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With the global pandemic and the continuous mutations of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the need for effective and broadly neutralizing treatments has become increasingly urgent. This study introduces a novel strategy that targets two aspects simultaneously, using bifunctional antibodies to inhibit both the attachment of SARS-CoV-2 to host cell membranes and viral fusion. We developed pioneering IgG4-(HR2)4 bifunctional antibodies by creating immunoglobulin G4-based and phage display-derived human monoclonal antibodies (mAbs) that specifically bind to the SARS-CoV-2 receptor-binding domain, engineered with four heptad repeat 2 (HR2) peptides. Our in vitro experiments demonstrate the superior neutralization efficacy of these engineered antibodies against various SARS-CoV-2 variants, ranging from original SARS-CoV-2 strain to the recently emerged Omicron variants, as well as SARS-CoV, outperforming the parental mAb. Notably, intravenous monotherapy with the bifunctional antibody neutralizes a SARS-CoV-2 variant in a murine model without causing significant toxicity. In summary, this study unveils the significant potential of HR2 peptide-driven bifunctional antibodies as a potent and versatile strategy for mitigating SARS-CoV-2 infections. This approach offers a promising avenue for rapid development and management in the face of the continuously evolving SARS-CoV-2 variants, holding substantial promise for pandemic control.
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Anticuerpos Biespecíficos , COVID-19 , Humanos , Animales , Ratones , SARS-CoV-2/genética , Anticuerpos Monoclonales/uso terapéutico , Inmunoglobulina G , Péptidos/genética , Poder PsicológicoRESUMEN
Antibody phage display is a key technology for the discovery and development of target-specific monoclonal antibodies (mAbs) for use in research, diagnostics, and therapy. The construction of a high-quality antibody library, with larger and more diverse antibody repertoires, is essential for the successful development of phage display-derived mAbs. In this study, a large human combinatorial single-chain variable fragment library (1.5 × 1011 colonies) was constructed from Epstein-Barr virus-infected human peripheral blood mononuclear cells stimulated with a combination of two of the activators of human B cells, the Toll-like receptor 7/8 agonist R848 and interleukin-2. Next-generation sequencing analysis with approximately 1.9 × 106 and 2.7 × 106 full-length sequences of heavy chain variable (VH) and κ light chain variable (Vκ) domains, respectively, revealed that the library consists of unique VH (approximately 94%) and Vκ (approximately 91%) sequences with greater diversity than germline sequences. Lastly, multiple unique mAbs with high affinity and broad cross-species reactivity could be isolated from the library against two therapeutically relevant target antigens, validating the library quality. These findings suggest that the novel antibody library we have developed may be useful for the rapid development of target-specific phage display-derived recombinant human mAbs for use in therapeutic and diagnostic applications.
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Infecciones por Virus de Epstein-Barr , Biblioteca de Péptidos , Humanos , Leucocitos Mononucleares , Herpesvirus Humano 4 , Anticuerpos Monoclonales/genética , Secuenciación de Nucleótidos de Alto RendimientoRESUMEN
Cancer is the second leading cause of death worldwide after cardiovascular diseases. Harnessing the power of immune cells is a promising strategy to improve the antitumor effect of cancer immunotherapy. Recent progress in recombinant DNA technology and antibody engineering has ushered in a new era of bispecific antibody (bsAb)-based immune-cell engagers (ICEs), including T- and natural-killer-cell engagers. Since the first approval of blinatumomab by the United States Food and Drug Administration (US FDA), various bsAb-based ICEs have been developed for the effective treatment of patients with cancer. Simultaneously, several potential therapeutic targets of bsAb-based ICEs have been identified in various cancers. Therefore, this review focused on not only highlighting the action mechanism, design and structure, and status of bsAb-based ICEs in clinical development and their approval by the US FDA for human malignancy treatment, but also on summarizing the currently known and emerging therapeutic targets in cancer. This review provides insights into practical considerations for developing next-generation ICEs.
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Inmunoterapia , Neoplasias , Humanos , Neoplasias/tratamiento farmacológico , Estados UnidosRESUMEN
Gastric cancer (GC), a leading cause of cancer-related mortality, remains a significant challenge despite recent therapeutic advancements. In this study, we explore the potential of targeting cell surface glucose-regulated protein 94 (GRP94) with antibodies as a novel therapeutic approach for GC. Our comprehensive analysis of GRP94 expression across various cancer types, with a specific focus on GC, revealed a substantial overexpression of GRP94, highlighting its potential as a promising target. Through in vitro and in vivo efficacy assessments, as well as toxicological analyses, we found that K101.1, a fully human monoclonal antibody designed to specifically target cell surface GRP94, effectively inhibits GC growth and angiogenesis without causing in vivo toxicity. Furthermore, our findings indicate that K101.1 promotes the internalization and concurrent downregulation of cell surface GRP94 on GC cells. In conclusion, our study suggests that cell surface GRP94 may be a potential therapeutic target in GC, and that antibody-based targeting of cell surface GRP94 may be an effective strategy for inhibiting GRP94-mediated GC growth and angiogenesis. [BMB Reports 2024; 57(4): 188-193].
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Anticuerpos Monoclonales , Neoplasias Gástricas , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patología , Neoplasias Gástricas/inmunología , Humanos , Anticuerpos Monoclonales/farmacología , Línea Celular Tumoral , Animales , Ratones , Proliferación Celular/efectos de los fármacos , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/inmunología , Neovascularización Patológica/metabolismo , Ratones DesnudosRESUMEN
The rapid emergence of new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants has resulted in the ongoing global coronavirus disease 2019 (COVID-19) pandemic. Thus, the rapid development of a platform to detect a broad range of SARS-CoV-2 variants is essential for successful COVID-19 management. In this study, four SARS-CoV-2 spike protein-specific single-chain variable fragments (scFvs) were isolated from a synthetic antibody library using phage display technology. Following the conversion of these scFvs into monoclonal antibodies (mAbs) (K104.1-K104.4) and production and purification of the mAbs, the antibody pair (K104.1 and K104.2) that exhibited the highest binding affinity (K104.1 and K104.2, 1.3 nM and 1.9 nM) was selected. Biochemical analyses revealed that this antibody pair specifically bound to different sites on the S2 subunit of the spike protein. Furthermore, we developed a highly sensitive sandwich immunoassay using this antibody pair that accurately and quantitatively detected the spike proteins of wild-type SARS-CoV-2 and multiple variants, including Alpha, Beta, Gamma, Delta, Kappa, and Omicron, in the picomolar range. Conclusively, the novel phage display-derived mAbs we have developed may be useful for the rapid and efficient detection of the fast-evolving SARS-CoV-2.
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Anticuerpos Monoclonales , Bacteriófagos , SARS-CoV-2 , Anticuerpos de Cadena Única , Humanos , Anticuerpos Monoclonales/genética , Anticuerpos Neutralizantes , Anticuerpos Antivirales , COVID-19 , SARS-CoV-2/genética , Anticuerpos de Cadena Única/genética , Glicoproteína de la Espiga del CoronavirusRESUMEN
Introduction: The emergence of new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants has caused unprecedented health and socioeconomic crises, necessitating the immediate development of highly effective neutralizing antibodies. Despite recent advancements in anti-SARS-CoV-2 receptor-binding domain (RBD)-specific monoclonal antibodies (mAbs) derived from convalescent patient samples, their efficacy against emerging variants has been limited. In this study, we present a novel dual-targeting strategy using bispecific antibodies (bsAbs) that specifically recognize both the SARS-CoV-2 RBD and fusion peptide (FP), crucial domains for viral attachment to the host cell membrane and fusion in SARS-CoV-2 infection. Methods: Using phage display technology, we rapidly isolated FP-specific mAbs from an established human recombinant antibody library, identifying K107.1 with a nanomolar affinity for SARS-CoV-2 FP. Furthermore, we generated K203.A, a new bsAb built in immunoglobulin G4-(single-chain variable fragment)2 forms and demonstrating a high manufacturing yield and nanomolar affinity to both the RBD and FP, by fusing K102.1, our previously reported RBD-specific mAb, with K107.1. Results: Our comprehensive in vitro functional analyses revealed that the K203.A bsAb significantly outperformed the parental RBD-specific mAb in terms of neutralization efficacy against SARS-CoV-2 variants. Furthermore, intravenous monotherapy with K203.A demonstrated potent in vivo neutralizing activity without significant in vivo toxicity in a mouse model infected with a SARS-CoV-2 variant. Conclusion: These findings present a novel bsAb dual-targeting strategy, directed at SARS-CoV-2 RBD and FP, as an effective approach for rapid development and management against continuously evolving SARS-CoV-2 variants.
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Anticuerpos Biespecíficos , COVID-19 , Animales , Ratones , Humanos , SARS-CoV-2 , Anticuerpos Biespecíficos/uso terapéutico , Anticuerpos Neutralizantes , Anticuerpos AntiviralesRESUMEN
Rapid emergence of new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has prompted an urgent need for the development of broadly applicable and potently neutralizing antibody platform against the SARS-CoV-2, which can be used for combatting the coronavirus disease 2019 (COVID-19). In this study, based on a noncompeting pair of phage display-derived human monoclonal antibodies (mAbs) specific to the receptor-binding domain (RBD) of SARS-CoV-2 isolated from human synthetic antibody library, we generated K202.B, a novel engineered bispecific antibody with an immunoglobulin G4-single-chain variable fragment design, with sub- or low nanomolar antigen-binding avidity. Compared with the parental mAbs or mAb cocktail, the K202.B antibody showed superior neutralizing potential against a variety of SARS-CoV-2 variants in vitro. Furthermore, structural analysis of bispecific antibody-antigen complexes using cryo-electron microscopy revealed the mode of action of K202.B complexed with a fully open three-RBD-up conformation of SARS-CoV-2 trimeric spike proteins by simultaneously interconnecting two independent epitopes of the SARS-CoV-2 RBD via inter-protomer interactions. Intravenous monotherapy using K202.B exhibited potent neutralizing activity in SARS-CoV-2 wild-type- and B.1.617.2 variant-infected mouse models, without significant toxicity in vivo. The results indicate that this novel approach of development of immunoglobulin G4-based bispecific antibody from an established human recombinant antibody library is likely to be an effective strategy for the rapid development of bispecific antibodies, and timely management against fast-evolving SARS-CoV-2 variants.
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Anticuerpos Biespecíficos , COVID-19 , Animales , Ratones , Humanos , SARS-CoV-2/metabolismo , Anticuerpos Antivirales , Anticuerpos Biespecíficos/farmacología , Microscopía por Crioelectrón , Anticuerpos Neutralizantes , Glicoproteína de la Espiga del CoronavirusRESUMEN
Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has resulted in an ongoing global pandemic crisis, caused by the life-threatening illness coronavirus disease 2019 (COVID-19). Thus, the rapid development of monoclonal antibodies (mAbs) to cope with COVID-19 is urgently necessary. In this study, we used phage display to develop four human mAbs specific to the receptor-binding domain (RBD) of SARS-CoV-2. Our intensive in vitro functional analyses demonstrated that K102.1, an anti-SARS-CoV-2 RBD-specific mAb, exerted potent neutralizing activity against pseudoviral and live viral infection and the interaction between SARS-CoV-2 RBD and human angiotensin-converting enzyme 2. Monotherapy with K102.1 also revealed the therapeutic potential against SARS-CoV-2 infection in vivo. Further, this study developed a sandwich enzyme-linked immunosorbent assay with a non-competing mAb pair, K102.1 and K102.2, that accurately detected the RBDs of SARS-CoV-2 wild-type and variants with high sensitivity in the picomolar range. These findings suggest that the phage-display-based mAb selection from an established antibody library may be an effective strategy for the rapid development of mAbs against the constantly evolving SARS-CoV-2.
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Colorectal cancer (CRC) is one of the life-threatening malignancies worldwide. Thus, novel potential therapeutic targets and therapeutics for the treatment of CRC need to be identified to improve the clinical outcomes of patients with CRC. In this study, we found that glucose-regulated protein 94 (GRP94) is overexpressed in CRC tissues, and its high expression is correlated with increased microvessel density. Next, through phage display technology and consecutive in vitro functional isolations, we generated a novel human monoclonal antibody that specifically targets cell surface GRP94 and shows superior internalizing activity comparable to trastuzumab. We found that this antibody specifically inhibits endothelial cell tube formation and simultaneously promotes the downregulation of GRP94 expression on the endothelial cell surface. Finally, we demonstrated that this antibody effectively suppresses tumor growth and angiogenesis of HCT116 human CRC cells without causing severe toxicity in vivo. Collectively, these findings suggest that cell surface GRP94 is a novel potential anti-angiogenic target in CRC and that antibody targeting of GRP94 on the endothelial cell surface is an effective strategy to suppress CRC tumor angiogenesis.