Isolation and characterization of human antibodies targeting human aspartyl (asparaginyl) beta-hydroxylase.
Over-expression of the enzyme human aspartyl (asparaginyl) beta-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.
Yeung
Y A
YA
Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Finney
A H
AH
Koyrakh
I A
IA
Lebowitz
M S
MS
Ghanbari
H A
HA
Wands
J R
JR
Wittrup
K D
KD
D000595:Amino Acid Sequence; D000920:Antibody-Dependent Cell Cytotoxicity; D045744:Cell Line, Tumor; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007128:Immunoglobulin Fragments / Q000096:biosynthesis / Q000276:immunology* / Q000627:therapeutic use; D007074:Immunoglobulin G / Q000276:immunology*; D007150:Immunohistochemistry; D000922:Immunotoxins / Q000627:therapeutic use; D006899:Mixed Function Oxygenases / Q000276:immunology*; D008969:Molecular Sequence Data
D007128:Immunoglobulin Fragments; D007074:Immunoglobulin G; D000922:Immunotoxins; C055966:immunoglobulin Fv; D006899:Mixed Function Oxygenases; C058291:aspartic acid 2-oxoglutarate-dependent dioxygenase