The complete nucleotide sequence of the Pseudomonas gene coding for carboxypeptidase G2

Minton, Nigel P.; Atkinson, Tony; Bruton, Chris J.; Sherwood, Roger F.

doi:10.1016/0378-1119(84)90192-6

Cited by 103 publications

(71 citation statements)

References 20 publications

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“…This change in absorbance of MTX after CP hydrolysis was measured by spectrophotometry at 320 nm (Beckman DU-64 spectrophotometer, Soft Pac Module KINETICS Software Package, Beckman Instruments Ltd, Bucks, UK) as previously described (Minton et al, 1984). Measurements were performed in duplicate, 10 ml of sample were incubated in 1 ml of assay buffer (100 mM Tris-HCl, pH 7.3, 0.2 mM ZnSO4 and 60 mM MTX) at 371C.…”

Section: Catalytic Activity Of Cpmentioning

confidence: 99%

Modifying an immunogenic epitope on a therapeutic protein: a step towards an improved system for antibody-directed enzyme prodrug therapy (ADEPT)

et al. 2004

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Carboxypeptidase G2 (CP) is a bacterial enzyme, which is targeted to tumours by an antitumour antibody for local prodrug activation in antibody-directed enzyme prodrug therapy (ADEPT). Repeated cycles of ADEPT are desirable but are hampered by human antibody response to CP (HACA). To address this, we aimed to identify and modify clinically important immunogenic sites on MFECP, a recombinant fusion protein of CP with MFE-23, a single chain Fv (scFv) antibody. A discontinuous conformational epitope at the C-terminus of the CP previously identified by the CM79 scFv antibody (CM79-identified epitope) was chosen for study. Modification of MFECP was achieved by mutations of the CM79-identified epitope or by addition of a hexahistidine tag (His-tag) to the C-terminus of MFECP, which forms part of the epitope. Murine immunisation experiments with modified MFECP showed no significant antibody response to the CM79-identified epitope compared to A5CP, an unmodified version of CP chemically conjugated to an F(ab) 2 antibody. Success of modification was also demonstrated in humans because patients treated with His-tagged MFECP had a significantly reduced antibody response to the CM79-identified epitope, compared to patients given A5CP. Moreover, the polyclonal antibody response to CP was delayed in both mice and patients given modified MFECP. This increases the prospect of repeated treatment with ADEPT for effective cancer treatment.

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Section: Catalytic Activity Of Cpmentioning

confidence: 99%

Modifying an immunogenic epitope on a therapeutic protein: a step towards an improved system for antibody-directed enzyme prodrug therapy (ADEPT)

et al. 2004

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“…As CPG2 is not glycosylated (Minton et al, 1984) this form of hepatic clearance mechanism is not a likely complication, but, as already mentioned, antibody-antigen complexes would tend to alter distribution of the anti-hCGbound enzyme towards the liver. It is evident that the exact distribution of a given antibody-protein conjugate will be determined by a number of competing physiological factors.…”

Section: Molecular Weight Analysis Of Blood Samplesmentioning

confidence: 93%

The potential of carboxypeptidase G2: antibody conjugates as anti-tumour agents. II. In vivo localising and clearance properties in a choriocarcinoma model

Melton

Searle²,

Sherwood³

et al. 1990

Br J Cancer

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Summary The in vivo localising and clearance properties of conjugates of the folate-degrading enzyme carboxypeptidase G2 (CPG2) with anti-human chorionic gonadotrophin (W14A) were measured in nude mice bearing CC3 choriocarcinoma xenografts. Conjugates of W14A-F (ab')2 fragment coupled to CPG2 localised in tumour as effectively as native antibody alone but showed lower uptake in other major tissues. The clearance rates of conjugates prepared with intact antibody or F (ab')2 fragment were shown to be up to five-fold faster than for native antibody and two-fold compared to F (ab')2 fragment. Molecular weight analysis of residual conjugate in the blood showed that no degradation of conjugate to its component molecules occurred during circulation. It was concluded that F (ab')2: CPG2 conjugates offered the greatest potential for targeting applications.

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“…The CPG2 structural gene encoding amino acid residue Q26 -K415 inclusive, which had been synthesized from the published sequence, 8 was cloned into the AstraZeneca plasmid pNG3 -VKss -HuCK ( NCIMB deposit no. 40798 ) that contained the human cytomegalovirus (hCMV ) promoter and SV40pA.…”

Section: The Cpg2 Enzymementioning

confidence: 99%

“…bacteria. 8 To target CPG2 to the mammalian secretory pathway, we used the mammalian kappa antibody light chain secretory signal sequence ( amino acid sequence KLAATMDFQVQIFSFLLISASVIMSR ), with enzyme expression driven by a hCMV promoter.…”

Section: Generation Of Adenoviral Vectors Expressing Secreted and Gpimentioning

confidence: 99%

Adenovirus vector–mediated delivery of the prodrug-converting enzyme carboxypeptidase G2 in a secreted or GPI-anchored form: High-level expression of this active conditional cytotoxic enzyme at the plasma membrane

et al. 2002

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Carboxypeptidase G2 ( CPG2 ) is a powerful prodrug -converting enzyme. Without a requirement for endogenous enzymes or cofactors, it can directly activate mustard alkylating prodrugs to cytotoxic species, killing both quiescent and dividing cells. This paper provides the first report of its use in the context of a clinically relevant delivery vehicle using adenovirus vectors. To strengthen the efficacy of the prodrug -activating system, the enzyme has been engineered to be secreted or glycosylphosphatidylinositol ( GPI ) anchored to the extracellular membrane of tumor cells, resulting in an enhanced bystander effect by facilitating diffusion of the active drug through extracellular, rather than intracellular, activation. Using the vectors, we have achieved expression of functional secreted or GPI -anchored CPG2 in a panel of tumor cell lines demonstrating no loss in efficacy as a result of GPI anchor retention. Despite variable transduction efficiencies inherent to these vectors, greater than 50% cell kill was achievable in all of the cell lines tested following only a single exposure to the prodrug ZD2767P. Even in cell lines refractive to infection with the vectors, substantial cell death was recorded, indicative of the enhanced bystander effect generated following extracellular prodrug activation. A direct evaluation of the efficacy of our system has been made against adenoviral delivery of herpes simples virus thymidine kinase plus ganciclovir ( GCV ), a suicide gene therapy approach already in the clinic. In a short -term human glioma culture ( IN1760 ) resistant to the clinical chemotherapeutic drug CCNU ( 1 -( 2 -chloroethyl ) -3 -cyclohexyl -1 -nitrosourea ), thymidine kinase / GCV effected no cell killing compared to 70% cell killing with our system.

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The complete nucleotide sequence of the Pseudomonas gene coding for carboxypeptidase G2

Cited by 103 publications

References 20 publications

Modifying an immunogenic epitope on a therapeutic protein: a step towards an improved system for antibody-directed enzyme prodrug therapy (ADEPT)

Modifying an immunogenic epitope on a therapeutic protein: a step towards an improved system for antibody-directed enzyme prodrug therapy (ADEPT)

The potential of carboxypeptidase G2: antibody conjugates as anti-tumour agents. II. In vivo localising and clearance properties in a choriocarcinoma model

Adenovirus vector–mediated delivery of the prodrug-converting enzyme carboxypeptidase G2 in a secreted or GPI-anchored form: High-level expression of this active conditional cytotoxic enzyme at the plasma membrane

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