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Principal Investigator: Mitchell Ho
Organization: DIVISION OF BASIC SCIENCES - NCI
Fiscal Year: 2023
Award: $502,510
Funding agency: National Cancer Institute
While antibody-based therapeutics have emerged as a major component in cancer treatment, the generation of antibodies to important targets such as cell surface receptors and ion channels remains difficult. These proteins contain buried functional sites that are often unreachable by classical mouse or human IgG-based antibodies. Single-domain antibodies have shown a promising ability to target difficult antigens and hidden epitopes. Dr. Mitchell Ho at the NCI has demonstrated that single-domain antibodies are capable of targeting buried functional sites in cancer signaling complexes [Feng et al. PNAS, 2013; Gao et al Nature Communications, 2015; Li et al. PNAS, 2017; Li et al. Hepatology, 2019]. The Ho lab has constructed large shark and camel single-domain ('nanobody') libraries and isolated binders to a wide range of antigens [Feng et al. 2013; Gao et al 2015; Li et al. 2017; Hong et al. 2022; Li et al. 2023; Buffington et al. 2023], indicating that the phage-displayed single domain antibody libraries can be a valuable source to isolate therapeutic antibodies. In FY23, the AEP published one research article [Buffington et al. FASEB Journal, 2023], one method paper [Zhang et al. Current Protocols, 2023], and one collaborative paper [Fayn et al. Journal Nuclear Medicine 2023]. SARS-CoV-2 is the etiological agent of the COVID-19 pandemic. Antibody-based therapeutics targeting the spike protein, specifically the S1 subunit or the receptor binding domain (RBD) of SARS-CoV-2, have gained attention due to their clinical efficacy in treating patients diagnosed with COVID-19. An alternative to conventional antibody therapeutics is the use of shark new antigen variable receptor domain (VNAR) antibodies. VNAR s are small (15 kDa) and can reach deep into the pockets or grooves of the target antigen. In FY2023, the AEP isolated 53 VNAR s that bind to the S2 subunit by phage panning from a naive nurse shark VNAR phage display library constructed and provided by Dr. Mitchell Ho's laboratory. Among those binders, S2A9 showed the best neutralization activity against the original pseudotyped SARS-CoV-2 virus. Several binders, including S2A9, showed cross-reactivity against S2 subunits from other beta coronaviruses. Furthermore, S2A9 showed neutralization activity against all variants of concern (VOCs) from alpha to omicron (including BA1, BA2, BA4, and BA5) in both pseudovirus and live virus neutralization assays. Our findings suggest that S2A9 could be a promising lead molecule for the development of broadly neutralizing antibodies against SARS-CoV-2 and emerging variants. The nurse shark VNAR phage library created by the Ho lab offers a novel platform that can be used to rapidly isolate single-domain antibodies against emerging viral pathogens. One critical obstacle to getting a shark VNAR single domain to work in the clinic is its potential immunogenicity in humans. The VNAR of shark single domain antibodies is evolutionarily distant from the variable regions (VH) of mammalian immunoglobulins, yet we hypothesize that it may still have complementarity-determining regions (CDRs) that are involved in antigen recognition, at least for the most part of the antigen recognition, therefore making it possible to humanize by grafting these CDRs to the framework of human VH homologs. For testing this hypothesis, we show the VNAR CDR based on an analysis of currently available VNAR -antigen structure complexes in the global Protein Data Bank archive of 3D structure data, and describe the detailed protocol to humanize VNAR by CDR grafting, using B6 (an anti-Pseudomonas exotoxin VNAR), the most common type (Type II) of shark VNAR s, as an example. We published the methods in Current Protocols (Buffington J, Duan Z, Kwon HJ, Hong J, Li D, Feng M, Xie H, Ho M. Identification of nurse shark VNAR single-domain antibodies targeting the spike S2 subunit of SARS-CoV-2. FASEB J. 2023 Jun;37(6):e22973. doi: 10.1096/fj.202202099RR.) so other scientists can also test our methodology. Ongoing efforts will further optimize the protocol for engineering shark VNARs for treating cancer and other human diseases. In FY2023, the AEP also published one research paper as co-authors in collaboration with Dr. Freddy Escorcia in Molecular Imaging Branch at the NCI CCR [Fayne et al. J Nucl Med 2023]. Ongoing AEP collaborations aim to develop therapeutic antibodies and engineer immune protein and cells with Dr. Ira Pastan, Dr. James Gulley (NCI), Dr. Glenn Merlino (NCI), Dr. Peter Kwong (NIAID), Dr. Eytan Ruppin (NCI) and Dr. Christian Hinrichs (Rutgers University).
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