Engineering pan–HIV-1 neutralization potency through multispecific antibody avidity

Deep mining of B cell repertoires of HIV-1-infected individuals has resulted in the isolation of dozens of HIV-1 broadly neutralizing antibodies (bNAbs). Yet, it remains uncertain whether any such bNAbs alone are sufficiently broad and potent to deploy therapeutically. Here, we engineered HIV-1 bNAbs for their combination on a single multispecific and avid molecule via direct genetic fusion of their Fab fragments to the human apoferritin light chain. The resulting molecule demonstrated a remarkable median IC50 value of 0.0009 g/mL and 100% neutralization coverage of a broad HIV-1 pseudovirus panel (118 isolates) at a 4 g/mL cutoff-a 32-fold enhancement in viral neutralization potency compared to a mixture of the corresponding HIV-1 bNAbs. Importantly, Fc incorporation on the molecule and engineering to modulate Fc receptor binding resulted in IgG-like bioavailability invivo. This robust plug-and-play antibody design is relevant against indications where multispecificity and avidity are leveraged simultaneously to mediate optimal biological activity.The following reagents were obtained through the NIH AIDS Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases: TZM-bl cells (ARP-8129; contributed by Dr. John C. Kappes and Dr. Xiaoyun Wu); anti–HIV-1 gp160 monoclonal antibody (N6/ PGDM1400x10E8v4) (ARP-13390; contributed by Drs. Ling Xu and Gary Nabel); HIV-1 NL4-3 ΔEnv Vpr luciferase reporter vector (pNL4-3.Luc.R-E-) (ARP-3418; contributed by Dr. Nathaniel Landau and Aaron Diamond); plasmids pcDNA3.1 D/V5-His TOPO-expressing HIV-1 Env/Rev (ARP-11017, ARP-11018, ARP-11024, and ARP-11022; contributed by Drs. David Montefiori, Feng Gao, and Ming Li); plasmid pcDNA3.1(+)-expressing HIV-1 Env/Rev (ARP-11037; contributed by Drs. B. H. Hahn and D. L. Kothe); plasmid pcDNA3.1 D/V5-His TOPO-expressing HIV-1 Env/Rev (ARP-11308; contributed by Drs. D. Montefiori, F. Gao, C. Wil- liamson, and S. Abdool Karim); plasmid pcDNA3.1 V5-His TOPO-expressing HIV-1 Env/Rev (ARP-11309; contributed by Drs. B. H. Hahn, Y. Li, and J. F. Sala- zar-Gonzalez); HIV-1 BG505 Env expression vector (BG505.W6M.ENV.C2) (ARP- 11518; contributed by Dr. Julie Overbaugh); HIV-1 Env expression vector (CRF02_AG clone 257) (ARP-11599; contributed by Drs. D. Ellenberger, B. Li, M. Callahan, and S. Butera); plasmid pcDNA3.1 V5-His TOPO-expressing HIV-1 CNE8 Env (ARP-12653; contributed by Drs. Linqi Zhang, Hong Shang, David Montefiori, Tsinghua University (Beijing, China), China Medical University (Bei- jing, China), and Duke University (Durham, NC); HIV-1 SF162 gp160 expression vector (ARP-10463; contributed by Drs. Leonidas Stamatatos and Cecilia Cheng- Mayer); plasmid pcDNA3.1 V5-His TOPO-expressing HIV-1 Env/Rev (ARP-11034; contributed by Drs. B. H. Hahn, X. Wei, and G. M. Shaw); plasmid pcDNA3.1/V5- His TOPO-expressing HIV Env/Rev (ARP-11038; contributed by Drs. B. H. Hahn and D. L. Kothe); plasmid pcDNA3.1 V5-His TOPO-expressing HIV-1 Env/Rev (ARP-11310; contributed by Drs. B. H. Hahn, Y. Li, and J. F. Salazar-Gonzalez); HIV-1 Env expression vector (p16845 env) (ARP-11503; contributed by Drs. R. Paranjape, S. Kulkarni, and D. Montefiori); HIV-1 1054 Env expression vector (p1054.TC4.1499) (ARP-11561) and 6244 Env expression vector (p6244_13.B5.4576) (ARP-11566; contributed by Drs. Beatrice H. Hahn, Brandon F. Keele, and George M. Shaw); HIV-1 ZM246F Env expression vector (pZM246F_C1G) (ARP-11830; contributed by Dr. Beatrice Hahn); HIV-1 Env expression vector (CRF02_AG clone 278) (ARP-11605; contributed by Drs. Michael Thomson, Ana Revilla, Elena Delgado, David Montefiori, Sonia P erez Castro, Centro Nacional de Microbiologia, Instituto de Salud Carlos III (Majada- honda, Madrid, Spain), Complejo Hospitalario Santa Mar ıa Madre (Orense, Spain), Duke University (Durham, NC), and the CAVD; and NL4-3 Env expression vector (pDOLHIVenv) (from Dr. Eric Freed and Dr. Rex Risser). The following reagents were kindly provided by CAVD: X2988, ZM106.9, and 3817. We thank S. Tabruyn and F. Arbogast for their assistance with in vivo studies. We thank the SickKids-University Health Network Flow Cytometry Facility. This work wassupported by Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant 6280100058 (J.-P.J.) and by Operating Grant PJ4- 169662 from the Canadian Institutes of Health Research (CIHR; B.T. and J.-P.J.). This research was also supported by the European Union’s Horizon 2020 research and innovation program under Marie Sklodowska-Curie Grant 790012 (E.R.), a Hospital for Sick Children Restracomp Postdoctoral Fellowship (C.B.A.), an NSERC postgraduate doctoral scholarship (T.Z.), a predoctoral fel- lowship from the Basque Government (PRE_2019_2_0046) (S.I.), the Canadian Institute for Advanced Research (CIFAR) Azrieli Global Scholar program (J.-P.J.), the Ontario Early Researcher Awards program (J.-P.J.), and the CanadaResearch Chairs program (B.T. and J.-P.J.). This work was supported, in part, by NSERC Discovery Grant RGPIN-2019-06442 and CIHR Project Grant–Priority Announcement PJH-175379 to C.G., and a CIHR Canada Graduate Scholarship (CGS-M) to J.B. Further support was obtained from the Spanish Ministry of Sci- ence, Innovation and Universities (MCIU) with the support of the Spanish Research Agency/The European Regional Development Fund (AEI/FEDER) (RTI2018-095624-B-C21) (J.L.N.) and the Basque Government (IT1196-19) (J.L.N.). Biophysical data were collected at the Structural & Biophysical Core facility supported by the Canada Foundation for Innovation and Ontario Research Fun

Engineering pan-HIV-1 neutralization potency through multispecific antibody avidity

Deep mining of B cell repertoires of HIV-1–infected individuals has resulted in the isolation of dozens of HIV-1 broadly neutralizing antibodies (bNAbs). Yet, it remains uncertain whether any such bNAbs alone are sufficiently broad and potent to deploy therapeutically. Here, we engineered HIV-1 bNAbs for their combination on a single multispecific and avid molecule via direct genetic fusion of their Fab fragments to the human apoferritin light chain. The resulting molecule demonstrated a remarkable median IC50 value of 0.0009 µg/mL and 100% neutralization coverage of a broad HIV-1 pseudovirus panel (118 isolates) at a 4 µg/mL cutoff—a 32-fold enhancement in viral neutralization potency compared to a mixture of the corresponding HIV-1 bNAbs. Importantly, Fc incorporation on the molecule and engineering to modulate Fc receptor binding resulted in IgG-like bioavailability in vivo. This robust plug-and-play antibody design is relevant against indications where multispecificity and avidity are leveraged simultaneously to mediate optimal biological activity.This work was supported by Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant 6280100058 (J.-P.J.) and by Operating Grant PJ4-169662 from the Canadian Institutes of Health Research (CIHR; B.T. and J.-P.J.). This research was also supported by the European Union’s Horizon 2020 research and innovation program under Marie Sklodowska-Curie Grant 790012 (E.R.), a Hospital for Sick Children Restracomp Postdoctoral Fellowship (C.B.A.), an NSERC postgraduate doctoral scholarship (T.Z.), a predoctoral fellowship from the Basque Government (PRE_2019_2_0046) (S.I.), the Canadian Institute for Advanced Research (CIFAR) Azrieli Global Scholar program (J.-P.J.), the Ontario Early Researcher Awards program (J.-P.J.), and the Canada Research Chairs program (B.T. and J.-P.J.). This work was supported, in part, by NSERC Discovery Grant RGPIN-2019-06442 and CIHR Project Grant–Priority Announcement PJH-175379 to C.G., and a CIHR Canada Graduate Scholarship (CGS-M) to J.B. Further support was obtained from the Spanish Ministry of Science, Innovation and Universities (MCIU) with the support of the Spanish Research Agency/The European Regional Development Fund (AEI/FEDER) (RTI2018-095624-B-C21) (J.L.N.) and the Basque Government (IT1196-19) (J.L.N.). Biophysical data were collected at the Structural & Biophysical Core facility supported by the Canada Foundation for Innovation and Ontario Research Fund.Peer reviewe

A multi-specific, multi-affinity antibody platform neutralizes sarbecoviruses and confers protection against SARS-CoV-2 in vivo

© 2023 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution License 4.0 (CC BY). https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), has been responsible for a global pandemic. Monoclonal antibodies (mAbs) have been used as antiviral therapeutics; however, these therapeutics have been limited in efficacy by viral sequence variability in emerging variants of concern (VOCs) and in deployment by the need for high doses. In this study, we leveraged the multi-specific, multi-affinity antibody (Multabody, MB) platform, derived from the human apoferritin protomer, to enable the multimerization of antibody fragments. MBs were shown to be highly potent, neutralizing SARS-CoV-2 at lower concentrations than their corresponding mAb counterparts. In mice infected with SARS-CoV-2, a tri-specific MB targeting three regions within the SARS-CoV-2 receptor binding domain was protective at a 30-fold lower dose than a cocktail of the corresponding mAbs. Furthermore, we showed in vitro that mono-specific MBs potently neutralize SARS-CoV-2 VOCs by leveraging augmented avidity, even when corresponding mAbs lose their ability to neutralize potently, and that tri-specific MBs expanded the neutralization breadth beyond SARS-CoV-2 to other sarbecoviruses. Our work demonstrates how avidity and multi-specificity combined can be leveraged to confer protection and resilience against viral diversity that exceeds that of traditional monoclonal antibody therapies.This work was supportedby Natural Sciences and Engineering Research Council of Canada discovery grant 6280100058 (to J.-P.J.), operating grant PJ4-169662 from the Canadian Institutes of Health Research (CIHR; to B.T. and J.-P.J.), COVID-19 Research Fund C-094-2424972-JULIEN (to J.-P.J.) from the Province of Ontario Ministry of Colleges and Universities, the Bill and Melinda Gates Foundation INV-023398 (to J.-P.J.), and the Hospital for Sick Children Foundation. This research was also supported by Hospital for Sick Children Restracomp Postdoctoral Fellowships (to C.B.A.and I.K.), an Ontario Graduate Scholarship (OGS; to K.M.), a Banting Postdoctoral Fellowship (to C.B.A.), the CIFAR Azrieli Global Scholar program (to J.-P.J.), the Ontario Early Researcher Awards program (to J.-P.J.), and the Canada Research Chairs program (to J.L.R., B.T., and J.-P.J.). Cryo-EM data were collected at the Toronto High-Resolution High-Throughput cryo-EM facility, and biophysical data were collected at the Structural and Biophysical Core Facility, both supported by the Canada Foundation for Innovation and Ontario Research Fund. X-ray diffraction experiments were performed at GM/CA@APS,which has been funded in wholeor in part with federal funds from the National Cancer Institute (ACB-12002) and the National Institute of General Medical Sciences (AGM-12006). The EIGER16M detector at GM/CA-XSD was funded by NIH grant S10OD012289.This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science user facility operated for the U.S. DOE Office of Science by Argonne National Laboratory under contract DE-AC02-06CH11357.Peer reviewe

Greater Durability and Protection against Herpes Simplex Viral Disease following Immunization of Mice with Single-Cycle ΔgD-2 Compared to an Adjuvanted Glycoprotein D Protein Vaccine

Herpes simplex viruses (HSV) cause chronic infections with significant morbidity. Prior vaccines, designed to generate neutralizing antibodies (nAbs) targeting glycoprotein D (gD), failed to provide durable protection. We adopted a different strategy and evaluated a single-cycle virus deleted in gD (ΔgD-2). ΔgD-2elicits antibodies that primarily mediate antibody-dependent cell mediated cytolysis (ADCC) and provides complete protection against clinical isolates of HSV in multiple lethal mouse models. To assess durability, we vaccinated mice (2 doses administered intramuscularly) with ΔgD-2, adjuvanted recombinant gD-2 (rgD-2/Alum-MPL), or uninfected cells as a control, and quantified antibody responses over one year. Mice (n = 5/group) were lethally challenged at 2, 4, 6, 8, and 10-months post-boost. ΔgD-2-vaccinated mice elicited a durable ADCC-mediating response, which provided complete protection against challenge at all timepoints. In contrast, rgD-2/Alum-MPL elicited only nAbs, which declined significantly within 6 months, provided only partial protection at early timepoints, and no protection after 6 months. Serum sampling after viral challenge showed that infection elicited low levels of ADCC-mediating antibodies in rgD-2/Alum-MPL-vaccinated mice and boosted the nAb response, but only after 6 months. Conversely, infection significantly and consistently boosted both the ADCC and nAbs responses in ΔgD-2-vaccinated mice. Results recapitulate clinical trial outcomes with gD vaccines, highlight the importance of ADCC, and predict that ΔgD-2 will elicit durable responses in humans

Molecular and functional properties of human Plasmodium falciparum CSP C‐terminus antibodies

Abstract Human monoclonal antibodies (mAbs) against the central repeat and junction domain of Plasmodium falciparum circumsporozoite protein (PfCSP) have been studied extensively to guide malaria vaccine design compared to antibodies against the PfCSP C terminus. Here, we describe the molecular characteristics and protective potential of 73 germline and mutated human mAbs against the highly immunogenic PfCSP C‐terminal domain. Two mAbs recognized linear epitopes in the C‐terminal linker with sequence similarity to repeat and junction motifs, whereas all others targeted conformational epitopes in the α‐thrombospondin repeat (α‐TSR) domain. Specificity for the polymorphic Th2R/Th3R but not the conserved RII+/CS.T3 region in the α‐TSR was associated with IGHV3‐21/IGVL3‐21 or IGLV3‐1 gene usage. Although the C terminus specific mAbs showed signs of more efficient affinity maturation and class‐switching compared to anti‐repeat mAbs, live sporozoite binding and inhibitory activity was limited to a single C‐linker reactive mAb with cross‐reactivity to the central repeat and junction. The data provide novel insights in the human anti‐C‐linker and anti‐α‐TSR antibody response that support exclusion of the PfCSP C terminus from malaria vaccine designs

Multivalency transforms SARS-CoV-2 antibodies into ultrapotent neutralizers

Here, the authors combine three different antibody specificities and an Fc domain on a single multivalent molecule, resulting in high neutralization activity despite viral sequence variability