As the culmination of several years of experiments, we propose a complete amino-acid sequence for porcine pepsin, an enzyme containing 327 amino-acid residues in a single polypeptide chain. In the sequence determination, the enzyme was treated with cyanogen bromide. Five resulting fragments were purified. The amino-acid sequence of four of the fragments accounted for 290 residues. Because the structure of a 37-residue carboxyl-terminal fragment was already known, it wag not studied. The alignment of these fragments was determined from the sequence of methionyl-peptides we had previously reported. We also discovered the locations of activesite aspartyl residues, as well as the pairing of the three disulfide bridges. A minor component of commercial crystalline pepsin was found to contain two extra aminoacid residues, Ala-Leu-, at the amino-terminus of the molecule. This minor component was apparently derived from a different site of cleavage during the activation of porcine pepsinogen.Although pepsin (EC 4.3.3.1) was one of the first enzymes to be discovered and purified in crystalline form, its structurefunction relationship is understood only superficially. In recent years, workers have realized that the complete elucidation of the mechanism of action of an enzyme depends upon the detailed knowledge of the chemical and three-dimensional structure of its molecule. For this reason, we undertook a long-term study of the complete amino-acid sequence of porcine pepsin.A number of studies have contributed to our knowledge of the partial sequence of porcine pepsin. They include investigations of the amino-acid sequences at the regions near the carboxyl-terminus (1-4), the amino-terminus (5, 6), the three disulfide bonds (7), the tryptophanyl residues (8, 9), the phosphoseryl residue (7, 10), two separate active-site aspartyl residues (11,12), and a number of small peptides (13,14). We have confirmed most of these previous findings and now present the complete amino-acid sequence of this enzyme. METHODSPorcine pepsin (three-times crystallized) was obtained from Worthington. The enzyme was either alkali-denatured (7) or reduced and aminoethylated (15) and subjected to cyanogenbromide cleavage (16). Preliminary experiments indicated that at room temperature one of the methionyl bonds (MetThr at residues 80-81) was cleaved less than 5% by cyanogen * Present address:
CPT-11 is a clinically important prodrug that requires conversion into the active metabolite SN-38, a potent topoisomerase I poison, for antitumor activity. However, SN-38 is rapidly metabolized to the inactive SN-38 glucuronide (SN-38G) in the liver, which reduces the amount of SN-38 available for killing cancer cells. Here, we investigated if local expression of b-glucuronidase (bG) on cancer cells to catalytically convert SN38G to SN38 could enhance the antitumor activity of CPT-11. bG was tethered on the plasma membrane of three different human cancer cell lines: human colon carcinoma (LS174T), lung adenocarcinoma (CL1-5) and bladder carcinoma (EJ). Surface b-glucuronidase-expressing cells were 20 to 80-fold more sensitive to SN-38G than the parental cells. Intravenous CPT-11 produced significantly greater suppression of CL1-5 and LS174 T tumors that expressed bG as compared with unmodified tumors. Furthermore, an adenoviral vector expressing membrane-tethered bG (Ad.bG) increased the sensitivity of cancer cells to SN-38G even at multiplicity of infections as low as 0.16, indicating bystander killing of non-transduced cancer cells. Importantly, intratumoral injection of Ad.bG significantly enhanced the in vivo antitumor activity of CPT-11 as compared with treatment with CPT-11 or Ad vectors alone. This study shows that Ad.bG has potential to boost the therapeutic index of CPT-11.
Antibody-directed enzyme prodrug therapy (ADEPT) utilizing β-glucuronidase is a promising method to enhance the therapeutic index of cancer chemotherapy. In this approach, an immunoenzyme (antibody-β-glucuronidase fusion protein) is employed to selectively activate anticancer glucuronide prodrugs in the tumor microenvironment. A major roadblock to the clinical translation of this therapeutic strategy, however, is the low enzymatic activity and strong immunogenicity of the current generation of immunoenzymes. To overcome this problem, we fused a humanized single-chain antibody (scFv) of mAb CC49 to S2, a human β-glucuronidase (hβG) variant that displays enhanced catalytic activity for prodrug hydrolysis. Here, we show that hcc49-S2 displayed 100-fold greater binding avidity than hcc49 scFv, possessed greater enzymatic activity than wild-type hβG, and more effectively killed antigen-positive cancer cells exposed to an anticancer glucuronide prodrug as compared to an analogous hβG immunoenzyme. Treatment of tumor-bearing mice with hcc49-S2 followed by prodrug significantly delayed tumor growth as compared to hcc49-hβG. Our study shows that hcc49-S2 is a promising targeted enzyme for cancer treatment and demonstrates that enhancement of human enzyme catalytic activity is a powerful approach to improve immunoenzyme efficacy.
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