title: Identification of high-affinity anti-CD16A allotype-independent human antibody domains.
abstract: CD16A (FcγRIIIA) is an activating receptor mostly expressed on natural killer (NK) cells and monocytes/macrophages. It can mediate antibody-dependent cell-mediated cytotoxicity (ADCC) through low-affinity interaction with human immunoglobulin G (IgG) Fc. It can also mediate cell lysis if NK cells are guided by bispecific killer cells engagers (BiKEs). BiKEs showed some success in clinical trials of cancer and are promising candidate therapeutics. However, currently reported BiKEs are based on antibody fragments (scFvs) of relatively large size. The CD16A-specific antibodies are also typically from animal origin. Decreasing the BiKE size could result in enhanced penetration into solid tumor and normal tissues, and using fully human antibodies could decrease the likelihood of immunogenicity. Here we report the identification and characterization of two antibody domains, D6 and E11, isolated from a very large human VH antibody domain library displayed on phage. D6 and E11 bound CD16A with EC50of 4nM and 8nM, respectively, but not other Fc gamma receptors (FcγRs) such as CD64 (FcγRI), CD32 (FcγRII) and CD16B (FcγRIIIB). They bound to both CD16A allotypes (158F,V) with equal affinity and competed with each other as well as with human IgG1 and the mouse anti-CD16A antibody 3G8. These and other results were used to build a molecular docking model predicting that D6 and E11 may bind to the CD16A membrane proximal D2 domain by interacting with its BC, C'E and EF loops. Importantly, cross-linked (bivalent) D6 and E11 induced secretion of IL-2 after binding to CD16A-expressing Jurkat T cells. The small size of these antibody domains combined with their high-affinity, specific, allotype-independent, activating interactions with CD16A could allow generation of novel highly effective BiKEs and other candidate protein therapeutics.
authors.affiliation: Protein Interactions Section, Cancer and Inflammation Program, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD 21702, USA. Electronic address: wei.li3@nih.gov.
authors.forename: Wei
authors.initials: W
authors.lastname: Li
authors.affiliation: Palisades Charter High School, 15777 Bowdoin St, Pacific Palisades, CA 90272, USA.
authors.forename: Hongjia
authors.initials: H
authors.lastname: Yang
authors.affiliation: Protein Interactions Section, Cancer and Inflammation Program, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD 21702, USA. Electronic address: dimitrdi@mail.nih.gov.
authors.forename: Dimiter S
authors.initials: DS
authors.lastname: Dimitrov
mesh_terms: D000915:Antibody Affinity / Q000276:immunology*; D000918:Antibody Specificity / Q000276:immunology; D002462:Cell Membrane / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D007126:Immunoglobulin Allotypes / Q000737:chemistry* / Q000276:immunology*; D007376:Interleukin-2 / Q000378:metabolism; D019169:Jurkat Cells; D007694:Killer Cells, Natural / Q000276:immunology; D008213:Lymphocyte Activation; D062105:Molecular Docking Simulation; D011485:Protein Binding; D000072417:Protein Domains; D017452:Receptors, IgG / Q000276:immunology*; D011994:Recombinant Proteins / Q000378:metabolism; D013601:T-Lymphocytes / Q000276:immunology
chemical_list: D000939:Epitopes; C496124:FCGR3A protein, human; D007126:Immunoglobulin Allotypes; D007376:Interleukin-2; D017452:Receptors, IgG; D011994:Recombinant Proteins
keywords: ADCC; Antibody domains; BiKE; CD16A; Natural killer (NK) cells