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1.
Cell ; 184(5): 1232-1244.e16, 2021 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-33626330

RESUMO

Human cytomegalovirus (HCMV) infects the majority of the human population and represents the leading viral cause of congenital birth defects. HCMV utilizes the glycoproteins gHgLgO (Trimer) to bind to platelet-derived growth factor receptor alpha (PDGFRα) and transforming growth factor beta receptor 3 (TGFßR3) to gain entry into multiple cell types. This complex is targeted by potent neutralizing antibodies and represents an important candidate for therapeutics against HCMV. Here, we determine three cryogenic electron microscopy (cryo-EM) structures of the trimer and the details of its interactions with four binding partners: the receptor proteins PDGFRα and TGFßR3 as well as two broadly neutralizing antibodies. Trimer binding to PDGFRα and TGFßR3 is mutually exclusive, suggesting that they function as independent entry receptors. In addition, Trimer-PDGFRα interaction has an inhibitory effect on PDGFRα signaling. Our results provide a framework for understanding HCMV receptor engagement, neutralization, and the development of anti-viral strategies against HCMV.


Assuntos
Citomegalovirus/química , Glicoproteínas de Membrana/química , Proteínas do Envelope Viral/química , Internalização do Vírus , Microscopia Crioeletrônica , Citomegalovirus/fisiologia , Glicoproteínas de Membrana/metabolismo , Modelos Moleculares , Proteoglicanas/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/química , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Proteínas do Envelope Viral/metabolismo
2.
Nature ; 610(7930): 182-189, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36131013

RESUMO

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.


Assuntos
Anticorpos , Especificidade de Anticorpos , Proteínas de Membrana , Proteólise , Ubiquitina-Proteína Ligases , Animais , Anticorpos/imunologia , Anticorpos/metabolismo , Neoplasias Colorretais/metabolismo , Ligantes , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Receptores de Superfície Celular/imunologia , Receptores de Superfície Celular/metabolismo , Especificidade por Substrato , Ubiquitina-Proteína Ligases/imunologia , Ubiquitina-Proteína Ligases/metabolismo
3.
J Biol Chem ; 299(5): 104685, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-37031819

RESUMO

The exquisite specificity, natural biological functions, and favorable development properties of antibodies make them highly effective agents as drugs. Monoclonal antibodies are particularly strong as inhibitors of systemically accessible targets where trough-level concentrations can sustain full target occupancy. Yet beyond this pharmacologic wheelhouse, antibodies perform suboptimally for targets of high abundance and those not easily accessible from circulation. Fundamentally, this restraint on broader application is due largely to the stoichiometric nature of their activity-one drug molecule is generally able to inhibit a maximum of two target molecules at a time. Enzymes in contrast are able to catalytically turnover multiple substrates, making them a natural sub-stoichiometric solution for targets of high abundance or in poorly accessible sites of action. However, enzymes have their own limitations as drugs, including, in particular, the polypharmacology and broad specificity often seen with native enzymes. In this study, we introduce antibody-guided proteolytic enzymes to enable selective sub-stoichiometric turnover of therapeutic targets. We demonstrate that antibody-mediated substrate targeting can enhance enzyme activity and specificity, with proof of concept for two challenging target proteins, amyloid-ß and immunoglobulin G. This work advances a new biotherapeutic platform that combines the favorable properties of antibodies and proteolytic enzymes to more effectively suppress high-bar therapeutic targets.


Assuntos
Anticorpos Monoclonais , Terapia Biológica , Endopeptidases , Peptídeo Hidrolases , Imunoglobulina G , Peptídeo Hidrolases/metabolismo , Terapia Biológica/métodos
4.
Nat Commun ; 15(1): 642, 2024 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-38245524

RESUMO

The ability to leverage antibodies to agonize disease relevant biological pathways has tremendous potential for clinical investigation. Yet while antibodies have been successful as antagonists, immune mediators, and targeting agents, they are not readily effective at recapitulating the biology of natural ligands. Among the important determinants of antibody agonist activity is the geometry of target receptor engagement. Here, we describe an engineering approach inspired by a naturally occurring Fab-Fab homotypic interaction that constrains IgG in a unique i-shaped conformation. i-shaped antibody (iAb) engineering enables potent intrinsic agonism of five tumor necrosis factor receptor superfamily (TNFRSF) targets. When applied to bispecific antibodies against the heterodimeric IL-2 receptor pair, constrained bispecific IgG formats recapitulate IL-2 agonist activity. iAb engineering provides a tool to tune agonist antibody function and this work provides a framework for the development of intrinsic antibody agonists with the potential for generalization across broad receptor classes.


Assuntos
Anticorpos Biespecíficos , Receptores do Fator de Necrose Tumoral , Imunoglobulina G/genética , Engenharia de Proteínas
5.
MAbs ; 11(7): 1254-1265, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31286843

RESUMO

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 BsIgG1without Fab engineering to be a common occurrence, i.e., observed for 33 of the 99 different antibody pairs evaluated. Installing charge mutations at both CH1/CL interfaces was sufficient for near quantitative yield (>90%) of BsIgG1 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 BsIgG1 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 BsIgG1 yield in most cases. Mutational analysis revealed that the disulfide bond between heavy and light chains did not affect the yield of BsIgG1. 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.


Assuntos
Anticorpos Biespecíficos/química , Imunoglobulina G/química , Cadeias Pesadas de Imunoglobulinas/química , Cadeias Leves de Imunoglobulina/química , Anticorpos Biespecíficos/genética , Regiões Determinantes de Complementaridade/genética , Dimerização , Células HEK293 , Humanos , Fragmentos Fab das Imunoglobulinas/genética , Imunoglobulina G/genética , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Leves de Imunoglobulina/genética , Ligação Proteica , Engenharia de Proteínas , Análise de Célula Única
6.
Protein Eng Des Sel ; 30(9): 627-637, 2017 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-28985411

RESUMO

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-ß 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.


Assuntos
Anticorpos Biespecíficos/biossíntese , Anticorpos Monoclonais/biossíntese , Ensaios de Triagem em Larga Escala , Imunoglobulina G/biossíntese , Engenharia de Proteínas/métodos , Animais , Anticorpos Biespecíficos/química , Anticorpos Biespecíficos/genética , Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Células CHO , Clonagem Molecular , Cricetulus , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/imunologia , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Células HEK293 , Humanos , Imunoglobulina G/química , Imunoglobulina G/genética , Proteínas Klotho , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/imunologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
7.
MAbs ; 8(8): 1536-1547, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27606571

RESUMO

Antigen-binding fragments (Fab) and F(ab')2 antibodies serve as alternative formats to full-length anti-bodies 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')2 C-terminus does not have detectable binding of pre-existing AHA, providing a practical route to produce F(ab')2 in vitro by proteolytic digestion when the binding of pre-existing 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 T225L variant and the natural C-terminal D221 as solutions with minimal serum reactivity. Our work now enables the production of Fab and F(ab')2 for therapeutic and diagnostic immune assays that have minimal reactivity toward pre-existing AHA.


Assuntos
Autoanticorpos/imunologia , Epitopos de Linfócito B/imunologia , Fragmentos Fab das Imunoglobulinas/imunologia , Imunoglobulina G/imunologia , Anticorpos Monoclonais , Especificidade de Anticorpos , Mapeamento de Epitopos , Humanos , Engenharia de Proteínas
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