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Principal Investigator: Mitchell Ho
Organization: DIVISION OF BASIC SCIENCES - NCI
Fiscal Year: 2020
Award: $582,613
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 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. Antibody Therapeutics, 2019], indicating that the phage-displayed single domain antibody libraries can be a valuable source to isolate therapeutic antibodies. In FY20, the AEP published two articles and collaborated in several other manuscripts in preparation. One is a review article that analyzes the shark library developed by the Ho lab [English et al. Antibody Therapeutics 2020; PMID: 32118195]. The other is a method article that describes the isolation of rabbit single domain antibodies for the cancer target B7-H3 (CD276) [Feng et al. Antibody Therapeutics 2020; PMID: 32166218]. The antigen binding variable domain (VNAR) of the shark immunoglobulin new antigen receptor (IgNAR) evolved approximately 500 million years ago and it is one of the smallest antibody fragments in the animal kingdom with sizes of 12-15 kDa. In the review article, the AEP evaluates the current knowledge of the shark VNAR single domain sequences and ongoing development of shark VNARs as research tools as well as potential therapeutics, in particular highlighting the recent next-generation sequencing analysis of 1.2 million shark VNAR sequences and construction of a large phage displayed shark VNAR library from six adult nurse sharks (Ginglymostoma cirratum) [English et al. Antibody Therapeutics 2020; PMID: 32118195]. The large phage-displayed VNAR single domain library covers all the four known VNAR types (Types I-IV) and many previously unknown types. Ongoing preclinical development will help define the utility of shark VNAR single domains as a potentially new family of drug candidates for treating cancer and other human diseases. Rabbit antibodies can recognize diverse epitopes, including those that are poorly immunogenic in mice and humans. The AEP has established a method to isolate rabbit VH single domain antibodies for potential cancer therapy [Feng et al. Antibody Therapeutics 2020; PMID: 32166218]. We immunized rabbits with recombinant human B7-H3 (CD276) protein, made a phage-displayed rabbit VH single domain library with a diversity of 109, and isolated two binders (A1 and B1) from phage panning. Both rabbit VH single domains exhibited antigen-dependent binding to B7-H3-positive tumor cell lines but not B7-H3 knockout tumor cell lines. Our study shows that protein immunization followed by phage display screening can be used to isolate rabbit single domain antibodies. The two single domain antibodies reported by the AEP may have potential applications in cancer immunotherapy. We are also supporting the development of neutralizing nanobodies against SARS-CoV-2.
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