title: Antitumor therapy mediated by 5-fluorocytosine and a recombinant fusion protein containing TSG-6 hyaluronan binding domain and yeast cytosine deaminase.
abstract: Matrix attachment therapy (MAT) is an enzyme prodrug strategy that targets hyaluronan in the tumor extracellular matrix to deliver a prodrug converting enzyme near the tumor cells. A recombinant fusion protein containing the hyaluronan binding domain of TSG-6 (Link) and yeast cytosine deaminase (CD) with an N-terminal His(x6) tag was constructed to test MAT on the C26 colon adenocarcinoma in Balb/c mice that were given 5-fluorocytosine (5-FC) in the drinking water. LinkCD was expressed in Escherichia coli and purified by metal-chelation affinity chromatography. The purified LinkCD fusion protein exhibits a K(m) of 0.33 mM and V(max) of 15 microM/min/microg for the conversion of 5-FC to 5-fluorouracil (5-FU). The duration of the enzyme activity for LinkCD was longer than that of CD enzyme at 37 degrees C: the fusion protein retained 20% of its initial enzyme activity after 24 h, and 12% after 48 h. The LinkCD fusion protein can bind to a hyaluronan oligomer (12-mer) at a K(D) of 55 microM at pH 7.4 and a K(D) of 5.32 microM at pH 6.0 measured using surface plasmon resonance (SPR). To evaluate the antitumor effect of LinkCD/5-FC combination therapy in vivo, mice received intratumoral injections of LinkCD on days 11 and 14 after C26 tumor implantation and the drinking water containing 10 mg/mL of 5-FC starting on day 11. To examine if the Link domain by itself was able to reduce tumor growth, we included treatment groups that received LinkCD without 5-FC and Link-mtCD (a functional mutant that lacks cytosine deaminase activity) with 5-FC. Animals that received LinkCD/5-FC treatment showed significant tumor size reduction and increased survival compared to the CD/5-FC treatment group. Treatment groups that were unable to produce 5-FU had no effect on the tumor growth despite receiving the fusion protein that contained the Link domain. The results indicate that a treatment regime consisting of a fusion protein containing the Link domain, the active CD enzyme, and the prodrug 5-FC is sufficient to produce an antitumor effect. Thus, the LinkCD fusion protein is an alternative to antibody-directed prodrug enzyme therapy (ADEPT) approaches for cancer treatment.
authors.affiliation: Department of Pharmaceutical Chemistry and Biopharmaceutical Sciences, University of California, San Francisco, California 94143-0912, USA.
authors.forename: Joshua I
authors.initials: JI
authors.lastname: Park
authors.forename: Limin
authors.initials: L
authors.lastname: Cao
authors.forename: Virginia M
authors.initials: VM
authors.lastname: Platt
authors.forename: Zhaohua
authors.initials: Z
authors.lastname: Huang
authors.forename: Robert A
authors.initials: RA
authors.lastname: Stull
authors.forename: Edward E
authors.initials: EE
authors.lastname: Dy
authors.forename: Jeffrey J
authors.initials: JJ
authors.lastname: Sperinde
authors.forename: Jennifer S
authors.initials: JS
authors.lastname: Yokoyama
authors.forename: Francis C
authors.initials: FC
authors.lastname: Szoka
mesh_terms: D000230:Adenocarcinoma / Q000188:drug therapy*; D000818:Animals; D003110:Colonic Neoplasms / Q000188:drug therapy*; D043525:Cytosine Deaminase / Q000235:genetics / Q000378:metabolism*; D005260:Female; D005437:Flucytosine / Q000008:administration & dosage / Q000378:metabolism* / Q000627:therapeutic use*; D005472:Fluorouracil / Q000378:metabolism; D006820:Hyaluronic Acid / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D051379:Mice; D008807:Mice, Inbred BALB C; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000008:administration & dosage / Q000737:chemistry / Q000378:metabolism* / Q000627:therapeutic use*; D020349:Surface Plasmon Resonance
chemical_list: D011993:Recombinant Fusion Proteins; D006820:Hyaluronic Acid; D005437:Flucytosine; D043525:Cytosine Deaminase; D005472:Fluorouracil