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

Abstract

Bispecific antibodies can uniquely influence cellular responses, but selecting target combinations for optimal functional activity remains challenging. Here we describe a high-throughput, combinatorial, phenotypic screening approach using a new bispecific antibody target discovery format, allowing screening of hundreds of target combinations. Simple in vitro mixing of Fab-fusion proteins from a diverse library enables the generation of thousands of screen-ready bispecific antibodies for high-throughput, biologically relevant assays. We identified an obligate bispecific co-targeting CD79a/b and CD22 as a potent inhibitor of human B cell activation from a short-term flow cytometry signaling assay. A long-term, highcontent imaging assay identified anti-integrin bispecific inhibitors of human cell matrix accumulation targeting integrins β1 and β6 or αV and β1. In all cases, functional activity was conserved from the bispecific screening format to a therapeutically relevant format. We also introduce a broader type of mechanistic screen whereby functional modulation of different cell subsets in peripheral blood mononuclear cells was evaluated simultaneously. We identified bispecific antibodies capable of activating different T cell subsets of potential interest for applications in oncology or infectious disease, as well as bispecifics abrogating T cell activity of potential interest to autoimmune or inflammatory disease. The bispecific target pair discovery technology described herein offers access to new target biology and unique bispecific therapeutic opportunities in diverse disease indications.

Facilitating high throughput bispecific antibody production and potential applications within biopharmaceutical discovery workflows

“…Dashed grey lines represent disulfide bonds; full grey lines, peptide linkers; blue and red rectangular modules, immunoglobulin domains within protein chains binding target 1 or target 2 respectively; yellow rectangular modules, immunoglobulin domains within a common light chain; circular module connectors, a mutation set to drive correct HC-HC pairing; triangular and square module connectors, a mutation set to drive correct HC-LC pairing. (b) Six exemplar methods amenable to HTP bsAb production: (i)Recombinant Expression, (ii) Conjugation via peptide-dAb/scFv modules, 36 (iii) Conjugation via split inteins, 37 (iv) Chemical conjugation via bis maleimide linker, 38 (v) Chemical conjugation via ‘click’ chemistry 39 and (vi) Redox recombination. 40 The bsAb format classes that each method can generate are marked in tickboxes.…”

“…Smaller antigen-binding antibody fragments, such as Fabs, scFv or dAbs can be reliably produced in standard expression systems and isolated with single step purification processes and so are convenient starting modules. 36 , 89 An early ‘Dock and Lock’ bsAb production strategy relied on covalent disulfide bond formation to lock in binding between two fusion peptides derived from cAMP-dependent protein kinase and an A-kinase anchor protein. 89 , 90 The Fab-K D -Fab screening format developed by Bhatta et al contains a much simpler linkage between a peptide from the yeast transcription factor GCN4 and an anti-GCN4 scFv and has proven very amenable to HTP bsAb production ( Figure 2b (ii)).…”

ADCT-602, a Novel PBD Dimer–containing Antibody–Drug Conjugate for Treating CD22-positive Hematologic Malignancies

“…In addition, CD22 is almost universally expressed in B-cell acute lymphoblastic leukemia (B-ALL), including in the relapsed or refractory (R/R) setting ( 6–8 ). Various therapeutic modalities against CD22 have been developed, including naked or radiolabeled antibodies ( 9–11 ), bispecific antibodies ( 12, 13 ), and chimeric antigen receptor T-cell therapy ( 14, 15 ). CD22 is known to recycle and partially accumulates in the lysosome ( 16 ).…”

Rapid Production of Bispecific Antibodies from Off-the-Shelf IgGs with High Yield and Purity

“…4 The ability of BsAbs to bind two different epitopes on a cell surface simultaneously can also interfere with signaling pathways in unique ways that is not possible with either antibody alone or the combination of unlinked antibodies. 5 The cross-linking of cell-surface antigens can trigger, redirect, or inhibit cross-talk between signaling pathways. 6 The simultaneous binding of two receptors is especially valuable in angiogenesis inhibition and tumor treatments, where the biological signals are often redundant and target cells can evade inhibition of one receptor by upregulating another.…”

“…In one such case, bispecific T cell engagers (BiTEs), which contain tumor surface antigen-binding domains and T cell-specific CD3-binding domains, recruit T-cells to promote cytolysis of tumor cells. , BsAbs can also be used to preferentially bind cells that simultaneously express two molecular targets, compared to cells that express only one, due to increased avidity . The ability of BsAbs to bind two different epitopes on a cell surface simultaneously can also interfere with signaling pathways in unique ways that is not possible with either antibody alone or the combination of unlinked antibodies . The cross-linking of cell-surface antigens can trigger, redirect, or inhibit cross-talk between signaling pathways .…”