Production and characterization of a camelid single domain antibody-urease enzyme conjugate for the treatment of cancer.
A novel immunoconjugate (L-DOS47) was developed and characterized as a therapeutic agent for tumors expressing CEACAM6. The single domain antibody AFAIKL2, which targets CEACAM6, was expressed in the Escherichia coli BL21 (DE3) pT7-7 system. High purity urease (HPU) was extracted and purified from Jack bean meal. AFAIKL2 was activated using N-succinimidyl [4-iodoacetyl] aminobenzoate (SIAB) as the cross-linker and then conjugated to urease. The activation and conjugation reactions were controlled by altering pH. Under these conditions, the material ratio achieved conjugation ratios of 8-11 antibodies per urease molecule, the residual free urease content was practically negligible (<2%), and high purity (>95%) L-DOS47 conjugate was produced using only ultradiafiltration to remove unreacted antibody and hydrolyzed cross-linker. L-DOS47 was characterized by a panel of analytical techniques including SEC, IEC, Western blot, ELISA, and LC-MS(E) peptide mapping. As the antibody-urease conjugate ratio increased, a higher binding signal was observed. The specificity and cytotoxicity of L-DOS47 was confirmed by screening in four cell lines (BxPC-3, A549, MCF7, and CEACAM6-transfected H23). BxPC-3, a CEACAM6-expressing cell line was found to be most susceptible to L-DOS47. L-DOS47 is being investigated as a potential therapeutic agent in human phase I clinical studies for nonsmall cell lung cancer.
†Helix BioPharma Corporation, 3-305 Industrial Parkway South, Aurora, Ontario L4G 6X7 Canada.
Baomin
B
Tian
†Helix BioPharma Corporation, 3-305 Industrial Parkway South, Aurora, Ontario L4G 6X7 Canada.
Wah Yau
WY
Wong
‡Liquid Crystal Institute, Kent State University, Kent, Ohio 44242, United States.
Elda
E
Hegmann
†Helix BioPharma Corporation, 3-305 Industrial Parkway South, Aurora, Ontario L4G 6X7 Canada.
Kim
K
Gaspar
†Helix BioPharma Corporation, 3-305 Industrial Parkway South, Aurora, Ontario L4G 6X7 Canada.
Praveen
P
Kumar
†Helix BioPharma Corporation, 3-305 Industrial Parkway South, Aurora, Ontario L4G 6X7 Canada.
Heman
H
Chao
D000595:Amino Acid Sequence; D000818:Animals; D000970:Antineoplastic Agents / Q000737:chemistry / Q000627:therapeutic use*; D002161:Camelids, New World; D032449:Canavalia / Q000201:enzymology*; D045744:Cell Line, Tumor; D057487:Enzyme Therapy; D006801:Humans; D018796:Immunoconjugates / Q000737:chemistry / Q000627:therapeutic use*; D008297:Male; D008819:Mice, Nude; D008969:Molecular Sequence Data; D009369:Neoplasms / Q000188:drug therapy*; D061905:Single-Domain Antibodies / Q000737:chemistry / Q000627:therapeutic use*; D014510:Urease / Q000737:chemistry / Q000627:therapeutic use*
D000970:Antineoplastic Agents; D018796:Immunoconjugates; D061905:Single-Domain Antibodies; D014510:Urease