Astrid Mayer

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

Purpose: Antibody-directed enzyme prodrug therapy is a two-stage treatment whereby a tumor-targeted antibody-enzyme complex localizes in tumor for selective conversion of prodrug. The purpose of this study was to establish optimal variables for single administration of MFECP1, a recombinant antibody-enzyme fusion protein of an anti–carcinoembryonic antigen single-chain Fv antibody and the bacterial enzyme carboxypeptidase G2 followed by a bis-iodo phenol mustard prodrug. MFECP1 is manufactured in mannosylated form to facilitate normal tissue elimination. Experimental Design: Pharmacokinetic, biodistribution, and tumor localization studies were used to test the hypothesis that MFECP1 localizes in tumor and clears from normal tissue via the liver. Firstly, safety of MFECP1 and a blood concentration of MFECP1 that would avoid systemic prodrug activation were tested. Secondly, dose escalation of prodrug was done. Thirdly, the dose of MFECP1 and timing of prodrug administration were optimized. Results: MFECP1 was safe and well tolerated, cleared rapidly via the liver, and was less immunogenic than previously used products. Eighty-fold dose escalation from the starting dose of prodrug was carried out before dose-limiting toxicity occurred. Confirmation of the presence of enzyme in tumor and DNA interstrand cross-links indicating prodrug activation were obtained for the optimal dose and time point. A total of 28 of 31 patients was evaluable for response, the best response being a 10% reduction of tumor diameter, and 11 of 28 patients had stable disease. Conclusions: Optimal conditions for effective therapy were established. A study testing repeat treatment is currently being undertaken.

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

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.

Engineering Antibodies for Clinical Applications in Cancer

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.

Recombinant anti-carcinoembryonic antigen antibodies for targeting cancer

Abstract Antibodies can be used to target cancer therapies to malignant tissue; the approach is attractive because conventional treatments such as chemo- and radiotherapy are dose limited due to toxicity in normal tissues. Effective targeting relies on appropriate pharmacokinetics of antibody-based therapeutics, ideally showing maximum uptake and retention in tumor and rapid clearance from normal tissue. We have studied the factors influencing these dynamics for antibodies against carcinoembryonic antigen (CEA). Protein engineering of anti-CEA antibodies, in vivo biodistribution models, and mathematical models have been employed to improve understanding of targeting parameters, define optimal characteristics for the antibody-based molecules employed, and develop new therapies for the clinic. Engineering antibodies to obtain the desired therapeutic characteristics is most readily achieved using recombinant antibody technology, and we have taken the approach of immunizing mice to provide high-affinity anti-CEA single-chain Fv antibodies (sFvs) from filamentous bacteriophage libraries. MFE-23, the most characterized of these sFvs, has been expressed in bacteria and purified in our laboratory for two clinical trials: a gamma camera imaging trial using 123I-MFE-23 and a radioimmunoguided surgery trial using 125I-MFE-23, where tumor deposits are detected by a hand-held probe during surgery. Both these trials showed that MFE-23 is safe and effective in localizing tumor deposits in patients with cancer. We are now developing fusion proteins that use the MFE-23 antibody to deliver a therapeutic moiety; MFE-23:: carboxypeptidase G2 (CPG2) targets the enzyme CPG2 for use in the antibody-directed enzyme prodrug therapy system and MFE::tumor necrosis factor alpha (TNFα) aims to reduce sequestration and increase tumor concentrations of systemically administered TNFα.

Clinical Applications of Phage-Derived sFvs and sFv Fusion Proteins

Single chain Fv antibodies (sFvs) have been produced from filamentous bacteriophage libraries obtained from immunised mice. MFE-23, the most characterised of these sFvs, is reactive with carcinoembryonic antigen (CEA), a glycoprotein that is highly expressed in colorectal adenocarcinomas. MFE-23 has been expressed in bacteria and purified in our laboratory for two clinical trials; a gamma camera imaging trial using 123I-MFE-23 and a radioimmunoguided surgery trial using 125I-MFE-23, where tumour deposits are detected by a hand-held probe during surgery. Both these trials show MFE-23 is safe and effective in localising tumour deposits in patients with cancer. We are now developing fusion proteins which use MFE-23 to deliver a therapeutic moiety; MFE-23::CPG2 targets the enzyme carboxypeptidase G2 (CPG2) for use in the ADEPT (antibody directed enzyme prodrug therapy) system and MFE::TNFα aims to reduce sequestration and increase tumor concentrations of systemically administered TNFα.

Phase I trial combining gemcitabine and treosulfan in advanced cutaneous and uveal melanoma patients.

Gemcitabine and treosulfan are DNA-damaging agents. Preclinical studies suggest that synergism exists when melanoma cells are exposed to both drugs concurrently. We conducted a phase I trial in advanced melanoma patients to determine the optimal dose of gemcitabine to be combined with treosulfan. Cohorts of three patients received increasing doses of gemcitabine, commencing at 0.5 g m−2, followed by a fixed dose of 5.0 g m−2 treosulfan on day one of a 21-day cycle. Patients alternately received a first cycle of single-agent gemcitabine or treosulfan before subsequent cycles of both drugs. Peripheral blood lymphocytes were collected in cycles 1 and 2 at various time points until 48 h post-treatment. The single-cell gel electrophoresis (Comet) assay was used to measure chemotherapy-induced DNA damage. A total of 27 patients were enrolled, no objective responses were observed, but two uveal melanoma patients had minor responses. Dose-limiting myelosuppression was reached at 3.0 g m−2 gemcitabine. DNA single-strand breaks were detected 4 h post-gemcitabine, repaired by 24 h. DNA interstrand crosslinks were detected 4 h post-treosulfan, fully removed by 48 h. Following combination chemotherapy, treosulfan-induced DNA crosslinks persisted, still being detectable 48 h post-treatment, supporting the hypothesis that gemcitabine potentiates treosulfan-induced cytotoxicity. The recommended regimen for further study is 2.5 g m−2 gemcitabine combined with 5.0 g m−2 treosulfan.

Overcoming the immunologic response to foreign enzymes in cancer therapy

Foreign enzymes play a part in a variety of cancer treatments and have the potential for a much greater role. In most cases, repeated administration of the enzyme is required for effective therapy, however, this can be restricted by undesirable effects caused by immunogenicity. Repeated or prolonged treatment can lead to an antibody response, and this may neutralize the enzyme and prevent it from remaining in the circulation. Ultimately, this can lead to a diminished therapeutic effect and increase the risk of infusion reactions. More insidiously, there is a danger that antibodies will not only neutralize the foreign enzyme, but will crossreact with a vital component in normal tissue. Such responses can damage normal tissues and give rise to serious clinical syndromes. The aim for enzymes in cancer therapy is to allow repeated treatments without compromising safety or efficacy. This can potentially be achieved by understanding and controlling the immune response and by modifying the enzyme accordingly. A crucial requirement, and one that is particularly challenging for enzymes to achieve, is that the modifications introduced do not interfere with structure, function or stability.

Patients’ and carers’ experiences of interacting with home haemodialysis technology: implications for quality and safety

BackgroundLittle is known about patients’ and carers’ experiences of interacting with home haemodialysis (HHD) technology, in terms of user experience, how the design of the technology supports safety and fits with home use, and how the broader context of service provision impacts on patients’ use of the technology.MethodsData were gathered through ethnographic observations and interviews with 19 patients and their carers associated with four different hospitals in the UK, using five different HHD machines. All patients were managing their condition successfully on HHD. Data were analysed qualitatively, focusing on themes of how individuals used the machines and how they managed their own safety.ResultsFindings are organised by three themes: learning to use the technology, usability of the technology, and managing safety during dialysis. Home patients want to live their lives fully, and value the freedom and autonomy that HHD gives them; they adapt use of the technology to their lives and their home context. They also consider the machines to be safe; nevertheless, most participants reported feeling scared and having to learn through mistakes in the early months of dialysing at home. Home care nurses and technicians provide invaluable support. Although participants reported on strategies for anticipating problems and keeping safe, perceived limitations of the technology and of the broader system of care led some to trade off safety against immediate quality of life.ConclusionsEnhancing the quality and safety of the patient experience in HHD involves designing technology and the broader system of care to take account of how individuals manage their dialysis in the home. Possible design improvements to enhance the quality and safety of the patient experience include features to help patients manage their dialysis (e.g. providing timely reminders of next steps) and features to support communication between families and professionals (e.g. through remote monitoring).

Higher dose and dose-rate in smaller tumors result in improved tumor control

Small tumors are more sensitive to radioimmunotherapy (RIT) than larger ones. A greater proportion of viable radiosensitive areas in small tumors, higher antibody uptake, and radiation dose may be responsible. Six groups of mice with small (median tumor size 0.06 cm3) or large LoVo xenografts (median tumor size 0.38 cm3) received either RIT using a 131I-labeled anti-CEA antibody A5B7, 5-fluorouracil (5-FU) modulated with folinic acid (FA), or no treatment. The % injected activity/gram, antibody distribution in viable and necrotic areas, and dose distribution were determined. High-power microscopy images of the original section were reconstructed to estimate the proportion of viable areas. Mice with small and large tumors grew significantly less rapidly when treated with RIT compared to the control group (p<0.0004 and p<0.003, respectively), while 5-FU was ineffective. Small tumors treated with RIT grew less than large tumors (p<0.02). A higher amount of % injected activity/gram of tumor (median 26.6% vs. 8.1%,p=0.0007) and a higher dose-rate were found in small tumors at 24 hours post injection (viable areas: 56.2±23.7 vs. 13.3±7 cGy/h, necrosis 19.2±16.3 vs. 4.9±4.7 cGy/h,p=0.0007). It appears that as viable tumor masses grow the access to them decreases and this has a fourfold effect on dose delivered for RIT in this example. These data support the consideration of use of RIT for adjuvant treatment in colon cancer.

Exploring the Current Landscape of Intravenous Infusion Practices and Errors (ECLIPSE): protocol for a mixed-methods observational study

Introduction Intravenous medication is essential for many hospital inpatients. However, providing intravenous therapy is complex and errors are common. ‘Smart pumps’ incorporating dose error reduction software have been widely advocated to reduce error. However, little is known about their effect on patient safety, how they are used or their likely impact. This study will explore the landscape of intravenous medication infusion practices and errors in English hospitals and how smart pumps may relate to the prevalence of medication administration errors. Methods and analysis This is a mixed-methods study involving an observational quantitative point prevalence study to determine the frequency and types of errors that occur in the infusion of intravenous medication, and qualitative interviews with hospital staff to better understand infusion practices and the contexts in which errors occur. The study will involve 5 clinical areas (critical care, general medicine, general surgery, paediatrics and oncology), across 14 purposively sampled acute hospitals and 2 paediatric hospitals to cover a range of intravenous infusion practices. Data collectors will compare each infusion running at the time of data collection against the patients medication orders to identify any discrepancies. The potential clinical importance of errors will be assessed. Quantitative data will be analysed descriptively; interviews will be analysed using thematic analysis. Ethics and dissemination Ethical approval has been obtained from an NHS Research Ethics Committee (14/SC/0290); local approvals will be sought from each participating organisation. Findings will be published in peer-reviewed journals and presented at conferences for academic and health professional audiences. Results will also be fed back to participating organisations to inform local policy, training and procurement. Aggregated findings will inform the debate on costs and benefits of the NHS investing in smart pump technology, and what other changes may need to be made to ensure effectiveness of such an investment.