Construction, expression, and activities of L49-sFv-beta-lactamase, a single-chain antibody fusion protein for anticancer prodrug activation.
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.
Siemers
N O
NO
Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle, Washington 98121, USA.
Kerr
D E
DE
Yarnold
S
S
Stebbins
M R
MR
Vrudhula
V M
VM
Hellström
I
I
Hellström
K E
KE
Senter
P D
PD
D000818:Animals; D000970:Antineoplastic Agents / Q000493:pharmacokinetics*; D001711:Biotransformation; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D016972:Enterobacter cloacae / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007074:Immunoglobulin G / Q000235:genetics* / Q000494:pharmacology / Q000627:therapeutic use; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D009374:Neoplasms, Experimental / Q000628:therapy; D011355:Prodrugs / Q000493:pharmacokinetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000494:pharmacology / Q000627:therapeutic use; D014407:Tumor Cells, Cultured; D001618:beta-Lactamases / Q000235:genetics* / Q000494:pharmacology / Q000627:therapeutic use
D000970:Antineoplastic Agents; D007074:Immunoglobulin G; D011355:Prodrugs; D011993:Recombinant Fusion Proteins; C108272:L49-sFv-beta-lactamase; D001618:beta-Lactamases