”kwd-title” KEYWORDS: Antibody types, bispecific antibody, bispecific screening, crosslinked Fabs Intro Although the concept of bispecific antibodies and their potential benefits over monoclonal antibodies (mAbs) were described many years ago, manufacturing processes that allow multiple molecules to progress into clinical tests have only recently been developed.1 The success of bispecific antibodies is due in part to the fact that, in addition to targeting 2 antigens CHDI-390576 by a single molecule, bispecific antibodies enable novel mechanism of actions not accessible to monospecific antibodies.2,3 Examples include the recruitment of effector cells such as T cells, natural killer (NK) cells, macrophages; enabling transcytosis across the blood-brain barrier; the biomimetic alternative of one component of a protein complex (Element VIII) and the improvement of the uptake of antibody-drug conjugates by increasing the internalization of the prospective protein.2 These novel mechanisms are unavailable to mAbs and result from the physical connection of 2 different binding specificities in the same molecule. As with any other antibody-based therapeutic, the development of bispecific antibodies requires careful selection of the lead clones, a process that involves screening multiple antibodies to select the ones better matching the desired properties. Unlike mAbs that can be screened directly after hybridoma production, each component of a bispecific antibody needs to be tested in the context of the bispecific molecule. Here, we describe a method to produce heterodimeric (Fab)2 from any murine IgG isotype using chemical crosslinking. Using T-cell redirected cytolysis as a screening assay, we validated the use of the crosslinked F(ab)2 as a suitable tool for predicting the activity of hybridoma clones in the final full-length human IgG bispecific format. Results A general scheme of the strategy and the main steps involved in generating bispecific F(ab)2 (bisFabs) from murine IgGs is usually shown in Fig?1. First (a), antibodies are digested with proteolytic enzymes to generate F(ab)2. After purification, (b) the F(ab)2 are reduced to release the individual Fab molecules, and they are then (c) oxidized to reform the disulfide bond between the light and heavy chains. The resulting Fab molecules bearing CHDI-390576 a single reactive cysteine in the hinge are then (d) coupled to a bis-maleimide crosslinker, and finally (e) conjugated to another Fab with a different binding specificity. Open in a separate window Physique 1. Schematic diagram showing the main actions involved in producing bispecific bisFabs from murine IgGs. Purification actions are indicated in green font. The inter-chain disulfide bonds are indicated in orange. (a) The purified IgGs are digested with the proteases indicated in Table 1. to generate F(ab)2. (b) Reduction with TCEP releases the monomeric Fab molecules and reduces the heavy-light chain disulfide bond. (c) Re-oxidation of the thiol groups re-form the heavy-light disulfide bond while cyclizing a pair of cysteines in the hinge, leaving a single reduced cysteine. (d) The reoxidized Fab is usually reacted with an excess of bismaleimide crosslinker. (e) the resulting modified Fab is usually conjugated to a second Fab with a different specificity. Selection of digestion enzymes Because the first step in the process is to generate F(ab)2 from the intact hybridoma antibodies, we started optimizing the method for each murine isotype by screening and selecting enzymes capable of generating F(ab)2 with the desired structural properties. The factors taken into consideration to choose between different enzymes were: 1) complete, specific digestion to maximize the yield of Fab molecules, 2) limited heterogeneity in the cleavage site to facilitate the final quality control by mass spectrometry, and CR2 3) an odd number of cysteine residues in the hinge of the resulting Fab to facilitate the downstream actions. For mouse IgG1, methods for generating Fab molecules using pepsin or ficin have been described.4,5 However, since having an even or odd number of cysteines in the hinge region affects the methods used downstream and the resulting final yields, we wanted to.