Affiliations: [a] Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA | [b] Panacea Pharmaceuticals Inc., Gaithersburg, MD 20877, USA | [c] Department of Medicine, Liver Research Center, Rhode Island Hospital and Brown Medical School, Providence, RI 02903, USA | [d] Departments of Chemical Engineering & Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
Correspondence:
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Corresponding author: The Massachusetts Institute of Technology, Department of Chemical Engineering & Biological Engineering Division, Building 66-552, 77 Massachusetts Avenue, Cambridge, MA 02139, USA. Tel.: +1 617 253 4578; Fax: +1 617 258 5766; E-mail: [email protected].
Note: [1] Supported by NSF biotechnology process engineering center (BPEC). Research conducted, in part, by the Clayton Foundation for Research.
Abstract: Over-expression of the enzyme human aspartyl (asparaginyl) β-hydroxylase (HAAH) has been detected in a variety of cancers. It is proposed that upon cellular transformation, HAAH is overexpressed and translocated to the tumor cell surface, rendering it a specific surface antigen for tumor cells. In this work, twelve human single-chain Fv fragments (scFv) against HAAH were isolated from a human non-immune scFv library displayed on the surface of yeast. Five of the twelve were reformatted as human IgG1. Two of the five IgGs, 6–22 and 6–23, showed significant binding to recombinant HAAH in ELISA, tumor cell lines, and tumor tissues. The apparent dissociation constants of 6–22 and 6–23 IgG were 1.0 ± 0.2 nM and 20 ± 10 nM respectively. These two antibodies were shown to target different domains of HAAH, with 6–22 targeting the catalytic domain of HAAH and 6–23 targeting the N-terminal non-catalytic domain of HAAH. 6–22 IgG was further characterized, as it had high affinity and targeted the catalytic domain. 6–22 IgG alone does not exhibit significant cytotoxicity toward the tumor cells. However, 6–22 internalizes into tumor cells and can therefore be employed to deliver cytotoxic moieties. A goat anti-human IgG-saporin conjugate was delivered into tumor cells by 6–22 IgG and hence elicited cytotoxicity toward the tumor cells in vitro. These tumor-binding human antibodies can potentially be used in both diagnosis and immunotherapy targeting HAAH-expressing tumor cells.
Keywords: Human aspartyl asparaginyl β-hydroxylase, human antibody, immunotoxin, internalization, targeting
