In vitro and in vivo characterisation of a recombinant carboxypeptidase G2::anti-CEA scFv fusion protein

Michael, NP; Chester, KA; Rg, Melton; Robson, L; Nicholas, W. L.; Boden, JA; Pedley, RB; Rh, Begent; Sherwood, R.F.; Minton, Nigel P.

doi:10.1016/1380-2933(96)00033-4

Cited by 64 publications

(41 citation statements)

References 27 publications

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“…The MFE-CPG2 fusion protein gave modest yields in the bacterial expression system but was shown to localize well in human colon carcinoma xenografts in nude mice [24,25]. Clearance was more rapid than for the previously used the F(ab') 2 anti-CEA antibody chemically conjugated to CPG2, and tumor to normal tissue ratios were higher.…”

Section: Antibody-enzyme Moleculementioning

confidence: 99%

Engineering Antibodies for Clinical Applications in Cancer

Chester¹,

Pedley²,

Tolner³

et al. 2004

Tumor Biol

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The ‘magic bullet’ concept predicted over a century ago that antibodies would be used to target cancer therapy. Since then initial problems that were related to specificity, purity and immungenicity of antibody-based reagents have slowly been overcome due to developments in technology and increased knowledge. As a result, antibodies are in use for many clinical applications and now comprise the second largest category of medicines in clinical development after vaccines. For antibody-based cancer therapeutics the last 20 years have met with an explosion of knowledge about the biology of the disease and potential targets as well as new technology which allows cloning and manipulation of multifunctional antibody-based molecules. However, the focus still remains on developing therapeutics that will have potential for treating cancer in people and this is efficiently assessed in mechanistic clinical trials that feed back to the laboratory for further development. This review illustrates the mechanistic approach to making new molecules for antibody imaging and therapy of cancer. It is illustrated by examples of radioimmunotherapy and antibody-directed enzyme prodrug therapy developed by the authors.

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Section: Antibody-enzyme Moleculementioning

confidence: 99%

Engineering Antibodies for Clinical Applications in Cancer

Chester¹,

Pedley²,

Tolner³

et al. 2004

Tumor Biol

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“…Construction of pPM331 (encoding MFECP, a recombinant fusion protein of the anti-CEA scFv MFE-23 and CP, an enzyme derived from Variovorax paradoxus, formerly Pseudomonas sp RS 16) has been described previously (Michael et al, 1996). Plasmid pDP161 (encoding MFEdmCP) is identical to MFECP except for a mutation in both regions encoding the discontinuous immunodominant epitope of CP (R162A and G412A) (Spencer et al, 2002).…”

Section: Construction Expression and Purification Of Proteins In Escmentioning

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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“…25,30,31 Animal studies with xenograft models have shown that enzyme conjugates of smaller size directing molecules such as single-domain antibodies, single chain antibodies (scFv) and peptides may overcome some of the limitations related to large antibody molecules. [32][33][34][35][36][37][38] The present communication describes steps toward development of a novel b-lactamase molecular library in which the target-binding moiety is built into the Enterobacter cloacae P99 cephalosporinase (E.C. 3.5.2.6) enzyme molecule.…”

mentioning

confidence: 99%

Towards a ligand targeted enzyme prodrug therapy: Single round panning of a β‐lactamase scaffold library on human cancer cells

Shukla

Murray²,

Estabrook³

et al. 2007

Intl Journal of Cancer

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A novel b-lactamase scaffold library in which the target-binding moiety is built into the enzyme was generated using phage display technology. The binding element is composed of a fully randomized 8 amino acid loop inserted at position between Y34 and K37 on the outer surface of Enterobacter cloacae P99 cephalosporinase (b-lactamase, E.C. 3.5.2.6) with all library members retaining catalytic activity. The frequency and diversity of amino acids distributions in peptide inserts from library clones were analyzed. The complexity of the randomized loop appears consistent with standards of other types of phage display library systems. The library was panned against SKBR3 human breast cancer cells in 1 round using rolling circle amplification of phage DNA to recover bound phage. Individual b-lactamase clones, independent of phage, were rapidly assessed for their binding to SKBR3 cells using a simple high throughput screen based on cell-bound b-lactamase activity. SKBR3 cell-binding b-lactamase enzymes were also shown to bind specifically using an immunochemical method. Selected b-lactamase clones were further studied for their protein expression, enzyme activity and binding to nontumor celllines. Overall, the approach outlined here offers the opportunity of rapidly selecting targeted b-lactamase ligands that may have a potential for their use in enzyme prodrug therapy with cephalosporin-based prodrugs. It is expected that a similar approach will be useful in developing tumor-targeting molecules of several other enzyme candidates of cancer prodrug therapy. ' 2007 Wiley-Liss, Inc.Key words: phage display; b-lactamase; screening; cancer; targeted enzyme; prodrug therapyThe major problems associated with cytotoxic cancer therapeutic drugs are lack of selectivity for tumor cells over normal cells, insufficient drug concentrations in tumors, systemic toxicity and development of drug resistant cancer cells.1,2 Developing a successful strategy for a targeted delivery of drugs to cancer cells without harming the rest of the body is one of the most important challenges for cancer researchers today. The ability to selectively concentrate or deliver chemotherapeutic drugs to cancer cells may make chemotherapy both more effective and less likely to cause side effects. A variety of strategies are being evaluated to achieve this goal. [3][4][5][6] The majority of cancer-targeting agents developed over the last 2 decades are based on the use of monoclonal antibodies (mAb) to selectively deliver toxic agents such as conventional cytotoxic drugs, radioisotopes, or plantand bacteria-derived toxins for a direct tumor-killing.7-9 These approaches can reduce systemic toxicity; however, the therapeutic results have generally been less than ideal at least in part because of inadequate conjugate uptake into the tumor. 10Another approach that shows promise is targeted prodrug therapy. This is based on the concept that a systemically administered nontoxic prodrug can be converted locally to high concentrations of a cytotoxic drug by an enzyme p...

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In vitro and in vivo characterisation of a recombinant carboxypeptidase G2::anti-CEA scFv fusion protein

Cited by 64 publications

References 27 publications

Engineering Antibodies for Clinical Applications in Cancer

Engineering Antibodies for Clinical Applications in Cancer

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

Towards a ligand targeted enzyme prodrug therapy: Single round panning of a β‐lactamase scaffold library on human cancer cells

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