ADEPT

The objective of antibody directed enzyme prodrug therapy (ADEPT) is to selectively deliver chemotherapy to cancer sites. The basic principle of ADEPT (Fig 1) is to target an enzyme to tumours by attaching it to an antibody directed to a tumour associated antigen. After clearance of enzyme from blood, a non-toxic prodrug is given. The targeted enzyme converts a non-toxic prodrug into a potent cell killing agent within tumours to achieve effective therapy without normal tissue toxicity.

Pre-Clinical studies

I have been involved with ADEPT since its inception in the 1980’s and since then have been responsible for the design and execution of all the pre-clinical studies using in-vivo models. These studies have confirmed the theoretical considerations that underpin the ADEPT concept. In particular, the necessity for removing enzyme activity in blood by inactivation of the enzyme or by its rapid clearance before the prodrug is given. These studies have been the basis for 4 ADEPT clinical trials. The selective delivery of enzyme to tumours with rapid clearance from blood has been achieved both in in-vivo models (Fig 2A and 2B) and in patients (Fig 2C).
A single ADEPT cycle produces growth delay in in-vivo models, while multiple ADEPT cycles produce tumour regressions in both pre-clinical (Fig 3A) and clinical trials (Fig 3B).

CLINICAL TRIALS - ADEPT

The group has critical responsibilities in relation to clinical trials. We develop improved techniques for laboratory measurement of parameters that are important to the care, safety and management of patients. One essential feature of the ADEPT clinical trials has been the need to monitor patient’s blood enzyme levels to ensure that prodrug is given only when it is safe to do so. These measurements are performed by the group in real time.

Other clinical trials

We also perform pharmacokinetic and pharmacodynamic measurements that are an essential component of all Phase I/II clinical trials that are undertaken by the department. These clinical studies include a diverse range of molecules including radiolabelled monoclonal, single chain Fv and chimeric antibodies, as well as combination therapies with anti-vascular agents. In addition, laboratory support is provided for any external Phase II/III studies.

IMMUNOGENICITY OF BIOLOGICS

During the course of therapy with biologics, patients may develop an unwanted immune response to the therapeutic which may affect efficacy and safety. Therefore, the detection of immune response is an essential component of the pre-clinical and clinical studies with antibody-based molecules. In repeated cycles of treatment with radioimmunotherapy or ADEPT, we monitor patients for immune response to the antibody or antibody-enzyme constructs, in real time as the appearance of antibodies to the therapeutic determines the end point of treatment.

We have applied a number of approaches to reduce the immunogenicity of our molecules e.g. identification the immunodominat B cell epitopes on the enzyme and use of immunosuppressive agents. The immunogenicity of engineered new molecules is also studied in-vivo (Fig 4). New immunosuppressive agents as well as human enzymes are also been investigated. We apply a risk-based approach to the assessment and management of immunogenicity in our clinical trials. The group develops an appropriate assay strategy for measurement and confirmation of antibodies directed to the therapeutic. Neutralisation assays are also being developed.

DNA DAMAGE and REPAIR

The prodrugs utilised in our ADEPT studies are converted to bi-functional alkylating agents by the action of the targeted enzyme. The cytotoxicity of these drugs results from drug-induced cross-links in the tumour cell DNA (Fig 5). As these cross-links may be removed by various repair mechanisms, the rate of repair is of importance for therapeutic efficacy. We are investigating the DNA damage and repair effects related to ADEPT and other antibody based therapies, in vitro and in-vivo. A range of agents that inhibit tumour cell DNA repair are being explored. Tumour parameters related to therapeutic response, e.g. gamma H2AX formation, cell proliferation and apoptosis, are being investigated using quantitative, high resolution microscopy.

COMBINATION THERAPIES

We are currently exploring new combination therapies to enhance tumour cell death without affecting the normal tissues. These include studies of new prodrugs, small molecules including anti-vascular, anti-angiogenic agents and complimentary antibodies, which may synergise with ADEPT. Rationally designed successful combinations will then be studied in novel clinical trials.

GCLP Compliance

We lead the CRUK T & I group’s quality assurance programme for Good Clinical Laboratory Practice (GCLP), and closely liaise with Cancer Research-UK Drug development Office and the Quality Assurance management. The conditions for collecting, handling, processing and storing biological samples are standardized along with the appropriate training of clinical trial personnel to ensure the stability and integrity of analyte. We develop bioanalytical assays using different technologies to support pharmacokinetic and pharmacodynamic studies related to clinical trials. The appropriate assay is developed into a validated method which is then eligible for clinical trial application.

Funding:

ECMC (Experimental Cancer Medicine Centre Network)

Group Menebers:

Dr Surinder K Sharma PhD (Group Leader)

Natalie Griffin BSc

Dr Hassan Shahbakhti PhD

Fig1.The basic principle of ADEPT

Fig 2a. Fluorescence images of human colon carcinoma cells showing selective localisation of antibody-enzyme fusion protein (MFECP) in-vivo.
a) Expression of CEA, b) Localisation of i.v. given MFECP fusion protein, c) Co-localisation of CEA and MFECP (yellow) and DAPI (blue) showing tumour cell nuclei (x 400 magnification)
[Sharma et al, Clin Cancer Res., 2005]

Fig 2b. Selective retention of enzyme activity in tumour and rapid clearance of enzyme activity from blood with MFECP in-vivo.

Fig 2c. A liver biopsy specimen from an ADEPT patient showing localisation of antibody-enzyme (MFECP) in metastatic tumour cells.
[Mayer et al, Clin Cancer Res., 2006]

Fig 3a. Multiple cycles of ADEPT result in tumour regression in-vivo.

Fig 3b. Metastatic tumour deposits of colorectal cancer in Liver (arrows) e) before treatment
f) Tumour regression after ADEPT treatment
[Napier et al, Clin Cancer Res.,2000]

Fig 4. Immunogenicity in-vivo.
Second generation recombinant antibody-enzyme fusion protein (MFECP) showing reduced immunogenicity of enzyme [2/8 (M1 and M7) serum samples positive for anti-enzyme antibody] compared to the first generation chemically conjugated antibody-enzyme molecule [8/8 serum samples positive for anti-enzyme antibody].

Fig 5. Effect of ADEPT: A biopsy showing DNA damage in Tumour cells (short Comet tail).
No DNA damage within normal blood lymphocytes (long Comet tail).

Tolner B, Smith L, Hillyer T, Bhatia J, Beckett P, Robson L, Sharma S K, Griffin N, Vervecken W, Contreras R, Pedley RB, Begent RH, Chester KA. From laboratory to Phase I/II cancer trials with recombinant biotherapeutics. Eur J Cancer 2007 Nov;43(17):2515-22.

Kogelberg H, Tolner B, Sharma SK, Lowdell MW, Qureshi U, Robson M, Hillyer T, Pedley RB, Vervecken W, Contreras R, Begent RH, Chester KA (2007) Clearance mechanism of a mannosylated antibody-enzyme fusion protein used in experimental cancer therapy. Glycobiology 17: 36-45

Mayer A, Francis RJ, Sharma SK, Tolner B, Springer CJ, Martin J, Boxer GM, Bell J, Green AJ, Hartley JA, Cruickshank C, Wren J, Chester KA, Begent RH (2006) A phase I study of single administration of antibody-directed enzyme prodrug therapy with the recombinant anti-carcinoembryonic antigen antibody-enzyme fusion protein MFECP1 and a bis-iodo phenol mustard prodrug. Clin Cancer Res 12: 6509-6516

Sharma SK, Pedley RB, Bhatia J, Boxer GM, El-Emir E, Qureshi U, Tolner B, Lowe H, Michael NP, Minton N, Begent RH, Chester KA (2005) Sustained tumor regression of human colorectal cancer xenografts using a multifunctional mannosylated fusion protein in antibody-directed enzyme prodrug therapy. Clin Cancer Res 11: 814-825

Sharma SK, Bagshawe KD, Begent RH. Advances in antibody-directed enzyme prodrug therapy. Curr Opin Investig Drugs 2005 Jun;6(6):611-5.

Mayer A, Sharma SK, Tolner B, Minton NP, Purdy D, Amlot P, Tharakan G, Begent RH, Chester KA (2004) Modifying an immunogenic epitope on a therapeutic protein: a step towards an improved system for antibody-directed enzyme prodrug therapy (ADEPT). Br J Cancer 90: 2402-2410

Napier MP, Sharma SK, Springer CJ, Bagshawe KD, Green AJ, Martin J, Stribbling SM, Cushen N, O'Malley D, Begent RH (2000) Antibody-directed enzyme prodrug therapy: efficacy and mechanism of action in colorectal carcinoma. Clin Cancer Res 6: 765-772

Pedley RB, Sharma SK, Boxer GM, Boden R, Stribbling SM, Davies L, et al. Enhancement of antibody-directed enzyme prodrug therapy in colorectal xenografts by an antivascular agent. Cancer Res 1999 Aug 15;59(16):3998-4003.

Sharma SK, Bagshawe KD, Burke PJ, Boden JA, Rogers GT, Springer CJ, et al. Galactosylated antibodies and antibody-enzyme conjugates in antibody-directed enzyme prodrug therapy. Cancer 1994 Feb 1;73(3 Suppl):1114-20.

Sharma SK, Bagshawe KD, Melton RG, Sherwood RF. Human immune response to monoclonal antibody-enzyme conjugates in ADEPT pilot clinical trial. Cell Biophys 1992 Aug;21(1-3):109-20

Bagshawe KD, Sharma SK, Springer CJ, Antoniw P, Boden JA, Rogers GT, et al. Antibody directed enzyme prodrug therapy (ADEPT): clinical report. Dis Markers 1991 May;9(3-4):233-8.

Bagshawe KD, Springer CJ, Searle F, Antoniw P, Sharma SK, Melton RG, et al. A cytotoxic agent can be generated selectively at cancer sites. Br J Cancer 1988 Dec;58(6):700-3.

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