Most current therapies that target plasma membrane receptors function by antagonizing ligand binding or enzymatic activities. However, typical mammalian proteins comprise multiple domains that execute discrete but coordinated activities. Thus, inhibition of one domain often incompletely suppresses the function of a protein. Indeed, targeted protein degradation technologies, including proteolysis-targeting chimeras1 (PROTACs), have highlighted clinically important advantages of target degradation over inhibition2. However, the generation of heterobifunctional compounds binding to two targets with high affinity is complex, particularly when oral bioavailability is required3. Here we describe the development of proteolysis-targeting antibodies (PROTABs) that tether cell-surface E3 ubiquitin ligases to transmembrane proteins, resulting in target degradation both in vitro and in vivo. Focusing on zinc- and ring finger 3 (ZNRF3), a Wnt-responsive ligase, we show that this approach can enable colorectal cancer-specific degradation. Notably, by examining a matrix of additional cell-surface E3 ubiquitin ligases and transmembrane receptors, we demonstrate that this technology is amendable for ‘on-demand’ degradation. Furthermore, we offer insights on the ground rules governing target degradation by engineering optimized antibody formats. In summary, this work describes a strategy for the rapid development of potent, bioavailable and tissue-selective degraders of cell-surface proteins.
Multiple strategies have been developed to facilitate the efficient production of bispecific IgG (BsIgG) in single host cells. For example, we previously demonstrated near quantitative (≥90%) formation of BsIgG of different species and isotypes by combining 'knob-into-hole' mutations for heavy chain heterodimerization with engineered antigen-binding fragments (Fabs) for preferential cognate heavy/light chain pairing. Surprisingly, in this study we found high yield (>65%) of BsIgG 1 without Fab engineering to be a common occurrence, i.e., observed for 33 of the 99 different antibody pairs evaluated. Installing charge mutations at both C H 1/C L interfaces was sufficient for near quantitative yield (>90%) of BsIgG 1 for most (9 of 11) antibody pairs tested with this inherent cognate chain pairing preference. Mechanistically, we demonstrate that a strong cognate pairing preference in one Fab arm can be sufficient for high BsIgG 1 yield. These observed chain pairing preferences are apparently driven by variable domain sequences and can result from a few specific residues in the complementarity-determining region (CDR) L3 and H3. Transfer of these CDR residues into other antibodies increased BsIgG 1 yield in most cases. Mutational analysis revealed that the disulfide bond between heavy and light chains did not affect the yield of BsIgG 1. This study provides some mechanistic understanding of factors contributing to antibody heavy/light chain pairing preference and subsequently contributes to the efficient production of BsIgG in single host cells.
Bispecific antibodies offer a clinically validated platform for drug discovery. In generating functionally active bispecific antibodies, it is necessary to identify a unique parental antibody pair to merge into a single molecule. However, technologies that allow high-throughput production of bispecific immunoglobulin Gs (BsIgGs) for screening purposes are limited. Here, we describe a novel bispecific antibody format termed tethered-variable CLBsIgG (tcBsIgG) that allows robust production of intact BsIgG in a single cell line, concurrently ensuring cognate light chain pairing and preserving key antibody structural and functional properties. This technology is broadly applicable in the generation of BsIgG from a variety of antibody isotypes, including human BsIgG1, BsIgG2 and BsIgG4. The practicality of the tcBsIgG platform is demonstrated by screening BsIgGs generated from FGF21-mimetic anti-Klotho-β agonistic antibodies in a combinatorial manner. This screen identified multiple biepitopic combinations with enhanced agonistic activity relative to the parental monoclonal antibodies, thereby demonstrating that biepitopic antibodies can acquire enhanced functionality compared to monospecific parental antibodies. By design, the tcBsIgG format is amenable to high-throughput production of large panels of bispecific antibodies and thus can facilitate the identification of rare BsIgG combinations to enable the discovery of molecules with improved biological function.
Antigen-binding fragments (Fab) and F(ab 0 ) 2 antibodies serve as alternative formats to full-length antibodies in therapeutic and immune assays. They provide the advantage of small size, short serum half-life, and lack of effector function. Several proteases associated with invasive diseases are known to cleave antibodies in the hinge-region, and this results in anti-hinge antibodies (AHA) toward the neoepitopes. The AHA can act as surrogate Fc and reintroduce the properties of the Fc that are otherwise lacking in antibody fragments. While this response is desired during the natural process of fighting disease, it is commonly unwanted for therapeutic antibody fragments. In our study, we identify a truncation in the lower hinge region of the antibody that maintains efficient proteolytic cleavage by IdeS protease. The resulting neoepitope at the F(ab 0 ) 2 C-terminus does not have detectable binding of pre-existing AHA, providing a practical route to produce F(ab 0 ) 2 in vitro by proteolytic digestion when the binding of preexisting AHA is undesired. We extend our studies to the upper hinge region of the antibody and provide a detailed analysis of the contribution of C-terminal residues of the upper hinge of human IgG1, IgG2 and IgG4 to pre-existing AHA reactivity in human serum. While no pre-existing antibodies are observed toward the Fab of IgG2 and IgG4 isotype, a significant response is observed toward most residues of the upper hinge of human IgG1. We identify a T 225 L variant and the natural C-terminal D 221 as solutions with minimal serum reactivity. Our work now enables the production of Fab and F(ab 0 ) 2 for therapeutic and diagnostic immune assays that have minimal reactivity toward pre-existing AHA.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply.
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.