Construction, expression, and activities of L49-sFv-beta-lactamase, a single-chain antibody fusion protein for anticancer prodrug activation

Bioconjug Chem. Jul-Aug 1997;8(4):510-9. doi: 10.1021/bc9700751.

Abstract

The L49 (IgG1) monoclonal antibody binds to p97 (melanotransferrin), a tumor-selective antigen that is expressed on human melanomas and carcinomas. A recombinant fusion protein, L49-sFv-bL, that contains the antibody binding regions of L49 fused to the Enterobacter cloacae r2-1 beta-lactamase (bL) was constructed, expressed, and purified to homogeneity in an Escherichia coli soluble expression system. The variable regions of L49 were cloned by reverse transcription-polymerase chain reaction from L49 hybridoma mRNA using signal sequence and constant region primers. Construction of the gene encoding L49-sFv-bL was accomplished by hybridization insertion of VH, VL, and sFv linker sequences onto a pET phagemid template containing the bL gene fused to the pelB leader sequence. Optimal soluble expression of L49-sFv-bL in E. coli was found to take place at 23 degrees C with 50 microM isopropyl beta-D-thiogalactopyranoside induction and the use of the nonionic detergent Nonidet P-40 for isolation from the bacteria. Construction and expression of a soluble form of the p97 antigen in Chinese hamster ovary cells allowed affinity-based methods for analysis and purification of the fusion protein. Surface plasmon resonance, fluorescent activated cell sorting, and Michaelis-Menten kinetic analyses showed that L49-sFv-bL retained the antigen binding capability of monovalent L49 as well as the enzymatic activity of bL. In vitro experiments demonstrated that L49-sFv-bL bound to 3677 melanoma cells expressing the p97 antigen and effected the activation of 7-(4-carboxybutanamido)cephalosporin mustard (CCM), a cephalosporin nitrogen mustard prodrug. On the basis of these results, L49-sFv-bL was injected into nude mice with subcutaneous 3677 tumors, and localization was determined by measuring bL activity. Tumor to blood conjugate ratios of 13 and 150 were obtained 4 and 48 h post conjugate administration, respectively, and the tumor to liver, spleen, and kidney ratios were even higher. A chemically produced L49-Fab'-bL conjugate yielded a much lower tumor to blood ratio (5.6 at 72 h post administration) than L49-sFv-bL. Therapy experiments established that well-tolerated doses of L49-sFv-bL/CCM combinations resulted in cures of 3677 tumors in nude mice. The favorable pharmacokinetic properties of L49-sFv-bL allowed prodrug treatment to be initiated 12 h after the conjugate was administered. Thus, L49-sFv-bL appears to have promising characteristics for site-selective anticancer prodrug activation.

MeSH terms

  • Animals
  • Antineoplastic Agents / pharmacokinetics*
  • Biotransformation
  • CHO Cells
  • Cloning, Molecular
  • Cricetinae
  • Enterobacter cloacae / genetics
  • Escherichia coli / genetics
  • Humans
  • Immunoglobulin G / genetics*
  • Immunoglobulin G / pharmacology
  • Immunoglobulin G / therapeutic use
  • Mice
  • Mice, Inbred BALB C
  • Mice, Nude
  • Neoplasms, Experimental / therapy
  • Prodrugs / pharmacokinetics*
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / pharmacology
  • Recombinant Fusion Proteins / therapeutic use
  • Tumor Cells, Cultured
  • beta-Lactamases / genetics*
  • beta-Lactamases / pharmacology
  • beta-Lactamases / therapeutic use

Substances

  • Antineoplastic Agents
  • Immunoglobulin G
  • Prodrugs
  • Recombinant Fusion Proteins
  • L49-sFv-beta-lactamase
  • beta-Lactamases