Tumor Targeting and Prodrug Lab

Prodrug References

Huennekens-FM. Development of methotrexate alpha-peptides as prodrugs for activation by enzyme-monoclonal antibody conjugates. Adv-Enzyme-Regul. 1997; 37: 77-92

Selective delivery of lethal concentrations of drugs to tumors, allowing the latter to be eradicated without damage to other tissues, continues to be a major goal in cancer chemotherapy. Prodrugs (i.e. drugs that have been derivatized to prevent uptake into cells or interaction with targets), activated by enzyme-monoclonal antibody conjugates positioned at tumor sites, offer promise for achieving this objective. Methotrexate alpha-peptides (derivatives in which an amino acid is linked to the alpha-carboxyl group of the glutamate moiety) are ideal prodrugs, since they are not transported into cells and can be converted to the parent drug by carboxypeptidases. The L,L-diastereomer of MTX-alpha-Phe, synthesized in good yield by treatment of the p-nitrophenyl ester of 4-amino-4-deoxy-10-methylpteroic acid with Glu-alpha-Phe, was hydrolyzed readily by carboxypeptidase A (CP-A). Conjugate was prepared by derivatizing the enzyme and monoclonal antibody KS1/4 with linkers containing maleimide and sulfhydryl groups, respectively; interaction of these groups to form a stable thioether bond joined the proteins. When administered in vitro to UCLA-P3 human lung adenocarcinoma cells (ca. 5 x 10(4) antibody binding sites/cell) that had been pre-treated with the conjugate (whose antibody KS1/4 is targeted to these cells), and excess conjugate removed by extensive washing, MTX-Phe (ID50 = 6.3 x 10(-8) M) approached the toxicity of MTX (ID50 = 4.5 x 10(-8) M). In the absence of conjugate, MTX-Phe was much less toxic (ID50 = 2.2 x 10(-6) M).

Chen-BM; Chan-LY; Wang-SM; Wu-MF; Chern-JW; Roffler-SR. Cure of malignant ascites and generation of protective immunity by monoclonal antibody-targeted activation of a glucuronide prodrug in rats. Int-J-Cancer. 1997 Nov 4; 73(3): 392-402

We examined the in vivo efficacy of targeting beta-glucuronidase (betaG) to activate a glucuronide prodrug (BHAMG) of p-hydroxyaniline mustard (pHAM) at hepatoma ascites in Sprague-Dawley rats. Injection i.p. of 500 microg RH1-betaG, a conjugate formed between recombinant betaG and monoclonal antibody RH1 with specificity for an antigen expressed on AS-30D rat hepatoma cells, into rats bearing AS-30D ascites resulted in the accumulation of 54 microg conjugate per 10(9) tumor cells after 2 hr. Ascites fluid and serum contained 0.53 and 0 microg/ml, respectively, RH1-betaG 2 hr after injection of the conjugate. Conjugate binding to AS-30D cells was heterogeneous and non-saturated, as determined by flow cytometry. BHAMG was less toxic than pHAM to SD rats based on measures of animal mortality, weight loss and hematological toxicity. Treatment of rats bearing established hepatoma ascites with 500 microg RH1-betaG followed 2 hr later with a single i.p. injection of 30 mg/kg BHAMG or 3 i.p. injections of 10 mg/kg BHAMG 2, 3 and 4 hr later resulted in the cure of 6/8 and 8/8 animals, respectively. Treatment with BHAMG or pHAM alone did not produce cures, whereas treatment with a control antibody-betaG conjugate and BHAMG produced significantly greater hematological toxicity compared to treatment with RH1-betaG and BHAMG. All cured rats were completely protected from rechallenge with 2 x 10(7) AS-30D cells, indicating that successful treatment of animals induced protective immunity.

Haisma-HJ; Brakenhoff-RH; v.d.-Meulen-Muileman-I; Pinedo-HM; Boven-E. Construction and characterization of a fusion protein of single-chain anti-carcinoma antibody 323/A3 and human beta-glucuronidase. Cancer-Immunol-Immunother. 1998 Jan; 45(5): 266-72

We report the construction and expression of a fusion protein between a single-chain antibody specific for human carcinomas and human beta-glucuronidase by recombinant DNA technology. The sequences encoding the murine monoclonal antibody 323/A3 light- and heavy-chain variable genes were joined by a synthetic sequence encoding a 15-amino-acid linker and combined with human beta-glucuronidase by a synthetic sequence encoding a 6-amino-acid linker. The construct was placed under the control of the cytomegalovirus promotor and expressed in COS-7 cells. The yield of active fusion protein was 10 ng/ml transfectoma supernatant. Antibody affinity, antibody specificity and enzyme activity were fully retained by the fusion protein. Biochemical characterization of the fusion protein by sodium dodecyl sulfate/polyacrylamide gel electrophoresis showed a molecular mass of 100 kDa under denaturing conditions. Gel-filtration analysis indicated that the enzymatically active form is a tetramer of approximately 400 kDa. The non-toxic prodrug N-[4-doxorubicin-N-carbonyl(oxymethyl)phenyl]-O-beta-glucuronyl carbamate was activated to the cytotoxic drug doxorubicin by the fusion protein with a hydrolysis rate similar to that of human beta-glucuronidase. The growth inhibition of tumor cells coated with the fusion protein and exposed to prodrug was similar to that obtained with doxorubicin. This study shows the feasibility of constructing eukaryotic fusion proteins consisting of a single-chain antibody and human beta-glucuronidase for use in the specific activation of anticancer prodrugs.

Bagshawe-KD. Antibody-directed enzyme prodrug therapy for cancer: its theoretical basis and application. Mol-Med-Today. 1995 Dec; 1(9): 424-31

Agents that can be administered systemically but that act selectively against cancer cells have been intensively sought but have thus far proved elusive. Nonselective cytotoxic drugs have the potential to eradicate cancer if they can be delivered selectively in sufficient concentration to cancer sites. In the approach described here, the cytotoxic agent is generated at cancer sites from a low-toxicity prodrug by the action of an enzyme delivered by an antibody to the cancer site. The feasibility of the approach has been demonstrated with a variety of enzyme-prodrug combinations.

Rodrigues-ML; Carter-P; Wirth-C; Mullins-S; Lee-A; Blackburn-BK. Synthesis and beta-lactamase-mediated activation of a cephalosporin-taxol prodrug. Chem-Biol. 1995 Apr; 2(4): 223-7

BACKGROUND: Enzyme-activatable prodrugs in conjunction with antibody-enzyme fusion proteins may enhance the anti-tumor efficacy of antibodies and reduce the toxic side effects of conventional chemotherapeutics. Cephalosporins have proven to be highly versatile triggers for the enzymatic activation of such prodrugs. RESULTS: A cephem prodrug of taxol (PROTAX) was synthesized by substituting the C-3' position of cephalothin with 2'-(gamma-aminobutyryl) taxol. Hydrolysis of PROTAX by beta-lactamase rapidly released 2'-(gamma-aminobutyryl) taxol (kcat/K(M) = (1.4 +/- 0.1) x 10(5) s-1 M-1), which yielded taxol following intramolecular displacement. PROTAX is inactive in a microtubule assembly assay in vitro but has similar activity to taxol following prolonged activation with beta-lactamase. PROTAX is approximately 10-fold less toxic than taxol against SK-BR-3 breast tumor cells in vitro but has activity approaching that of taxol following prolonged activation with a fusion protein comprising beta-lactamase fused to a tumor-targeting antibody fragment. CONCLUSIONS: Tubulin polymerization activity is abolished and cytotoxicity is reduced in the PROTAX prodrug compared to taxol. Activation of PROTAX by beta-lactamase followed by self-immolation restores the activity of PROTAX to that of free taxol.

Siim-BG; Denny-WA; Wilson-WR. Nitro reduction as an electronic switch for bioreductive drug activation. Oncol-Res. 1997; 9(6-7): 357-69

It is well known that the reduction of aromatic nitro groups can give rise to toxic species, and that net nitro reduction by one-electron reductases can usually be inhibited by oxygen. There has been much interest in utilizing this biotransformation to activate drugs in hypoxic regions of tumors, but no clinically useful compound has yet resulted. Nitroreductive activation of prodrugs by oxygen-insensitive (and oxygen-sensitive) reductases is also of current interest because of new methods for introducing specific nitroreductases into tumors (e.g., as antibody-enzyme conjugates or by gene therapy). In most of the compounds investigated previously, cytotoxicity appears to be due to reactive nitroso or hydroxylamine reduction products arising from the nitro group itself. It is argued that there is greater scope for designing potent and selective nitro compounds by using the nitro group as an electronic switch to activate a latent reactive moiety elsewhere in the molecule. Examples of this approach include the nitro(hetero)aromatic mustards (e.g., SN 23816, NSC 646394) in which the nitro group controls the reactivity of a nitrogen mustard to which it is directly conjugated, and the nitro(hetero)aromatic methylquaternary (NMQ) mustards (e.g., SN 25341, NSC 658926) in which reduction of the nitro group triggers fragmentation of the molecule to release a reactive aliphatic nitrogen mustard. Many of these compounds show very high selectivity for hypoxic cells in culture. Some are also active against hypoxic cells in tumors, and provide large tumor growth delays when combined with tumor blood flow inhibitors such as 5,6-dimethylxanthenone-4-acetic acid (DMXAA). These prodrug designs also have potential for releasing effectors other than nitrogen mustards, which opens up many possibilities for use of nitro compounds as tumor-selective prodrugs.

Bakina-E; Wu-Z; Rosenblum-M; Farquhar-D. Intensely cytotoxic anthracycline prodrugs: glucuronides. J-Med-Chem. 1997 Dec 5; 40(25): 4013-8

We previously reported the synthesis of a series of doxorubicin analogue prodrugs that give rise to intensely cytotoxic metabolites in the presence of carboxylate esterases. We now report studies on structurally related beta-glucuronide prodrugs that are converted to similar potent metabolites in the presence of beta-glucuronidases. These prodrugs were prepared by reductive condensation of daunomycin or doxorubicin with methyl 1-O-[(1'RS)-1'-ethoxy-4'-oxobutyl]-2,3,4-tri-O-acetyl-beta-D- glucopyranosyluronate in the presence of sodium cyanoborohydride followed by base-mediated cleavage of the glucuronate protective groups. The doxorubicin derivatives were isolated in very low yield, most likely because of the inherent base lability of the parent aglycone. By contrast, fairly good yields of the more base-stable daunomycin analogues were obtained. The target daunomycin glucuronide, N-[(4"RS)-4"-ethoxy-4"-(sodium 1"'-O-beta-D-glucopyranuronate)butyl]daunorubicin (6a), had a half-life of 30 h when incubated at a concentration of 12 microM in aqueous 0.05 M phosphate buffer, pH 7.4, at 37 degrees C. Under identical conditions in the presence of 197 units/mumol of Escherichia coli beta-glucuronidase, 6a was hydrolyzed with a half-life of 1.7 h. The single metabolite observed was chromatographically identical with that formed from the hydrolysis of N-(4,4-diacetoxybut-1-yl)daunomycin by carboxylate esterases. 6a was approximately 10,000-fold more toxic to human A375 melanoma cells in the presence of E. coli beta-glucuronidase than in the absence of the enzyme. These findings indicate the therapeutic potential of anthracycline glucuronide prodrugs as independent entities or four use in conjunction with enzyme tissue-targeting strategies such as antibody-directed enzyme prodrug therapy (ADEPT) or gene-directed enzyme prodrug therapy (GDEPT).

Siemers-NO; Kerr-DE; Yarnold-S; Stebbins-MR; Vrudhula-VM; Hellstrom-I; Hellstrom-KE; Senter-PD. Construction, expression, and activities of L49-sFv-beta-lactamase, a single-chain antibody fusion protein for anticancer prodrug activation. Bioconjug-Chem. 1997 Jul-Aug; 8(4): 510-9

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.

Cheng-TL; Chen-BM; Chan-LY; Wu-PY; Chern-JW; Roffler-SR. Poly(ethylene glycol) modification of beta-glucuronidase-antibody conjugates for solid-tumor therapy by targeted activation of glucuronide prodrugs. Cancer-Immunol-Immunother. 1997 Aug; 44(6): 305-15

Methoxypoly(ethylene glycol) (PEG) modification of Escherichia coli beta-glucuronidase (betaG) was examined as a method to improve the stability and pharmacokinetics of antibody-betaG conjugates for the targeted activation of glucuronide prodrugs at tumor cells. Introduction of 3 PEG molecules did not affect betaG activity whereas higher degrees of PEG modification produced progressively greater loss of enzymatic activity. The enzyme was found to be stable in serum regardless of PEG modification. PEG-modified betaG was coupled via a thioether bond to mAb RH1, an IgG2a antibody that binds to the surface of AS-30D hepatoma cells, to produce conjugates with 3 (RH1-betaG-3PEG), 5.2 (RH1-betaG-5PEG) or 9.8 (RH1-betaG-10PEG) PEG molecules per betaG with retention of 75%, 45% and 40% of the combined antigen-binding and enzymatic activity of the unmodified conjugate RH1-betaG. In contrast to the rapid serum clearance of RH1-betaG observed in mice, the PEG-modified conjugates displayed extended serum half-lives. RH1-betaG-3PEG and RH1-betaG-5PEG also exhibited reduced spleen uptake and greater tumor accumulation than RH1-betaG. BHAMG, the glucuronide prodrug of p-hydroxyaniline mustard (pHAM), was relatively nontoxic in vivo. Injection of 6 mg/kg or 12 mg/kg pHAM i.v. depressed white blood cell numbers by 46% and 71% whereas 80 mg/kg BHAMG reduced these levels by 22%. Although the tumor/blood ratio of RH1-betaG-5PEG was adversely affected by slow clearance from serum, combined therapy of small solid hepatoma tumors with this conjugate, followed 4 and 5 days later with i.v. injections of BHAMG, cured all of seven mice with severe combined immunodeficiency. Combined treatment with a control antibody-betaG conjugate and BHAMG delayed tumor growth and cured two of six mice while treatment with pHAM or BHAMG alone was ineffective.

Vrudhula-VM; Svensson-HP; Senter-PD. Immunologically specific activation of a cephalosporin derivative of mitomycin C by monoclonal antibody beta-lactamase conjugates. J-Med-Chem. 1997 Aug 15; 40(17): 2788-92

The syntheses of two cephalosporin derivatives 2 and 3 of mitomycin C (1) containing 7-phenylacetamido and 7-delta-carboxybutanamido side chains, respectively, are described. These compounds were prepared for evaluation as cephalosporin prodrugs capable of being activated by mAb-beta-lactamase conjugates. In vitro cytotoxicity assays performed on H2987 lung adenocarcinoma and clone 62 melanoma cell lines indicated that compound 2 was comparable in cytotoxicity to the parent drug. In an effort to improve upon the cytotoxic differential of 2, an alternative prodrug 3 containing a polar carboxyl group in the side chain of the cephalosporin moiety was prepared. Compound 3 consistently behaved as a prodrug and was approximately 40- and 10-fold less toxic than 1 toward H2987 and clone 62, respectively. Determination of kinetic constants for hydrolysis by beta-lactamase from Enterobacter cloacae P99 indicated kcat values of 476 +/- 170 and 248 +/- 15.1 s-1 for 2 and 3, respectively. The kcat/Km ratios for 2 and 3 were found to be approximately 9.7 and 2.1 microM/s, respectively. Comparison of these kcat/Km values with those obtained for similar cephalosporin derivatives of other antitumor agents demonstrated that compounds with delta-carboxybutanamido side chains generally have slightly diminished efficiency of enzymatic hydrolysis compared to the corresponding 7-phenylacetamido analog. It was also demonstrated that the less toxic prodrug 3 was activated in an immunologically specific manner by L6-F(ab')-beta-lactamase and 96.5-F(ab')-beta-lactamase conjugates, selective for H2987 and clone 62 cells, respectively.

Crosasso-P; Brusa-P; Dosio-F; Arpicco-S; Pacchioni-D; Schuber-F; Cattel-L. Antitumoral activity of liposomes and immunoliposomes containing 5-fluorouridine prodrugs. J-Pharm-Sci. 1997 Jul; 86(7): 832-9

Liposomes and immunoliposomes containing cytotoxic agents may be highly efficacious in intracavity therapy of malignancies confined principally to the peritoneal cavity. To assess the feasibility of this locoregional treatment, we prepared two derivatives of 5-fluorouridine (5-FUR), a highly cytotoxic metabolite of 5-fluorouracile, and incorporated them into REV liposomes, prepared with the reverse phase evaporation method. Encapsulation efficiency, drug leakage, and stability were determined, and size analysis and differential scanning calorimetry were carried out to evaluate the drug delivery potential of liposomes containing 5'-palmitoyl-5-FUR, 5'-succinyl-5-FUR, or the parent drug 5-FUR. The most suitable drug for encapsulation, in terms of minimum leakage and encapsulation efficiency, was 5'-palmitoyl-5-FUR, which differential scanning calorimetry indicated as being firmly anchored to the lipid bilayer. Thus, 5'-palmitoyl-5-FUR was chosen to prepare a chemotherapeutic liposome-monoclonal antibody conjugate (immunoliposome). The covalent linkage between antibody and liposome was realized by coupling the thiolated monoclonal antibody AR-3 with REV liposomes, containing N-[4-(p-maleimidophenyl)butyryl]phosphatidylethanolamine. The cytotoxic activity of drug-bearing liposomes and immunoliposomes was evaluated on the HT-29 human colon adenocarcinoma cell line; the immunoliposomes had higher cytotoxicity than liposomes or 5-FUR. To explore the potential of these drug formulations in anticancer therapy, we ip injected liposomes or immunoliposomes into athymic mice ip grafted with human HT-29 cell line. In this mouse model, the immunoliposome containing 5'-palmitoyl-5-FUR displayed the best antitumoral activity, since on day 27 postgraft only 5% of residual tumor mass was present, compared to control mice; there was a close relationship between exposure time of tumor tissue to the drug and antitumor potency.

Stribbling-SM; Martin-J; Pedley-RB; Boden-JA; Sharma-SK; Springer-CJ. Biodistribution of an antibody-enzyme conjugate for antibody-directed enzyme prodrug therapy in nude mice bearing a human colon adenocarcinoma xenograft. Cancer-Chemother-Pharmacol. 1997; 40(4): 277-84

The enzyme carboxypeptidase G2 (CPG2) can be targeted to tumors by antibodies and used to activate prodrugs in a treatment called antibody-directed enzyme prodrug therapy (ADEPT). Different doses of CPG2 conjugated to the anti-CEA antibody A5B7 were administered i.v. to nude mice bearing the LS174T human colon adenocarcinoma xenograft, and the biodistribution of conjugate activity 48 and 72 h later was determined using a novel high-performance liquid chromatography (HPLC) method. Conjugate doses of 2,500 and 625 U/kg gave tumor enzyme levels of 0.5-0.6 U/g. Lower doses of 300 and 150 U/kg gave tumor enzyme levels of 0.1-0.3 U/g. Intriguingly, the best tumor:blood ratio of conjugate activity at both 48 and 72 h was achieved after administration of the 625-U/kg dose, not the 2,500-U/kg dose. After 48 h this ratio was 3.8, whereas after 72 h the value was 5.5. This conjugate dose also gave the greatest tumor:tissue ratios in all other tissues examined. After 72 h the tumor:colon ratio was 105, whereas the tumor:kidney ratio was 36. In ADEPT, to obtain maximal tumor damage to LS174T xenografts in nude mice with minimal systemic toxicity using the A5B7-CPG2 conjugate, prodrug should therefore be administered at least 72 h after a conjugate dose of 625 U/kg.

Martin-J; Stribbling-SM; Poon-GK; Begent-RH; Napier-M; Sharma-SK; Springer-CJ. Antibody-directed enzyme prodrug therapy: pharmacokinetics and plasma levels of prodrug and drug in a phase I clinical trial. Cancer-Chemother-Pharmacol. 1997; 40(3): 189-201

Antibody-directed enzyme prodrug therapy (ADEPT) was administered to ten patients in a phase I clinical trial. The aim was to measure plasma levels of the prodrug 4-[(2-chloroethyl)(2-mesyloxyethyl) amino] benzoyl-L-glutamic acid (CMDA) and the bifunctional alkylating drug (CJS11) released from it by the action of tumour-localised carboxypeptidase G2 (CPG2) enzyme. New techniques were developed to extract the prodrug and drug from plasma by solid-phase absorption and elution and to measure CPG2 activity in plasma and tissue. All extracts were analysed by high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS). CPG2 activity was found in metastatic tumour biopsies but not in normal tissue, indicating that localisation had been successful. The clearing agent SB43-gal, given at 46.5 mg/m2, achieved the aim of clearing non-tumour-localised enzyme in the circulation, indicating that conversion of prodrug to drug could take place only at the site of localised conjugate. Plasma prodrug did not always remain above its required threshold of 3 microM for the "therapeutic window" of 120 min after dosing, but the presence of residual prodrug after the first administration of each day indicated that this could be achieved during the remaining four doses over the following 8 h. Despite considerable inter-patient prodrug plasma concentration variability, the elimination half-life of the prodrug was remarkably reproducible at 18 +/- 8 min. Rapid appearance of the drug in plasma indicated that successful conversion from the prodrug had taken place, but also undesirable leakback from the site of localisation into the bloodstream. However, drug plasma levels fell rapidly by at least 50% at between 10 and 60 min with a half-life of 36 +/- 14 min. Analysis of the plasma extracts by LC/MS indicated that this technique might be used to confirm qualitatively the presence of prodrug, drug and their metabolites.

Rihova-B. Targeting of drugs to cell surface receptors. Crit-Rev-Biotechnol. 1997; 17(2): 149-69

The new approach to the treatment of cancer or to immunomodulation is drug targeting. The effort to achieve either an absolute or a relative amplification of the tumoricidal effect of anticancer drugs through increased generation or acquisition of reactive molecules at the tumor site or a reduction of the toxic molecules available to the periphery has led to a number of strategies. Among them are (1) targeting using antibodies to their fragments, hormones, carbohydrates, and growth factors; (2) retargeting using bispecific antibodies; (3) construction of chimeric genes; (4) streptavidin-biotin based immunotherapy; (5) prodrug activation strategies (ADEPT); (6) antibody-targeted superantigens; and (7) gene delivery for the purpose of gene therapy.

Smith-GK; Banks-S; Blumenkopf-TA; Cory-M; Humphreys-J; Laethem-RM; Miller-J; Moxham-CP; Mullin-R; Ray-PH; Walton-LM; Wolfe-LA-3rd. Toward antibody-directed enzyme prodrug therapy with the T268G mutant of human carboxypeptidase A1 and novel in vivo stable prodrugs of methotrexate. J-Biol-Chem. 1997 Jun 20; 272(25): 15804-16

Antibody-directed enzyme prodrug therapy (ADEPT) has the potential of greatly enhancing antitumor selectivity of cancer therapy by synthesizing chemotherapeutic agents selectively at tumor sites. This therapy is based upon targeting a prodrug-activating enzyme to a tumor by attaching the enzyme to a tumor-selective antibody and dosing the enzyme-antibody conjugate systemically. After the enzyme-antibody conjugate is localized to the tumor, the prodrug is then also dosed systemically, and the previously targeted enzyme converts it to the active drug selectively at the tumor. Unfortunately, most enzymes capable of this specific, tumor site generation of drugs are foreign to the human body and as such are expected to raise an immune response when injected, which will limit their repeated administration. We reasoned that with the power of crystallography, molecular modeling and site-directed mutagenesis, this problem could be addressed through the development of a human enzyme that is capable of catalyzing a reaction that is otherwise not carried out in the human body. This would then allow use of prodrugs that are otherwise stable in vivo but that are substrates for a tumor-targeted mutant human enzyme. We report here the first test of this concept using the human enzyme carboxypeptidase A1 (hCPA1) and prodrugs of methotrexate (MTX). Based upon a computer model of the human enzyme built from the well known crystal structure of bovine carboxypeptidase A, we have designed and synthesized novel bulky phenylalanine- and tyrosine-based prodrugs of MTX that are metabolically stable in vivo and are not substrates for wild type human carboxypeptidases A. Two of these analogs are MTX-alpha-3-cyclobutylphenylalanine and MTX-alpha-3-cyclopentyltyrosine. Also based upon the computer model, we have designed and produced a mutant of human carboxypeptidase A1, changed at position 268 from the wild type threonine to a glycine (hCPA1-T268G). This novel enzyme is capable of using the in vivo stable prodrugs, which are not substrates for the wild type hCPA1, as efficiently as the wild type hCPA1 uses its best substrates (i.e. MTX-alpha-phenylalanine). Thus, the kcat/Km value for the wild type hCPA1 with MTX-alpha-phenylalanine is 0.44 microM-1 s-1, and kcat/Km values for hCPA1-T268G with MTX-alpha-3-cyclobutylphenylalanine and MTX-alpha-3-cyclopentyltyrosine are 1.8 and 0.16 microM-1 s-1, respectively. The cytotoxic efficiency of hCPA1-268G was tested in an in vitro ADEPT model. For this experiment, hCPA1-T268G was chemically conjugated to ING-1, an antibody that binds to the tumor antigen Ep-Cam, or to Campath-1H, an antibody that binds to the T and B cell antigen CDw52. These conjugates were then incubated with HT-29 human colon adenocarcinoma cells (which express Ep-Cam but not the Campath 1H antigen) followed by incubation of the cells with the in vivo stable prodrugs. The results showed that the targeted ING-1:hCPA1-T268G conjugate produced excellent activation of the MTX prodrugs to kill HT-29 cells as efficiently as MTX itself. By contrast, the enzyme-Campath 1H conjugate was without effect. These data strongly support the feasibility of ADEPT using a mutated human enzyme with a single amino acid change.

Rowsell-S; Pauptit-RA; Tucker-AD; Melton-RG; Blow-DM; Brick-P. Crystal structure of carboxypeptidase G2, a bacterial enzyme with applications in cancer therapy. Structure. 1997 Mar 15; 5(3): 337-47

BACKGROUND: Carboxypeptidase G enzymes hydrolyze the C-terminal glutamate moiety from folic acid and its analogues, such as methotrexate. The enzyme studied here, carboxypeptidase G2 (CPG2), is a dimeric zinc-dependent exopeptidase produced by Pseudomonas sp. strain RS-16. CPG2 has applications in cancer therapy: following its administration as an immunoconjugate, in which CPG2 is linked to an antibody to a tumour-specific antigen, it can enzymatically convert subsequently administered inactive prodrugs to cytotoxic drugs selectively at the tumour site. CPG2 has no significant amino acid sequence homology with proteins of known structure. Hence, structure determination of CPG2 was undertaken to identify active-site residues, which may in turn provide ideas for protein and/or substrate modification with a view to improving its therapeutic usefulness. RESULTS: We have determined the crystal structure of CPG2 at 2.5 A resolution using multiple isomorphous replacement methods and non-crystallographic symmetry averaging. Each subunit of the molecular dimer consists of a larger catalytic domain containing two zinc ions at the active site, and a separate smaller domain that forms the dimer interface. The two active sites in the dimer are more than 60 A apart and are presumed to be independent; each contains a symmetric distribution of carboxylate and histidine ligands around two zinc ions which are 3.3 A apart. This distance is bridged by two shared zinc ligands, an aspartic acid residue and a hydroxyl ion. CONCLUSIONS: We find that the CPG2 catalytic domain has structural homology with other zinc-dependent exopeptidases, both those with a single zinc ion and those with a pair of zinc ions in the active site. The closest structural homology is with the aminopeptidase from Aeromonas proteolytica, where the similarity includes superposable zinc ligands but does not extend to the rest of the active-site residues, consistent with the different substrate specificities. The mechanism of peptide cleavage is likely to be very similar in these two enzymes and may involve the bridging hydroxyl ion ligand acting as a primary nucleophile.

de-Bont-DB; Leenders-RG; Haisma-HJ; van-der-Meulen-Muileman-I; Scheeren-HW. Synthesis and biological activity of beta-glucuronyl carbamate-based prodrugs of paclitaxel as potential candidates for ADEPT. Bioorg-Med-Chem. 1997 Feb; 5(2): 405-14

The syntheses of prodrugs of paclitaxel, which can be used in ADEPT in order to target paclitaxel towards tumor cells, are described. The prodrugs 1 and 2a, b consist of a spacer molecule connected via a carbamate linkage to a beta-glucuronic acid. The spacer molecule is also connected via an ester linkage to the 2'-OH of paclitaxel. Enzyme-catalyzed hydrolysis of the glucuronic acid moiety by human beta-glucuronidase results in the liberation of the parent drug paclitaxel via gamma or delta lactam formation with half-lives of 45 min and 2 h (1 and 2b). The prodrugs 1 and 2b are two orders of magnitude less cytotoxic than paclitaxel.

Springer-CJ; Bavetsias-V; Jackman-AL; Boyle-FT; Marshall-D; Pedley-RB; Bisset-GM. Prodrugs of thymidylate synthase inhibitors: potential for antibody directed enzyme prodrug therapy (ADEPT). Anticancer-Drug-Des. 1996 Dec; 11(8): 625-36

Prodrugs of quinazoline antifolate thymidylate synthase (TS) inhibitors have been designed and synthesized for use in antibody-directed enzyme prodrug therapy (ADEPT). The syntheses of the alpha-linked dipeptides of two potent thymidylate synthase inhibitors, ZD1694 [N-[5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6- ylmethyl)-N-methylamino]-2-thenoyl]-L-glutamic acid] and ICI198583 {N-[4-[N-[(2-methyl-3,4-dihydro-4-oxo-6-quinazolinyl) methyl]-N-prop-2-ynylamino]benzoyl]-L-glutamic acid} are described. The alpha-carboxyl of the glutamic acid has been linked through an amide bond to an L-alanine or an L-glutamic acid. The alpha-linked L-dipeptide prodrugs were designed to be activated to their corresponding thymidylate synthase inhibitors at a tumour site by prior administration of a monoclonal antibody conjugated to the enzyme carboxypeptidase A (CPA). The viability of a colorectal cell line was monitored with the potential prodrugs in the presence or absence of CPA or with the parent drugs alone. All the dipeptides had greatly decreased cytotoxicity, with a deactivation of approximately 100-fold for the ZD1694 prodrugs and approximately 20-200-fold for the ICI198583 prodrugs. Activation of the alpha-linked L-alanine dipeptides with CPA led to a cytotoxicity enhancement of approximately 10-100 fold.

Baxter-LT; Jain-RK. Pharmacokinetic analysis of the microscopic distribution of enzyme-conjugated antibodies and prodrugs: comparison with experimental data. Br-J-Cancer. 1996 Feb; 73(4): 447-56

A mathematical model was developed to improve understanding of the biodistribution and microscopic profiles of drugs and prodrugs in a system using enzyme-conjugated antibodies as part of a two-step method for cancer treatment. The use of monoclonal antibodies alone may lead to heterogeneous uptake within the tumour tissue; the use of a second, low molecular weight agent may provide greater penetration into tumour tissue. This mathematical model was used to describe concentration profiles surrounding individual blood vessels within a tumour. From these profiles the area under the curve and specificity ratios were determined. By integrating these results spatially, average tissue concentrations were determined and compared with experimental results from three different systems in the literature; two using murine antibodies and one using humanised fusion proteins. The maximum enzyme conversion rate (Vmax) and the residual antibody concentration in the plasma and normal tissue were seen to be key determinants of drug concentration and drug-prodrug ratios in the tumour and other organs. Thus, longer time delays between the two injections, clearing the antibody from the blood stream and the use of 'weaker' enzymes (lower Vmax) will be important factors in improving this prodrug approach. Of these, the model found the effective clearance of the antibody outside of the tumour to be the most effective. The use of enzyme-conjugated antibodies may offer the following advantages over the bifunctional antibody-hapten system: (i) more uniform distribution of the active agent; (ii) higher concentrations possible for the active agent; and (iii) greater specificity (therapeutic index).

Perron-MJ; Page-M. Activation of methotrexate-phenylalanine by monoclonal antibody--carboxypeptidase A conjugate for the specific treatment of ovarian cancer in vitro. Br-J-Cancer. 1996 Feb; 73(3): 281-7

Monoclonal antibody 4E3 directed against a glycosylated surface protein on human ovarian teratocarcinoma cells (CRL-1572 cell line) was conjugated to bovine carboxypeptidase A (CPA) using a 3400 Da polyethylene glycol chain bearing an N-hydroxysuccinimide group at both ends. The conjugate preparation was purified by fast protein liquid chromatography on a Superose 12/30 HR column. The 4E3-CPA conjugate was recovered in the third fraction by SDS-PAGE analysis. The specific binding of the 4E3-CPA conjugate to CRL-1572 cells was confirmed by a FACS analysis and the enzymatic activity of the conjugate remained while tested with hippuryl-L-phenylalanine. In vitro cytotoxic assays on CRL-1572 cells showed that the prodrug methotrexate-phenylalanine (MTX-Phe) alone was non-toxic (ID50 > 1000 ng ml-1) but was selectively converted to MTX when the cells were pretreated with 50 micrograms ml-1 4E3-CPA conjugate, which enhanced considerably the pharmacological activity of the prodrug with an ID50 of 70 ng ml-1. The co-culture assays with CRL-1572 and MRC-5 cells (human normal lung diploid fibroblast cell lines) demonstrated the specificity of the 4E3-CPA conjugate for the CRL-1572 cells since no cytotoxicity was observed on the MRC-5 cells. When both cell lines were mixed in ratios ranging from 1:10,000 to 1:5 (CRL-1572:MRC-5), the significant increase in the ID25 was correlated with the proportion of tumoral cells present in the cell inoculum. These results suggest that MTX-Phe combined with 4E3-CPA conjugate is a promising model for a more selective and localised anti-cancer chemotherapy based on the ADEPT concept.

Schumacher-U; Adam-E; Zangemeister-Wittke-U; Gossrau-R. Histochemistry of therapeutically relevant enzymes in human tumours transplanted into severe combined immunodeficient (SCID) mice: nitric oxide synthase-associated diaphorase, beta-D-glucuronidase and non-specific alkaline phosphatase. Acta-Histochem. 1996 Nov; 98(4): 381-7

Human breast (MCF-7, HBL 100, T47D, BT20, HS578T), colon (HT29, CACO2, SW620, SW480, COLO320DM) and small cell lung cancer (NCI-N417, OH3, SW2) cell lines were transplanted subcutaneously into severe combined immunodeficient (SCID) mice. When sizeable tumours developed, the mice were sacrificed and the following enzyme activities were detected histochemically: presumed nitric oxide synthase-associated diaphorase (NOSaD), beta-D-glucuronidase (beta-Gluc) and non-specific alkaline phosphatase (alP). Except for HT29 and MCF-7 presumed NOSaD activity was not detected in the tumour itself or in the neo-vasculature of the tumours. beta-Gluc activity was found in all tumour cells (except N417 and COLO 320), in the necrotic parts of the tumours and in stromal cells of the tumour bed. AlP activity was present in all tumours including their necrotic areas. However, the activities of beta-Gluc and alP varied considerably even within one tumour, ranging from very weak to very strong. Principally the results show that the human/SCID mouse tumour model is well suited to test modern applications of tumour therapy involving the enzymes NOSaD, beta-Gluc and alP. In particular, antibody directed enzyme prodrug therapy concepts and activation of prodrugs by enzymes released from tumour cells into the necrotic areas of the tumour can be evaluated in this in vivo model.

Houba-PH; Boven-E; Haisma-HJ. Improved characteristics of a human beta-glucuronidase-antibody conjugate after deglycosylation for use in antibody-directed enzyme prodrug therapy. Bioconjug-Chem. 1996 Sep-Oct; 7(5): 606-11

Antibody-directed enzyme prodrug therapy (ADEPT) aims at the specific activation of relatively nontoxic prodrugs into active drugs at the tumor site. One of the enzymes described to be useful in ADEPT is human beta-glucuronidase (GUS), which is expected to have low immunogenicity in patients. A major obstacle for the use of GUS, however, is its rapid glycan-specific hepatic clearance. The carbohydrates of GUS have been modified by subsequent treatment with NaIO4 and NaBH4 to improve its retention in the circulation. The modification of GUS did not decrease the enzyme activity. In vitro it was demonstrated that a conjugate prepared with a pancarcinoma specific monoclonal antibody (mAb) 323/A3 and the modified enzyme (mGUS), when bound to tumor cells, was capable of complete prodrug activation. In vivo, the 323/A3-mGUS conjugate was cleared faster from the circulation of BALB/c mice (t1/2 = 9 h) than mAb 323/A3 (t1/2 = 32 h), but it was retained in the circulation much longer than an immunoconjugate prepared with native GUS (t1/2 = 24 min). In nude mice bearing subcutaneous OVCAR-3 tumors the distribution of 323/A3-mGUS was qualitatively comparable to that of mAb 323/A3. The 323/A3-mGUS conjugate showed specific localization in the tumor but to a lesser extent than mAb 323/A3 (2.7% vs 6.4% injected dose per gram at 1 day after iv injection). A favorable tumor-to-blood ratio of > 2 was observed for the conjugate at 7 days after administration, which is necessary for tumor-specific prodrug activation.

Atwell-GJ; Boyd-M; Palmer-BD; Anderson-RF; Pullen-SM; Wilson-WR; Denny-WA. Synthesis and evaluation of 4-substituted analogues of 5-[N,N-bis (2-chloroethyl)amino]-2-nitrobenzamide as bioreductively activated prodrugs using an Escherichia coli nitroreductase. Anticancer-Drug-Des. 1996 Oct; 11(7): 553-67

2,4-Dinitrobenzamide mustards, exemplified by the parent compound SN 23862 (2) are activated under aerobic conditions by an Escherichia coli nitroreductase enzyme (NR2) via selective reduction of the 2-nitro group, and are thus of interest as prodrugs for antibody-directed enzyme-prodrug therapy (ADEPT). A series of related compounds 12a-12d, where the 4-nitro group of 2 was replaced by other substituents of varying electronic properties, were prepared and evaluated as potential ADEPT prodrugs. One-electron reduction potentials of the compounds correlated well with the substituent sigma m values, with the exception of the unsubstituted (4-H) analogue 13, which had a much lower value than expected on electronic grounds, due to a coplanar conformation of the mustard. The cytotoxicities of the compounds towards aerobic UV4 cells correlated positively with the electron-donating ability of the 4-substituent (measured by sigma p values), indicating that the cytotoxicities of the compounds in the absence of the NR2 enzyme are due substantially to the parent (unreduced) compounds. A positive, although less strong, correlation was seen between the electronic properties of the 4-substituent and their cytotoxicities in the presence of the NR2 enzyme, suggesting that, in this closely related series, the degree of activation by the enzyme is significantly dependent on the reduction potential of the 2-nitro group. While the 4-SO2Me derivative 12d was the next most preferred substrate after the parent 2, it was considerably less so (degree of activation as measured by IC50 ratio of 26 compared with 145), despite the similar electronic properties of the two 4-substituents.

Laethem-RM; Blumenkopf-TA; Cory-M; Elwell-L; Moxham-CP; Ray-PH; Walton-LM; Smith-GK. Expression and characterization of human pancreatic preprocarboxypeptidase A1 and preprocarboxypeptidase A2. Arch-Biochem-Biophys. 1996 Aug 1; 332(1): 8-18

We are investigating the potential utility of human carboxypeptidases A in antibody-directed enzyme prodrug therapy (ADEPT). Hybridization screening of a human pancreatic cDNA library with cDNA probes that encoded either rat carboxypeptidase A1 (rCPA1) or carboxypeptidase A2 (rCPA2) was used to clone the human prepro-CPA homologs. After expression of the respective pro-hCPA cDNA in Saccharomyces cerevisiae, the enzymes were purified to homogeneity by a combination of hydrophobic and ion-exchange chromatography. Purified hCPA1 and hCPA2 migrate as a single protein band with M(r) 34,000 when subjected to gel electrophoresis in the presence of sodium dodecyl sulfate under reducing conditions. Kinetic studies of the purified enzymes with hippuryl-L-phenylalanine resulted in kcat/Km values of 57,000 and 19,000 M-1 s-1 for hCPA1 and hCPA2, respectively. Using the ester substrate, hippuryl-D, L-phenyllactate, we found unique esterase/ peptidase specific activity ratios among hCPA1, hCPA2, rCPA1, and bovine CPA (bCPA) ranging from 13 to 325. Two potential ADEPT substrates, methotrexate-alpha-phenylalanine (MTX-Phe) and methotrexate-alpha-(1-naphthyl)alanine (MTX-naphthylAla) were also analyzed. The kcat/Km values for MTX-Phe were 440,000 and 90,000 M-1 s-1 for hCPA1 and hCPA2, respectively, and for MTX-naphthylAla these values were 1400 and 1,400,000 M-1 s-1 for hCPA1 and hCPA2, respectively. The kinetic data show that hCPA2 has a larger substrate binding site than the hCPA1 enzyme. Differences between hCPA1 and hCPA2 were also observed in thermal stability experiments at 60 degrees C where the half-life for thermal denaturation of hCPA2 is eightfold longer than that for hCPA1. These experiments indicate that hCPA1 and hCPA2 are potential candidates for use in a human-based ADEPT approach.

Blakey-DC; Burke-PJ; Davies-DH; Dowell-RI; East-SJ; Eckersley-KP; Fitton-JE; McDaid-J; Melton-RG; Niculescu-Duvaz-IA; Pinder-PE; Sharma-SK; Wright-AF; Springer-CJ ZD2767, an improved system for antibody-directed enzyme prodrug therapy that results in tumor regressions in colorectal tumor xenografts. Cancer-Res. 1996 Jul 15; 56(14): 3287-92

ZD2767 represents an improved version of antibody-directed enzyme prodrug therapy. It consists of a conjugate of the F(ab')2 A5B7 antibody fragment and carboxypeptidase G2 (CPG2) and a prodrug, 4-[N,N-bis(2-iodoethyl)amino]phenoxycarbonyl L-glutamic acid. The IC50 of the prodrug against LoVo colorectal tumor cells was 47 microM, and cleavage by CPG2 released the potent bis-iodo phenol mustard drug (IC50 = 0.34 microM). The drug killed both proliferating and quiescent LoVo cells. Administration of the ZD2767 conjugate to nude mice bearing LoVo colorectal xenografts resulted in approximately 1% of injected ZD2767 conjugate localizing/g of tumor after 72 h, and blood and normal tissue levels of the conjugate were 10-50-fold lower. A single round of therapy involving the administration of the prodrug 72 h after the conjugate to athymic mice bearing established LoVo xenografts resulted in approximately 50% of the tumors undergoing complete regressions, tumor growth delays greater than 30 days, and little toxicity (as judged by body-weight loss). Similar studies using a control antibody-CPG2 conjugate that does not bind to LoVo tumor cells resulted in a growth delay of less than 5 days, confirming the tumor specificity of this approach. These studies demonstrate the potential of ZD2767 for the treatment of colorectal cancer.

Vingerhoeds-MH; Haisma-HJ; Belliot-SO; Smit-RH; Crommelin-DJ; Storm-G. Immunoliposomes as enzyme-carriers (immuno-enzymosomes) for antibody-directed enzyme prodrug therapy (ADEPT): optimization of prodrug activating capacity. Pharm-Res. 1996 Apr; 13(4): 604-10

PURPOSE. Immuno-enzymosomes are tumor-specific immunoliposomes bearing enzymes on their surface. These enzymes are capable of converting relatively nontoxic prodrugs into active cytostatic agents. The enzyme beta-glucuronidase (GUS)4 was coupled to the external surface of immunoliposomes directed against ovarian carcinoma cells. This study aimed at optimization of the prodrug-activating capacity of these immuno-enzymosomes by increasing the enzyme density on the immunoliposomal surface. METHODS. To achieve coupling of GUS to the liposomes, introduction of extra thiol groups was required. Two thiolating agents were examined: iminothiolane and SATA. RESULTS. When iminothiolane was used, aggregation of enzymosomes was observed above enzyme densities of 10 micrograms GUS/mumol lipid (TL). An increased electrostatic repulsion of the enzymosomes, created by inclusion of additional negatively charged lipids and by lowering the ionic strength of the external aqueous medium resulted in enzyme densities > or = 20 micrograms GUS/mumol TL without aggregation. Utilizing SATA, > or = 30 micrograms GUS/mumol TL could be coupled without aggregation, even at physiological ionic strength. It was shown that the enzyme density on immuno-enzymosomes, and thus on the tumor cell surface, strongly influences the antitumor effect of the prodrug daunorubicin-glucuronide against in vitro cultured ovarian cancer cells. The antitumor effect of immuno-enzymosomes with enzyme densities of about 20 micrograms GUS/mumol TL was similar to that of the parent drug daunorubicin. CONCLUSIONS. SATA-mediated thiolation of GUS-molecules enabled the preparation of immuno-enzymosomes with high enzyme densities while avoiding spontaneous aggregation. In vitro antitumor activity experiments showed that the improved immuno-enzymosome system is able to completely convert the prodrug daunorubicin-glucuronide into its parent compound.

Houba-PH; Leenders-RG; Boven-E; Scheeren-JW; Pinedo-HM; Haisma-HJ. Characterization of novel anthracycline prodrugs activated by human beta-glucuronidase for use in antibody-directed enzyme prodrug therapy. Biochem-Pharmacol. 1996 Aug 9; 52(3): 455-63

Antibody-directed enzyme prodrug therapy (ADEPT) aims at the specific activation of a prodrug by an enzyme-immunoconjugate localized in tumor tissue. The use of an enzyme of human origin is preferable in ADEPT because it might not be immunogenic when administered to patients. In the case of human beta-glucuronidase, prodrugs should be designed that are rapidly and completely activated at a neutral pH. Four new daunorubicin glucuronides were synthesized by coupling a glucuronide group to daunorubicin via an aliphatic (GA1 and GB1) or an aromatic (GA3, GB6) carbamate spacer, to be released by electron shift (A-type) or by ring closure (B-type). These prodrugs were characterized in vitro for their usefulness in ADEPT and were compared with the previously described prodrugs epirubicin-glucuronide and doxorubicin-nitrophenyl-glucuronide. The four new prodrugs were stable in serum, hydrophilic when compared to the lipophilic daunorubicin, and at least 20-fold less toxic than the parent compound. The hydrolysis rate at clinically relevant enzyme and prodrug concentrations (1 microgram/mL human beta-glucuronidase, 100 microM prodrug) at pH 6.8 were similar for GA3 (T1/2 160 min) and higher for GB6 (T1/2 40 min) when compared to that of doxorubicin-nitrophenyl-glucuronide (T1/2 170 min). Epirubicin-glucuronide, GA1, and GB1 showed a low hydrolysis rate (T1/2 > 400 min). GA1 and GA3, but not GB1 or GB6, were activated to the parent compound. Complete activation was confirmed in OVCAR-3 cells pretreated with a specific antibody-human beta-glucuronidase conjugate, where GA3 had similar antiproliferative effects to those of daunorubicin.

Dowell-RI; Springer-CJ; Davies-DH; Hadley-EM; Burke-PJ; Boyle-FT; Melton-RG; Connors-TA; Blakey-DC; Mauger-AB. New mustard prodrugs for antibody-directed enzyme prodrug therapy: alternatives to the amide link. J-Med-Chem. 1996 Mar 1; 39(5): 1100-5

Antibody-directed enzyme prodrug therapy (ADEPT) is a two-step approach for the treatment of cancer which seeks to generate a potent cytotoxic agent selectively at a tumor site. In this work described the cytotoxic agent is generated by the action of an enzyme CPG2 on a relatively nontoxic prodrug. The prodrug 1 currently on clinical trial is a benzamide and is cleaved by CPG2 to a benzoic acid mustard drug 1a. We have synthesized a series of new prodrugs 3-8 where the benzamide link has been replaced by, for example, carbamate or ureido. Some of these alternative links have been shown to be good substrates for CPG2 and therefore new candidates for ADEPT. The active drugs 3a and 4a derived from the best of these prodrugs are potent cytotoxic agents (1-2 microM) some 100 times more than 1a. The prodrugs 3 and 4 are some 100-200-fold less cytotoxic, in a proliferating cell assay, than their corresponding active drugs 3a and 4a.

Melton-RG; Sherwood-RF. Antibody-enzyme conjugates for cancer therapy. J-Natl-Cancer-Inst. 1996 Feb 21; 88(3-4): 153-65

The use of antibody-enzyme conjugates directed at tumor-associated antigens to achieve site-specific activation of prodrugs to potent cytotoxic species, termed "antibody-directed enzyme prodrug therapy" (ADEPT), has attracted considerable interest since the concept was first described in 1987. Prodrug forms of both clinically used anticancer agents and novel cytotoxic compounds have been developed to take advantage of potential prodrug-generating technology employing a variety of enzymes with widely differing substrate specificities. A particular advantage of the ADEPT approach is that it may allow the use of extremely potent agents such as nitrogen mustards and palytoxin, which are too toxic to be readily used in conventional chemotherapy. Preliminary studies using an antibody-enzyme conjugate constructed with a bacterial enzyme and a murine monoclonal antibody not only have established the value of the ADEPT technique, but also have highlighted the potential problem of immunogenicity of proteins of nonhuman origin. This problem has been tackled in the first instance by the use of immunosuppressive agents, but long-term solutions are being investigated in the development of second-generation ADEPT systems, including the development of human antibody-human enzyme fusion proteins and catalytic antibodies. Such improvements, coupled with further refinement of the prodrug-drug element of the system and the wide variety of antibody-enzyme-drug combinations available, should mean that ADEPT-based approaches will form an important element of the search for the anticancer drugs of the future.

Wentworth-P; Datta-A; Blakey-D; Boyle-T; Partridge-LJ; Blackburn-GM Toward antibody-directed "abzyme" prodrug therapy, ADAPT: carbamate prodrug activation by a catalytic antibody and its in vitro application to human tumor cell killing. Proc-Natl-Acad-Sci-U-S-A. 1996 Jan 23; 93(2): 799-803

Antibody-directed enzyme prodrug therapy, ADEPT, is a recent approach to targeted cancer chemotherapy intended to diminish the nonspecific toxicity associated with many commonly used chemotherapeutic agents. Most ADEPT systems incorporate a bacterial enzyme, and thus their potential is reduced because of the immunogenicity of that component of the conjugate. This limitation can be circumvented by the use of a catalytic antibody, which can be "humanized," in place of the bacterial enzyme catalyst. We have explored the scope of such antibody-directed "abzyme" prodrug therapy, ADAPT, to evaluate the potential for a repeatable targeted cancer chemotherapy. We report the production of a catalytic antibody that can hydrolyze the carbamate prodrug 4-[N,N-bis(2-chloroethyl)]aminophenyl-N-[(1S)-(1,3- dicarboxy)propyl]carbamate (1) to generate the corresponding cytotoxic nitrogen mustard (Km = 201 microM, kcat = 1.88 min-1). In vitro studies with this abzyme, EA11-D7, and prodrug 1 lead to a marked reduction in viability of cultured human colonic carcinoma (LoVo) cells relative to appropriate controls. In addition, we have found a good correlation between antibody catalysis as determined by this cytotoxicity assay in vitro and competitive binding studies of candidate abzymes to the truncated transition-state analogue ethyl 4-nitrophenylmethylphosphonate. This cell-kill assay heralds a general approach to direct and rapid screening of antibody libraries for catalysts.

Abraham-R; Aman-N; von-Borstel-R; Darsley-M; Kamireddy-B; Kenten-J; Morris-G; Titmas-R. Conjugates of COL-1 monoclonal antibody and beta-D-galactosidase can specifically kill tumor cells by generation of 5-fluorouridine from the prodrug beta-D-galactosyl-5-fluorouridine. Cell-Biophys. 1994; 24-25: 127-33

5'-O-beta-D-galactosyl-5-fluorouridine is a prodrug that can be converted by the enzyme beta-D-galactosidase to the potent antineoplastic drug 5-fluorouridine. The prodrug is more than 100x less toxic than the drug to bone marrow cells in Balb/c mice. The ratio of the IC50 of the prodrug to that of the drug determined on a variety of tumor cell lines in vitro ranged from 500:1-1000:1. An antibody-enzyme conjugate (AEC) was synthesized and purified. Maleimide-substituted COL-1 anti-CEA monoclonal antibody was linked to free thiol groups of beta-D-galactosidase. The conjugate was purified by size exclusion and ion exchange chromatography. It retained full immunoreactivity and enzyme activity. After binding to antigen-positive tumor cells, the conjugate was able to activate the prodrug and specifically kill the cells. We are continuing to investigate this model for its potential use in antibody-directed enzyme prodrug therapy (ADEPT). Previous Page

Bagshawe-KD. ADEPT and related concepts. Cell-Biophys. 1994; 24-25: 83-91

Antibody-based therapy has attracted interest because of its potential to improve selectivity. But the limitations of antibodies as delivery systems are well known and the objective of restricting action to tumor sites requires additional means. The ADEPT concept introduced two components, enzyme and prodrug, that have the advantage that they can be secondarily manipulated to augment the selectivity of the primary delivery systems. An antibody-enzyme conjugate (AEC) is no more selective as a delivery system than antibody itself and total catalytic capacity in tumor, plasma, and nontumor tissues is a function not only of concentration but also of volume. It is pointless giving a prodrug that is promptly activated by enzyme in blood. The ability to inactivate or clear plasma enzyme (PENCIL) by an antibody directed at its active site and modified to have low potential to penetrate the tumor is one of several ways of improving partition of enzyme between tumor and nontumor. A second opportunity for manipulation arises from structural differences between prodrug and active drug and the potential of enzymes to exploit that difference. However effective the enzyme delivery system, some leakage of active drug into plasma is likely and active drug access to hemopoietic tissues is dose limiting. An enzyme for which the active drug, but not the prodrug, is substrate, and which is conjugated to a macromolecule, is proposed. Some thymidylate synthetase inhibitors suggest themselves as ready agents for use in this intravascular inactivation of active drug (IVIAD). This approach is an alternative to inactivation of plasma enzyme. AECs may also be used advantageously in the context of antimetabolites used with rescue agents. Previous Page

Bagshawe-KD; Sharma-SK; Springer-CJ; Rogers-GT. Antibody directed enzyme prodrug therapy (ADEPT). A review of some theoretical, experimental and clinical aspects. Ann-Oncol. 1994 Dec; 5(10): 879-91

The concept of generating cytotoxic agents from non-toxic prodrugs at tumour sites by antibody vectored enzyme introduces a wide range of opportunities. Various prodrug-enzyme combinations have been described and encouraging results reported in xenograft models. Whilst the mouse model is a valuable tool in this approach translation to the human patient may expose more complex issues. The objective of restricting drug action to tumour sites and thus allowing greatly increased cytotoxic action requires more precise restriction of enzyme activity to tumour sites than has been achieved with an antibody vector and natural clearance alone. Assisted clearance mechanisms have been found effective. Alternatively, or additionally, the difference between prodrug and active drug creates the opportunity to degrade active drug selectively in blood and thus protect normal tissues. In order to give more than one cycle of treatment it will be necessary for the antibody-enzyme conjugate to be nonimmunogenic or for the concurrent administration of immunosuppressive agents. A pilot scale clinical trial with a prototype prodrug indicated the feasibility of antibody directed enzyme prodrug therapy (ADEPT). Previous Page

Blakey-DC; Davies-DH; Dowell-RI; East-SJ; Burke-PJ; Sharma-SK; Springer-CJ; Mauger-AB; Melton-RG. Anti-tumour effects of an antibody-carboxypeptidase G2 conjugate in combination with phenol mustard prodrugs. Br-J-Cancer. 1995 Nov; 72(5): 1083-8

ADEPT is an antibody-based targeting strategy for the treatment of cancer. We have developed two new prodrugs, 4-[N,N-bis(2-chloroethyl)amino]-phenoxycarbonyl-L- glutamic acid (PGP) and (S)-2-[N-[4-[N,N-bis(2-chloroethyl)amino]- phenoxycarbonyl]amino]-4-(5-tetrazoyl)butyric acid (PTP), which are cleaved by the bacterial enzyme CPG2 to release the 4-[N,N-bis(2-chloroethyl)amino] phenol drug. In vitro, both prodrugs are approximately 100- to 200-fold less potent than the parent drug (1 h IC50 = 1.4 microM) in LoVo colorectal tumour cells. These prodrugs have been evaluated for utility in ADEPT when used in combination with a conjugate of CPG2 and the F(ab')2 fragment of the anti-CEA monoclonal antibody, A5B7. The conjugate was shown to localise specifically to established LoVo tumour xenografts growing in nude mice and optimal tumour-normal tissue ratios were achieved after 72 h. Administration of either prodrug, at doses which cause 6-8% body weight loss, 72 h after administration of the A5B7-CPG2 conjugate to the LoVo tumour-bearing mice resulted in tumour regressions and growth delays of 14-28 days. The PTP prodrug in combination with a high dose of conjugate (10 mg kg-1) gave the best anti-tumour activity despite being a 10-fold worse substrate for CPG2 than PGP. Prodrug alone, active drug alone or prodrug in combination with a non-specific conjugate had minimal anti-tumour activity in this tumour model. Previous Page

Blakey-DC; Pinder-PE; Wright-AF. Comparison of the cellular internalization of antibodies used either as immunotoxins or in ADEPT. Cell-Biophys. 1994; 24-25: 175-83

The internalization into tumor cells of two antibodies (C242 and 454A12), which make potent immunotoxins when linked to ricin A-chain, and an antibody (A5B7), which does not make a potent immunotoxin but has proven useful in ADEPT, was evaluated. The 454A12 antibody was rapidly taken into the cells, 50% of the antibody being internalized after 2 h. The C242 antibody was internalized more slowly, approx 50% being taken up by the cells in 24 h. With A5B7, less than 10% of the antibody was internalized after 24 h. Internalization of the C242 antibody was accompanied by the appearance of antibody degradation products in the cell medium after 2 h, and this degradation could be inhibited by addition of a metabolic inhibitor that prevented cell internalization. In contrast, minimal degradation of the A5B7 antibody could be detected up to 24 h after binding to the cells. In conclusion, both 454A12 and C242 antibodies, which make potent immunotoxins, were internalized into tumor cells. The A5B7 antibody, which does not make a potent immunotoxin, was not internalized, and this property may be one reason why A5B7 has proved useful for delivery of enzymes in ADEPT. Previous Page

Knox-RJ; Friedlos-F; Jarman-M; Davies-LC; Goddard-P; Anlezark-GM; Melton-RG; Sherwood-RF. Virtual cofactors for an Escherichia coli nitroreductase enzyme: relevance to reductively activated prodrugs in antibody directed enzyme prodrug therapy (ADEPT). Biochem-Pharmacol. 1995 May 26; 49(11): 1641-7

A nitroreductase enzyme has been isolated from Escherichia coli that has the unusual property of being equally capable of using either NADH or NADPH as a cofactor for the reduction of its substrates which include menadione as well as 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954). This property is shared with the mammalian enzyme, DT diaphorase. The nitroreductase can, like DT diaphorase, also use simple reduced pyridinium compounds as virtual cofactors. The intact NAD(P)H molecule is not required and the simplest quaternary (and therefore reducible) derivative of nicotinamide, 1-methylnicotinamide (reduced), is as effective as NAD(P)H in its ability to act as an electron donor for the nitroreductase. The structure-activity relationship is not identical to that of DT diaphorase and nicotinic acid riboside (reduced) is selective, being active only for the nitroreductase. Irrespective of the virtual cofactor used, the nitroreductase formed the same reduction products of CB 1954 (the 2- and 4-hydroxylamino derivatives in equal proportions). Nicotinic acid riboside (reduced), unlike NADH, was stable to metabolism by serum enzymes and had a plasma half-life of seven minutes in the mouse after an i.v. bolus administration. NADH had an unmeasurably short half-life. Nicotinic acid riboside (reduced) could also be produced in vivo by administration of nicotinic acid 5'-O-benzoyl riboside (reduced). These results demonstrate that the requirement for a cofactor need not be a limitation in the use of reductive enzymes in antibody directed enzyme prodrug therapy (ADEPT). It is proposed that the E. coli nitroreductase would be a suitable enzyme for ADEPT in combination with CB 1954 and a synthetic, enzyme-selective, virtual cofactor such as nicotinic acid riboside (reduced). Previous Page

Sharma-SK; Boden-JA; Springer-CJ; Burke-PJ; Bagshawe-KD. Antibody-directed enzyme prodrug therapy (ADEPT). A three-phase study in ovarian tumor xenografts. Cell-Biophys. 1994; 24-25: 219-28

Antibody-directed enzyme prodrug therapy (ADEPT) has been studied in a human ovarian carcinoma xenograft grown subcutaneously in nude mice. Radioimmunoassay of supernatants obtained from tumor homogenates showed these to contain carcinoembryonic antigen (CEA). Biodistribution studies with 125I-labeled monoclonal anti-CEA antibody, A5B7, and its F(ab')2 fragment showed localization in these xenografts. The AB57-F(ab')2 fragment conjugated to a bacterial enzyme, carboxypeptidase G2 (CPG2), and, radiolabeled with 125iodine, also localized in the xenografts. The radiolabeled conjugate cleared from blood faster than the antibody alone. The percentage of injected dose per gram in tumor at 24 h postinjection was about fivefold lower than antibody alone. Tumor-to-blood ratio at 72 h after injection of the radiolabeled conjugate was 7 and the tumor-to-normal tissue ratios at this time point ranged from 20 (liver) to 75 (colon). A three-phase ADEPT antitumor study was carried out in which A5B7-F(ab')2-CPG2 was allowed to localize and was followed by accelerated inactivation/clearance of blood CPG2 by a galactosylated anti-CPG2 antibody (SB43gal). A benzoic acid mustard-derived prodrug was injected 24 h after the conjugate, which led to growth delay in this tumor compared to the control untreated group. Further antitumor studies in this model are in progress. Previous Page

Springer-CJ; Dowell-R; Burke-PJ; Hadley-E; Davis-DH; Blakey-DC; Melton-RG; Niculescu-Duvaz-I. Optimization of alkylating agent prodrugs derived from phenol and aniline mustards: a new clinical candidate prodrug (ZD2767) for antibody-directed enzyme prodrug therapy (ADEPT). J-Med-Chem. 1995 Dec 22; 38(26): 5051-65

Sixteen novel potential prodrugs derived from phenol or aniline mustards and their 16 corresponding drugs with ring substitution and/or different alkylating functionalities were designed. The [[[4-]bis(2-bromoethyl)-(1a), [[[4-[bis(2-iodoethyl)-(1b), and [[[4-[(2-chloroethyl)-[2-(mesyloxy)ethyl]amino]phenyl]oxy] carbonyl]-L-glutamic acids (1c), their [[[2- and 3-substituted-4-[bis(2-chloroethyl)amino]phenyl]oxy]carbonyl]-L- glutamic acids (1e-1), and the [[3-substituted-4-[bis(2-chloroethyl)amino]phenyl]carbamoyl]-L- glutamic acids (1o-r) were synthesized. They are bifunctional alkylating agents in which the activating effect of the phenolic hydroxyl or amino function is masked through an oxycarbonyl or a carbamoyl bond to a glutamic acid. These prodrugs were designed to be activated to their corresponding phenol and aniline nitrogen mustard drugs at a tumor site by prior administration of a monoclonal antibody conjugated to the bacterial enzyme carboxypeptidase G2 (CPG2) in antibody-directed enzyme prodrug therapy (ADEPT). The synthesis of the analogous novel parent drugs (2a-r) is also described. The viability of a colorectal cell line (LoVo) was monitored with the potential prodrugs and the parent drugs. The differential in the cytotoxicity between the potential prodrugs and their corresponding active drugs ranged between 12 and > 195 fold. Compounds 1b-d,f,o exhibited substantial prodrug activity, since a cytotoxicity differential of > 100 was achieved compared to 2b-d,f,o respectively. The ability of the potential prodrugs to act as substrates for CPG2 was determined (kinetic parameters KM and kcat), and the chemical stability was measured for all the compounds. The unsubstituted phenols with different alkylating functionalities (1a-c) proved to have the highest ratio of the substrates kcat:KM. From these studies [[[4-[bis(2-iodoethyl)amino]phenyl]oxy]carbonyl]-L-glutamic acid (1b) emerges as a new ADEPT clinical trial candidate due to its physicochemical and biological characteristics. Previous Page

Springer-CJ; Niculescu-Duvaz-I. Antibody-directed enzyme prodrug therapy (ADEPT) with mustard prodrugs. Anticancer-Drug-Des. 1995 Jul; 10(5): 361-72

Antibody-directed enzyme prodrug therapy (ADEPT) is a two-step targeting procedure designed to improve the selectivity of anti-tumour agents. The approach is based on the activation of specially designed prodrugs by enzyme-antibody conjugates targeted to tumour-associated antigens. This review concerns ADEPT using nitrogen mustard prodrugs and carboxypeptidase G2 (CPG2) as the activating enzyme. The specific structural features required of the nitrogen mustard prodrugs, their design, syntheses, physicochemical properties, biological characteristics and activation to the corresponding drugs are reviewed. The ADEPT clinical trial with a nitrogen mustard prodrug is also discussed.Previous Page

Springer-CJ; Poon-GK; Sharma-SK; Bagshawe-KD. Analysis of antibody-enzyme conjugate clearance by investigation of prodrug and active drug in an ADEPT clinical study. Cell-Biophys. 1994; 24-25: 193-207

Antibody-directed enzyme prodrug therapy (ADEPT) separates the cytotoxic function from the targeting function (5). An antibody-carboxypeptidase G2 (CPG2) enzyme is delivered prior to the nontoxic prodrug, CMDA, which is converted to a cytotoxic drug by the action of the localized conjugate at the tumor site. An indirect in vitro assay was developed to detect the presence of functional CPG2 in the plasma of patients in an ADEPT clinical trial. Compounds in the plasma of patients were characterized using liquid chromatography-mass spectrometry. Plasma at three different time points (prior to treatment, post-antibody-enzyme conjugate, and post-galactosylated anti-enzyme antibody clearing agent) was added to the CMDA prodrug and analyzed. Conversion of the CMDA prodrug to its active drug indicates that CPG2-conjugate remains in the plasma. This technique will provide essential data for the timing of prodrug administration in ADEPT. Previous Page

Connors-TA; Knox-RJ. Prodrugs in cancer chemotherapy. Stem-Cells-Dayt. 1995 Sep; 13(5): 501-11

At present, chemotherapy is not very effective against common solid cancers, especially once they have metastasized. However, laboratory experiments and studies on dose intensification in humans have indicated that some anticancer agents might be curative, but only if the dose given was very much higher than that attainable clinically. Prodrugs activated by enzymes expressed at a high level in tumors can deliver at least 50-fold the normal dose and can cure animals with tumors normally resistant to chemotherapy. The approach is not practicable clinically because of the rarity of human tumors expressing a high level of an activating enzyme. However, new therapies have been proposed that overcome this limitation of prodrug therapy. Enzymes that activate prodrugs can be directed to human tumor xenografts by conjugating them to tumor-associated antibodies. After allowing for the conjugate to clear from the blood a prodrug is administered which is normally inert, but which is activated by the enzyme delivered to the tumor. This procedure is referred to as ADEPT (antibody-directed enzyme prodrug therapy). Using different combinations of antibody, enzyme and prodrug, many classes of human tumor xenograft have been shown to be very sensitive to this procedure although in most cases they are quite resistant to conventional chemotherapy. Early clinical trials are promising and indicate that ADEPT may become an effective treatment for all solid cancers for which tumor-associated or tumor-specific antibodies are known. Tumors have also been targeted with the genes encoding for prodrug activating enzymes. This approach has been called virus-directed enzyme prodrug therapy (VDEPT) or more generally GDEPT (gene-directed enzyme prodrug therapy) and has shown good results in laboratory systems. These new therapies may finally realize the potential of prodrugs in cancer chemotherapy. Previous Page

Haisma-HJ; Van-Muijen-M; Scheffer-G; Scheper-RJ; Pinedo-HM; Boven-E. A monoclonal antibody against human beta-glucuronidase for application in antibody-directed enzyme prodrug therapy. Hybridoma. 1995 Aug; 14(4): 377-82

The selectivity of anticancer agents may be improved by antibody-directed enzyme prodrug therapy (ADEPT). The immunogenicity of antibody-enzyme conjugates and the low tumor to normal tissue ratio calls for the use of a human enzyme and the development of a monoclonal antibody (MAb) against that enzyme for rapid clearance of the conjugate from the circulation. We isolated beta-glucuronidase from human liver. BALB/c mice were immunized with the roughly purified human liver beta-glucuronidase and we obtained an MAb designated 105. Immunoblotting showed reactivity with native tetrameric human beta-glucuronidase. MAb 105 neither bound to enzyme from bovine liver, rat liver, or mouse liver nor reacted with other human lysosomal enzymes. The antibody appeared to be useful to further purify human beta-glucuronidase from human liver or human placenta to homogeneity by affinity chromatography. MAb 105 did not inhibit the activity of human beta-glucuronidase. When human beta-glucuronidase was injected i.v. into BALB/c mice, the newly generated MAb 105 could indeed accelerate the clearance of the enzyme with a 50% drop in its activity within 5 min. Previous Page

Haisma-HJ; van-Muijen-M; Pinedo-HM; Boven-E. Comparison of two anthracycline-based prodrugs for activation by a monoclonal antibody-beta-glucuronidase conjugate in the specific treatment of cancer. Cell-Biophys. 1994; 24-25: 185-92

Antibody-directed enzyme prodrug therapy (ADEPT) may improve the therapeutic index of cytostatic agents. We compared two prodrugs, epirubicin-glucuronide (Epi-glu) and doxorubicin-spacer-glucuronide (Dox-sp-glu), for their cytotoxicity on activation by a monoclonal antibody-enzyme conjugate bound to tumor cells. The results showed that the prodrugs were 10 (Dox-sp-glu) and 100 (Epi-glu) times less toxic than the parent drugs against OVCAR-3 cells. This difference was a result of the hydrophilic property of the prodrugs resulting in a reduced cellular uptake. The enzyme-catalyzed hydrolysis of Dox-sp-glu by E. coli-derived beta-glucuronidase (GUS) (Km 500 microM, Vmax 21,000 mumol/min/g) was much more efficient than that of Epi-glu (Km 10 microM, Vmax 40 mumol/min/g). Incubation of OVCAR-3 cells with an enzyme-immunoconjugate prepared from monoclonal antibody 323/A3 and E. coli-derived GUS before treatment with prodrugs completely restored the cytotoxicity of the prodrugs to the level of the parent drugs. Previous Page

Rogers-GT; Burke-PJ; Sharma-SK; Koodie-R; Boden-JA. Plasma clearance of an antibody--enzyme conjugate in ADEPT by monoclonal anti-enzyme: its effect on prodrug activation in vivo. Br-J-Cancer. 1995 Dec; 72(6): 1357-63

The effect of anti-enzyme antibody clearance on prodrug turnover in antibody-directed enzyme prodrug therapy (ADEPT) has been studied. Mice bearing LS174T xenografts were given localising carboxypeptidase G2 (CPG)2 conjugate (AEC) and 19 h later galactosylated anti-CPG2 antibody (SB43-GAL). In regimen I prodrug was injected 5 h after SB43-GAL as previously described. In regimen 2 and 3 a shortened and extended clearance time was used in which prodrug was administered 0.5 h or 53 h after SB43-GAL respectively. Regimen 1 resulted in similar tumour and normal tissue levels of active drug to those of the control in which prodrug was given 72 h after AEC. SB43-GAL therefore accelerated clearance of enzyme allowing early administration of prodrug. In regimen 2, very high active drug levels were found in the liver, showing removal of AEC from the blood followed by reactivation of enzyme and extensive and rapid prodrug turnover. Active drug levels in tumour and blood reached similar peak levels to those of the control. Regimen 3 resulted in lower active drug levels in tissues, consistent with degradation and excretion of enzyme. Regimen 3 also produced the best tumour to normal ratios for active drug. Residual prodrug in tumour was unaffected by SB43-GAL, showing the advantage of galactosylation in minimising inactivation of CPG2 in tumour. By contrast, residual prodrug in blood persisted for longer when SB43-GAL was used. Circulatory clearance of enzyme with SB43-GAL allows prodrug to be administered expediently with reduced toxicity and with the prospect of increasing the dosage. Previous Page

Wentworth-P; Datta-A; Blakey-D; Boyle-T; Partridge-LJ; Blackburn-GM. Toward antibody-directed "abzyme" prodrug therapy, ADAPT: carbamate prodrug activation by a catalytic antibody and its in vitro application to human tumor cell killing. Proc-Natl-Acad-Sci-U-S-A. 1996 Jan 23; 93(2): 799-803

Werlen-RC; Lankinen-M; Rose-K; Blakey-D; Shuttleworth-H; Melton-R; Offord-RE. Site-specific conjugation of an enzyme and an antibody fragment. Bioconjug-Chem. 1994 Sep-Oct; 5(5): 411-7

A site-specific immunoconjugate was prepared between an F(ab')2-like fragment of the monoclonal anti-CEA murine IgG1 A5B7 and a mutant of the dimeric enzyme carboxypeptidase G2 possessing an N-terminal Thr in place of Ala. First an aldehyde was introduced at the N-terminus of the enzyme by mild periodate oxidation and a residue of carbohydrazide was specifically introduced at the C-terminus of the truncated heavy chain of the F(ab')2-like fragment by reverse proteolysis. Then the two modified proteins were conjugated by the formation of a hydrazone bond between the hydrazide and the aldehyde groups. The conjugate obtained retained both enzymic activity and antigen-binding capacity. The antigen-binding capacity was better than that of a similar conjugate made conventionally by random reaction with side chains. Previous Page

Anlezark-GM; Melton-RG; Sherwood-RF; Wilson-WR; Denny-WA; Palmer-BD; Knox-RJ; Friedlos-F; Williams-A. Bioactivation of dinitrobenzamide mustards by an E. coli B nitroreductase. Biochem-Pharmacol. 1995 Aug 25; 50(5): 609-18

A nitroreductase isolated and purified from Escherichia coli B has been demonstrated to have potential applications in ADEPT (antibody-directed enzyme prodrug therapy) by its ability in vitro to reduce dinitrobenzamides (e.g. 5-aziridinyl 2,4-dinitrobenzamide, CB 1954 and its bischloroethylamino analogue, SN 23862) to form cytotoxic derivatives. In contrast to CB 1954, in which either nitro group is reducible to the corresponding hydroxylamine, SN 23862 is reduced by the nitroreductase to form only the 2-hydroxylamine. This hydroxylamine can react with S-acetylthiocholine to form a species capable of producing interstrand crosslinks in naked DNA. In terms of ADEPT, SN 23862 has a potential advantage over CB 1954 in that it is not reduced by mammalian DT diaphorases. Therefore, a series of compounds related to SN 23862 has been synthesized, and evaluated as potential prodrugs both by determination of kinetic parameters and by ratio of IC50 against UV4 cells when incubated in the presence of prodrug, with and without the E. coli enzyme and cofactor (NADH). Results from the two studies were generally in good agreement in that compounds showing no increase in cytotoxicity in presence of enzyme and cofactor were not substrates for the enzyme. None of the analogues were activated by DT diaphorase isolated from Walker 256 carcinoma cells. For those compounds which were substrates for the E. coli nitroreductase, there was a positive correlation between kcat and IC50 ratio. Two compounds showed advantageous properties: SN 25261 (with a dihydroxypropylcarboxamide ring substituent) which has a more than 10-fold greater aqueous solubility than SN 23862 whilst retaining similar kinetic characteristics and cytotoxic potency; and SN 25084, where a change in the position of the carboxamide group relative to the mustard resulted in an increased cytotoxicity ratio and kcat compared with SN 23862 (IC50 ratios 214 and 135; kcat values of 75 and 26.4 sec-1, respectively). An analogue (SN 25507) incorporating both these structural changes had an enhanced kcat of 576 sec-1. This study elucidates some of the structural requirements of the enzyme and aids identification of further directions in the search for suitable prodrugs for an ADEPT nitroreductase system. Previous Page