Markus Deckert - Academia.edu (original) (raw)
Papers by Markus Deckert
Molecular and Clinical Oncology, 2021
Whether a patient receives general or specialized outpatient palliative cancer care rarely follow... more Whether a patient receives general or specialized outpatient palliative cancer care rarely follows clear criteria, leading to undertreatment or overtreatment. Detailed scores exist to predict prognosis, but not treatment requirements, leaving caregivers to follow their intuition. As a phenomenological indicator incorporating possibly important subjective information, intuition may in fact be a helpful tool. In this prospective observational study, a score to estimate three global dimensions of patients' resources was applied: Medical prognosis, feeling of strength and feeling of support. The score results were correlated with the actual amount and effort of care required during the subsequent palliative care time. This phenomenological score correlated well with the performance index and the Hospice and Palliative care Evaluation score. Whilst various individual items correlated significantly with the score or its constituent parameters, there was no uniform coherent pattern, re...
International Journal of Hematology
Healthcare
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
Annals of Translational Medicine
Background: DNA double-strand breaks can be counted as discrete foci by imaging techniques. In pe... more Background: DNA double-strand breaks can be counted as discrete foci by imaging techniques. In personalized medicine and pharmacology, the analysis of counting data is relevant for numerous applications, e.g., for cancer and aging research and the evaluation of drug efficacy. By default, it is assumed to follow the Poisson distribution. This assumption, however, may lead to biased results and faulty conclusions in datasets with excess zero values (zero-inflation), a variance larger than the mean (overdispersion), or both. In such cases, the assumption of a Poisson distribution would skew the estimation of mean and variance, and other models like the negative binomial (NB), zero-inflated Poisson or zero-inflated NB distributions should be employed. The model chosen has an influence on the parameter estimation (mean value and confidence interval). Yet the choice of the suitable distribution model is not trivial. Methods: To support, simplify and objectify this process, we have developed the countfitteR software as an R package. We used a Bayesian approach for distribution model selection and the shiny web application framework for interactive data analysis. Results: We show the application of our software based on examples of DNA double-strand break count data from phenotypic imaging by multiplex fluorescence microscopy. In analyzing numerous datasets of molecular pharmacological markers (phosphorylated histone H2AX and p53 binding protein), countfitteR demonstrated an equal or superior statistical performance compared to the usually employed two-step procedure, with an overall power of up to 98%. In addition, it still gave information in cases with no result at all from the two-step procedure. In our data sample we found that the NB distribution was the most frequent, with the Poisson distribution taking second place. Conclusions: countfitteR can perform an automated distribution model selection and thus support the data analysis and lead to objective statistically verifiable estimated values. Originally designed for the analysis of foci in biomedical image data, countfitteR can be used in a variety of areas where non-Poisson distributed
Zeitschrift für Palliativmedizin
Journal of Laboratory and Precision Medicine
Diffuse large B-cell lymphoma (DLBCL) is a highly aggressive form of non-Hodgkin's lymphoma with ... more Diffuse large B-cell lymphoma (DLBCL) is a highly aggressive form of non-Hodgkin's lymphoma with a long-term survival rate of around 60% upon standard treatment with rituximab and chemotherapy. While this marks a great improvement over the pre-rituximab/pre-biologicals era, DLBCLs remain a heterogeneous disease with marked variability in clinical outcome. Efforts have been undertaken to predict its course based on characteristics such as the activated B-cell (ABC) versus germinal centre B-cell (GCB) types, but so far none of these has had much impact on treatment and outcome, and the international prognostic index, based on clinical and basic laboratory features, remains the best risk predictor. This review focuses on 13 biomarkers that have been described in the context of diagnostic tests, clinical studies or basic research related to solid and haematological malignancies in general or DLBCL in particular. These include biomarkers of the DNA repair response such as the phosphorylated histone variant 2AX (γH2AX), p53 binding protein (53BP1), Ataxia telangiectasia mutated protein (ATM), Ataxia telangiectasia and Rad3-related protein (ATR), the DNA-dependent protein kinase (DNA-PK) and Rad51 as well as proto-oncogene products like Myc, B-cell lymphoma 2 (Bcl-2), breast cancer 1 (BRCA1) and signal transducer and activator of transcription 3 (STAT3). We have also included 8-Hydroxy-2'-desoxyguanosine (8-OHdG), a marker for oxidative stress, and discuss a link between two proteins involved in apoptosis, the proteasome activator 28γ (PA28γ) and tumour suppressor p53. Our investigation revealed a connection between almost all biomarkers, which are based on methods such as indirect immunofluorescence, fluorescence in-situ hybridization or immunohistochemistry.
Journal of Cellular Biotechnology
Journal of Laboratory and Precision Medicine
Background: DNA double strand breaks (DSBs) are the most severe form of DNA damage in eukaryotic ... more Background: DNA double strand breaks (DSBs) are the most severe form of DNA damage in eukaryotic cells treated with ionizing radiation or chemotherapeutic drugs. They can be quantitatively assessed by fluorescence imaging of phosphorylated histone protein H2AX (γH2AX), where the number of γH2AX foci represents the number of DNA DSB. Real-time assessment of DSB could help tailoring cytotoxic therapies to individual patients regarding both response and adverse events. This would require reliable automated quantification technology not yet routinely available. Here we explore this concept in the context of malignant lymphoma. Methods: To investigate the DSB response to cytotoxic treatment in vitro, peripheral lymphocytes of healthy donors were incubated with bendamustine, an alkylating drug commonly used in lymphoma therapy. To mimic the clinical setting, the drug concentration per number of donor cells was either calculated as a standard dose or based on the body surface area of the individual donor. DNA DSB were quantified by an automatized immunofluorescence γH2AX assay using the AKLIDES NUK ® system. Results: Across all donors, the mean number of γH2AX foci per cell was 1.29, IQR (1.08) after bendamustine treatment as opposed to 0.04, IQR (0.125) in untreated freshly isolated peripheral blood mononuclear cells (PBMCs). The standardized incubation dosage resulted in a mean of 0.89, IQR (0.51) foci per cell, while individualized dose calculation yielded 1.57, IQR (0.5) foci per cell. The difference in γH2AX foci between the two dosage calculations was significant (P=0.036). In addition, we observed a trend towards a negative correlation between the donors' body surface area and the number of foci per cell. Between donors, no significant correlation of the number of foci in response to a given dose was observed. Dose titrations on the cells of individual donors demonstrated a significant response (P<0.05) between dose of bendamustine and the number of γH2AX foci per cell. Conclusions: The automatic AKLIDES analysis platform can assess γH2AX foci for routine use. The individual γH2AX foci response to in vitro bendamustine is dose-dependent and can be monitored timely. Calculating individual in vitro dosage from the donor's body surface area resulted in a broad variation of foci counts between individuals, suggesting that this dosage method does not result in equivalent biological effects among different individuals. Adjusting the dose individually based on biological responses such as DNA DSB could offer a way of personalized medicine with conventional substances, reducing toxicity while increasing therapeutic efficacy.
Encyclopedia of Cancer, 2014
Handbook of Therapeutic Antibodies, 2000
Dübel/Handbook of Therapeutic Antibodies, 2014
International journal of oncology, 2004
In antibody-directed enzyme-prodrug therapy (ADEPT), antibody-enzyme conjugates specifically acti... more In antibody-directed enzyme-prodrug therapy (ADEPT), antibody-enzyme conjugates specifically activate non-toxic prodrugs in tumour tissue. The A33 cognate antigen is a promising target for immunotherapy of gastrointestinal cancers. We have explored A33-based ADEPT with carboxypeptidase A (CPA) and the prodrug, methotrexate-phenylalanine (MTX-Phe). In A33-positive SW1222 cells, the toxicity of MTX-Phe was about 3 logarithms lower compared to MTX. Preincubation with a huA33 antibody-CPA conjugate (huA33-CPA), but not with an isotypic control conjugate, rendered MTX-Phe equally toxic to MTX. No toxicity was observed in mice receiving MTX-Phe in 8-fold the LD50 of MTX. Nude mice bearing A33-positive SW1222 colon carcinoma xenografts were injected intravenously (IV) with 125I-labeled huA33-CPA. The conjugate localised to the tumour with a maximum from 6-24 h. Pre-treating these mice with excess A33 substantially reduced subsequent conjugate uptake, demonstrating immunologic specificity o...
Cancer biotherapy & radiopharmaceuticals, 1996
Quinoline-3-carboxamide (Linomide) is a novel, synthetic immunomodulator acting via immunologic a... more Quinoline-3-carboxamide (Linomide) is a novel, synthetic immunomodulator acting via immunologic and non-immunologic mechanisms. It has shown efficacy against various malignancies, experimental autoimmune encephalomyelitis, and septic shock in animal models and has been investigated for clinical use in minimal residual myeloid leukemia with promising results. Interleukin-2 has shown considerable efficacy in palliative anti-tumor-treatment of advanced renal cell cancer, revealing remission rates of up to 40% in combination therapy regimens. Linomide is reported to exhibit synergistic effects with interleukin-2. Here we report on a clinical phase I/II study examining tolerance and efficacy of a combination therapy schedule of SQ interleukin-2 and PO Linomide in advanced renal cell cancer. Seventeen patients received 10 IU/m2 interleukin-2 per week for 8 weeks, resting interleukin-2 for another 8 weeks. In week 5 they started 5 mg Linomide daily, continued with 10 mg from week 7 to 16. ...
International Journal of Oncology, 2007
Antibody-directed enzyme-prodrug therapy (ADEPT) aims at improving the specificity of conventiona... more Antibody-directed enzyme-prodrug therapy (ADEPT) aims at improving the specificity of conventional chemotherapy by employing artificial antibody-enzyme constructs to convert a non-toxic prodrug into a cytotoxic agent specifically localized to the tumor site. The gpA33 antigen is a promising target for ADEPT in colon cancer, as it is expressed by >95% of human colon cancers, but is absent in all non-gastrointestinal tissues. We designed a recombinant fusion construct of a phage display-generated anti-gpA33 single chain fragment, A33scFv, with cytosine deaminase from yeast (CDy), which converts 5-fluorocytosine (5-FC) into 5-fluorouracil (5-FU). The resulting construct, A33scFv::CDy, was overexpressed in Pichia pastoris and secreted into culture supernatant. The fusion protein was purified by affinity chromatography on protein L. Silver-staining after SDSpolyacrylamide gel electrophoresis confirmed molecular mass and purity. Antibody binding and specificity were quantified by flow cytometry. The complete ADEPT system was applied in vitro on gpA33-positive LIM1215 cells, assessing cell survival by a fluorescein diacetate assay. Cytotoxicity of the prodrug 5-FC after A33scFv::CDy binding was equimolar to that of 5-FU, and this effect depended specifically on both antibody and enzyme function. These results demonstrate bifunctional activity of the heterogeneous Pichia-produced A33scFv::CDy fusion protein and proof of principle for the ADEPT system proposed herein.
![Research paper thumbnail of Biodistribution and efficacy of [131I]A33scFv::CDy, a recombinant antibody-enzyme protein for colon cancer](https://attachments.academia-assets.com/71369530/thumbnails/1.jpg)
](International Journal of Oncology, 2008
In antibody-directed enzyme-prodrug therapy (ADEPT), an antibody-bound enzyme localizes to tumor ... more In antibody-directed enzyme-prodrug therapy (ADEPT), an antibody-bound enzyme localizes to tumor tissue, where it selectively converts a subsequently administered non-toxic prodrug into a cytotoxic drug. A33scFv::CDy is a bifunctional fusion construct comprising a single chain antibody against the gpA33 antigen and the prodrug-converting enzyme cytosine deaminase. gpA33 is highly and homogeneously expressed in >95% of all colorectal cancers. Here we describe the biodistribution and tumor-targeting capacity of 131 I labeled A33scFv::CDy. 131 I labeling of A33scFv::CDy was performed by the chloramine-T method, and the properties of the resulting [ 131 I]A33scFv::CDy conjugate were determined in vivo and in vitro, including biodistribution studies in nude mice bearing human LIM1215 colon carcinoma xenografts. The [ 131 I]A33scFv::CDy conjugate bound specifically to colorectal cancer cells in vitro with KD = 15.8 nM as determined by a saturation assay. in vivo, the tumor uptake of [ 131 I]A33scFv::CDy peaked at 87% injected dose/g 47 h post injection. Normal tissue uptake was low, and activity in blood was lower than in tumor at all time-points studied (6-92 h). The tumor-to-blood ratio increased over time with a maximum of 8.1 at 67 h post injection. [ 131 I]A33scFv::CDy thus shows a biodistribution that makes it attractive for both radioimmunotherapy (RIT) and ADEPT. Preliminary therapeutic experiments showed a significant reduction of tumor size in mice treated with the A33scFv::CDy-5fluorocytosine/5-fluorouracil ADEPT system. This work demonstrates the feasibility of ADEPT and RIT based on the A33scFv::CDy recombinant construct.
Cancer Biotherapy, 1994
Serum concentrations of Interleukin-6 (IL-6) were determined in renal cell carcinoma patients tre... more Serum concentrations of Interleukin-6 (IL-6) were determined in renal cell carcinoma patients treated with low-dose subcutaneous human recombinant interleukin-2 (rIL-2). In all patients, administration of rIL-2 resulted in a significant increase in IL-6 serum levels to peak values within 4 to 6 hours as measured by enzyme-linked immunosorbent assays (ELISA). Repetitive administration of rIL-2 induced significantly lower IL-6 serum peaks when compared to the initial administration of rIL-2. Cumulative IL-6 release, as expressed by the area under the concentration curve (AUC), appeared to be independent of rIL-2 dose distribution (10 million IU rIL-2/m2 versus 20 million IU rIL-2/m2), and IL-6 serum peaks showed no direct dose dependency. Prior rIL-2 immunotherapy had no measurable effect on rIL-2 induced IL-6 release, while steroids resulted in a significant suppression of secondary IL-6 did not correlate with response to rIL-2 therapy or survival of rIL-2 treated renal cell carcinoma patients.
Tumor Biology, 1993
Natural killer (NK) cells are believed to contribute to the clinical efficacy of cancer immunothe... more Natural killer (NK) cells are believed to contribute to the clinical efficacy of cancer immunotherapy using recombinant interleukin-2 (rIL-2) in humans. In previous trials of high-dose i.v. rIL-2, however, no correlation has been established between circulating NK cells and treatment response. Between January 1989 and October 1990, we treated a total of 47 outpatients with advanced tumors using low-dose s.c. rIL-2 and interferon-alpha-2 (rIFN-alpha). Therapy consisted of a 2-day rIL-2 pulse at 18 million IU/m2/day, followed by 6 weeks of rIL-2 (3.6 x 10(6)-4.8 x 10(6) IU/m2/day x 5 days/week) and rIFN-alpha (5 x 10(6)-6 x 10(6) U/m2 x 3/week). Before and after therapy, we phenotypically evaluated circulating lymphocytes and correlated them with clinical response. During 6-week therapy, peripheral blood lymphocytes bearing the CD56 (NK-cell-associated) surface antigen were increased significantly (p < or = 0.005) in treatment responders [complete response (CR) and partial response (PR), n = 10; 3.8-fold] and stable disease (SD) patients (n = 20; 2.1-fold), while patients with progressive disease (PD, n = 17) exhibited no significant expansion of circulating NK cells (p > 0.1). After one 6-week treatment cycle, CR/PR patients had significantly more peripheral NK cells, when compared with patients in SD (1.6-fold) and PD (1.9-fold) (p < 0.04). The overall number of circulating lymphocytes was also increased upon therapy (1.6-fold; p < or = 0.001), but remained independent of response (p > 0.4). These data demonstrate that s.c. rIL-2 and s.c. rIFN-alpha produce a significant increase in peripheral blood NK cells; this expansion correlates significantly with treatment response in advanced tumor patients receiving long-term combination immunotherapy at outpatient doses.
Protein Engineering Design and Selection, 2007
Chemical conjugates of monoclonal antibodies with fluorophores or enzymes have long been used for... more Chemical conjugates of monoclonal antibodies with fluorophores or enzymes have long been used for diagnostic purposes and experimental therapeutic approaches. Recombinant technology allows for the design and expression of tailored genuine fusion proteins, providing defined molecules as to size, molar ratios of the functional components and stability. The production of functional protein, however, is often limited or impossible due to refolding and solubility problems. Here, we report on the production of a soluble recombinant fusion construct, A33scFv-green fluorescent protein (A33scFv::GFP) in Pichia pastoris. A33scFv is a single-chain antibody recognizing the A33 antigen, which is expressed by approximately 95% of colorectal carcinomas and has become a focus of pre-clinical and clinical investigation. The fusion partner GFP was selected both as an experimental tool for functional studies of the A33 antigen and as a potential diagnostic for colon cancer detection and therapy planning. Pichia pastoris yeast strains were transformed with A33scFv::GFP cDNA under the methanol-inducible AOX1 promotor. The construct was properly expressed and secreted into culture supernatants as a soluble protein, which was bifunctional without additional renaturation or solubilization steps. The crude protein solution was purified by affinity chromatography. Surface plasmon resonance, flow cytometry and fluorescence microscopy on sections of normal and cancerous colon tissue revealed specific binding and the applicability of this fusion protein for diagnostic purposes. In addition, the biodistribution of A33scFv::GFP was analyzed in mice bearing A33-positive tumor xenografts, confirming specific tumor targeting.
Molecular and Clinical Oncology, 2021
Whether a patient receives general or specialized outpatient palliative cancer care rarely follow... more Whether a patient receives general or specialized outpatient palliative cancer care rarely follows clear criteria, leading to undertreatment or overtreatment. Detailed scores exist to predict prognosis, but not treatment requirements, leaving caregivers to follow their intuition. As a phenomenological indicator incorporating possibly important subjective information, intuition may in fact be a helpful tool. In this prospective observational study, a score to estimate three global dimensions of patients' resources was applied: Medical prognosis, feeling of strength and feeling of support. The score results were correlated with the actual amount and effort of care required during the subsequent palliative care time. This phenomenological score correlated well with the performance index and the Hospice and Palliative care Evaluation score. Whilst various individual items correlated significantly with the score or its constituent parameters, there was no uniform coherent pattern, re...
International Journal of Hematology
Healthcare
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
Annals of Translational Medicine
Background: DNA double-strand breaks can be counted as discrete foci by imaging techniques. In pe... more Background: DNA double-strand breaks can be counted as discrete foci by imaging techniques. In personalized medicine and pharmacology, the analysis of counting data is relevant for numerous applications, e.g., for cancer and aging research and the evaluation of drug efficacy. By default, it is assumed to follow the Poisson distribution. This assumption, however, may lead to biased results and faulty conclusions in datasets with excess zero values (zero-inflation), a variance larger than the mean (overdispersion), or both. In such cases, the assumption of a Poisson distribution would skew the estimation of mean and variance, and other models like the negative binomial (NB), zero-inflated Poisson or zero-inflated NB distributions should be employed. The model chosen has an influence on the parameter estimation (mean value and confidence interval). Yet the choice of the suitable distribution model is not trivial. Methods: To support, simplify and objectify this process, we have developed the countfitteR software as an R package. We used a Bayesian approach for distribution model selection and the shiny web application framework for interactive data analysis. Results: We show the application of our software based on examples of DNA double-strand break count data from phenotypic imaging by multiplex fluorescence microscopy. In analyzing numerous datasets of molecular pharmacological markers (phosphorylated histone H2AX and p53 binding protein), countfitteR demonstrated an equal or superior statistical performance compared to the usually employed two-step procedure, with an overall power of up to 98%. In addition, it still gave information in cases with no result at all from the two-step procedure. In our data sample we found that the NB distribution was the most frequent, with the Poisson distribution taking second place. Conclusions: countfitteR can perform an automated distribution model selection and thus support the data analysis and lead to objective statistically verifiable estimated values. Originally designed for the analysis of foci in biomedical image data, countfitteR can be used in a variety of areas where non-Poisson distributed
Zeitschrift für Palliativmedizin
Journal of Laboratory and Precision Medicine
Diffuse large B-cell lymphoma (DLBCL) is a highly aggressive form of non-Hodgkin's lymphoma with ... more Diffuse large B-cell lymphoma (DLBCL) is a highly aggressive form of non-Hodgkin's lymphoma with a long-term survival rate of around 60% upon standard treatment with rituximab and chemotherapy. While this marks a great improvement over the pre-rituximab/pre-biologicals era, DLBCLs remain a heterogeneous disease with marked variability in clinical outcome. Efforts have been undertaken to predict its course based on characteristics such as the activated B-cell (ABC) versus germinal centre B-cell (GCB) types, but so far none of these has had much impact on treatment and outcome, and the international prognostic index, based on clinical and basic laboratory features, remains the best risk predictor. This review focuses on 13 biomarkers that have been described in the context of diagnostic tests, clinical studies or basic research related to solid and haematological malignancies in general or DLBCL in particular. These include biomarkers of the DNA repair response such as the phosphorylated histone variant 2AX (γH2AX), p53 binding protein (53BP1), Ataxia telangiectasia mutated protein (ATM), Ataxia telangiectasia and Rad3-related protein (ATR), the DNA-dependent protein kinase (DNA-PK) and Rad51 as well as proto-oncogene products like Myc, B-cell lymphoma 2 (Bcl-2), breast cancer 1 (BRCA1) and signal transducer and activator of transcription 3 (STAT3). We have also included 8-Hydroxy-2'-desoxyguanosine (8-OHdG), a marker for oxidative stress, and discuss a link between two proteins involved in apoptosis, the proteasome activator 28γ (PA28γ) and tumour suppressor p53. Our investigation revealed a connection between almost all biomarkers, which are based on methods such as indirect immunofluorescence, fluorescence in-situ hybridization or immunohistochemistry.
Journal of Cellular Biotechnology
Journal of Laboratory and Precision Medicine
Background: DNA double strand breaks (DSBs) are the most severe form of DNA damage in eukaryotic ... more Background: DNA double strand breaks (DSBs) are the most severe form of DNA damage in eukaryotic cells treated with ionizing radiation or chemotherapeutic drugs. They can be quantitatively assessed by fluorescence imaging of phosphorylated histone protein H2AX (γH2AX), where the number of γH2AX foci represents the number of DNA DSB. Real-time assessment of DSB could help tailoring cytotoxic therapies to individual patients regarding both response and adverse events. This would require reliable automated quantification technology not yet routinely available. Here we explore this concept in the context of malignant lymphoma. Methods: To investigate the DSB response to cytotoxic treatment in vitro, peripheral lymphocytes of healthy donors were incubated with bendamustine, an alkylating drug commonly used in lymphoma therapy. To mimic the clinical setting, the drug concentration per number of donor cells was either calculated as a standard dose or based on the body surface area of the individual donor. DNA DSB were quantified by an automatized immunofluorescence γH2AX assay using the AKLIDES NUK ® system. Results: Across all donors, the mean number of γH2AX foci per cell was 1.29, IQR (1.08) after bendamustine treatment as opposed to 0.04, IQR (0.125) in untreated freshly isolated peripheral blood mononuclear cells (PBMCs). The standardized incubation dosage resulted in a mean of 0.89, IQR (0.51) foci per cell, while individualized dose calculation yielded 1.57, IQR (0.5) foci per cell. The difference in γH2AX foci between the two dosage calculations was significant (P=0.036). In addition, we observed a trend towards a negative correlation between the donors' body surface area and the number of foci per cell. Between donors, no significant correlation of the number of foci in response to a given dose was observed. Dose titrations on the cells of individual donors demonstrated a significant response (P<0.05) between dose of bendamustine and the number of γH2AX foci per cell. Conclusions: The automatic AKLIDES analysis platform can assess γH2AX foci for routine use. The individual γH2AX foci response to in vitro bendamustine is dose-dependent and can be monitored timely. Calculating individual in vitro dosage from the donor's body surface area resulted in a broad variation of foci counts between individuals, suggesting that this dosage method does not result in equivalent biological effects among different individuals. Adjusting the dose individually based on biological responses such as DNA DSB could offer a way of personalized medicine with conventional substances, reducing toxicity while increasing therapeutic efficacy.
Encyclopedia of Cancer, 2014
Handbook of Therapeutic Antibodies, 2000
Dübel/Handbook of Therapeutic Antibodies, 2014
International journal of oncology, 2004
In antibody-directed enzyme-prodrug therapy (ADEPT), antibody-enzyme conjugates specifically acti... more In antibody-directed enzyme-prodrug therapy (ADEPT), antibody-enzyme conjugates specifically activate non-toxic prodrugs in tumour tissue. The A33 cognate antigen is a promising target for immunotherapy of gastrointestinal cancers. We have explored A33-based ADEPT with carboxypeptidase A (CPA) and the prodrug, methotrexate-phenylalanine (MTX-Phe). In A33-positive SW1222 cells, the toxicity of MTX-Phe was about 3 logarithms lower compared to MTX. Preincubation with a huA33 antibody-CPA conjugate (huA33-CPA), but not with an isotypic control conjugate, rendered MTX-Phe equally toxic to MTX. No toxicity was observed in mice receiving MTX-Phe in 8-fold the LD50 of MTX. Nude mice bearing A33-positive SW1222 colon carcinoma xenografts were injected intravenously (IV) with 125I-labeled huA33-CPA. The conjugate localised to the tumour with a maximum from 6-24 h. Pre-treating these mice with excess A33 substantially reduced subsequent conjugate uptake, demonstrating immunologic specificity o...
Cancer biotherapy & radiopharmaceuticals, 1996
Quinoline-3-carboxamide (Linomide) is a novel, synthetic immunomodulator acting via immunologic a... more Quinoline-3-carboxamide (Linomide) is a novel, synthetic immunomodulator acting via immunologic and non-immunologic mechanisms. It has shown efficacy against various malignancies, experimental autoimmune encephalomyelitis, and septic shock in animal models and has been investigated for clinical use in minimal residual myeloid leukemia with promising results. Interleukin-2 has shown considerable efficacy in palliative anti-tumor-treatment of advanced renal cell cancer, revealing remission rates of up to 40% in combination therapy regimens. Linomide is reported to exhibit synergistic effects with interleukin-2. Here we report on a clinical phase I/II study examining tolerance and efficacy of a combination therapy schedule of SQ interleukin-2 and PO Linomide in advanced renal cell cancer. Seventeen patients received 10 IU/m2 interleukin-2 per week for 8 weeks, resting interleukin-2 for another 8 weeks. In week 5 they started 5 mg Linomide daily, continued with 10 mg from week 7 to 16. ...
International Journal of Oncology, 2007
Antibody-directed enzyme-prodrug therapy (ADEPT) aims at improving the specificity of conventiona... more Antibody-directed enzyme-prodrug therapy (ADEPT) aims at improving the specificity of conventional chemotherapy by employing artificial antibody-enzyme constructs to convert a non-toxic prodrug into a cytotoxic agent specifically localized to the tumor site. The gpA33 antigen is a promising target for ADEPT in colon cancer, as it is expressed by >95% of human colon cancers, but is absent in all non-gastrointestinal tissues. We designed a recombinant fusion construct of a phage display-generated anti-gpA33 single chain fragment, A33scFv, with cytosine deaminase from yeast (CDy), which converts 5-fluorocytosine (5-FC) into 5-fluorouracil (5-FU). The resulting construct, A33scFv::CDy, was overexpressed in Pichia pastoris and secreted into culture supernatant. The fusion protein was purified by affinity chromatography on protein L. Silver-staining after SDSpolyacrylamide gel electrophoresis confirmed molecular mass and purity. Antibody binding and specificity were quantified by flow cytometry. The complete ADEPT system was applied in vitro on gpA33-positive LIM1215 cells, assessing cell survival by a fluorescein diacetate assay. Cytotoxicity of the prodrug 5-FC after A33scFv::CDy binding was equimolar to that of 5-FU, and this effect depended specifically on both antibody and enzyme function. These results demonstrate bifunctional activity of the heterogeneous Pichia-produced A33scFv::CDy fusion protein and proof of principle for the ADEPT system proposed herein.
International Journal of Oncology, 2008
In antibody-directed enzyme-prodrug therapy (ADEPT), an antibody-bound enzyme localizes to tumor ... more In antibody-directed enzyme-prodrug therapy (ADEPT), an antibody-bound enzyme localizes to tumor tissue, where it selectively converts a subsequently administered non-toxic prodrug into a cytotoxic drug. A33scFv::CDy is a bifunctional fusion construct comprising a single chain antibody against the gpA33 antigen and the prodrug-converting enzyme cytosine deaminase. gpA33 is highly and homogeneously expressed in >95% of all colorectal cancers. Here we describe the biodistribution and tumor-targeting capacity of 131 I labeled A33scFv::CDy. 131 I labeling of A33scFv::CDy was performed by the chloramine-T method, and the properties of the resulting [ 131 I]A33scFv::CDy conjugate were determined in vivo and in vitro, including biodistribution studies in nude mice bearing human LIM1215 colon carcinoma xenografts. The [ 131 I]A33scFv::CDy conjugate bound specifically to colorectal cancer cells in vitro with KD = 15.8 nM as determined by a saturation assay. in vivo, the tumor uptake of [ 131 I]A33scFv::CDy peaked at 87% injected dose/g 47 h post injection. Normal tissue uptake was low, and activity in blood was lower than in tumor at all time-points studied (6-92 h). The tumor-to-blood ratio increased over time with a maximum of 8.1 at 67 h post injection. [ 131 I]A33scFv::CDy thus shows a biodistribution that makes it attractive for both radioimmunotherapy (RIT) and ADEPT. Preliminary therapeutic experiments showed a significant reduction of tumor size in mice treated with the A33scFv::CDy-5fluorocytosine/5-fluorouracil ADEPT system. This work demonstrates the feasibility of ADEPT and RIT based on the A33scFv::CDy recombinant construct.
Cancer Biotherapy, 1994
Serum concentrations of Interleukin-6 (IL-6) were determined in renal cell carcinoma patients tre... more Serum concentrations of Interleukin-6 (IL-6) were determined in renal cell carcinoma patients treated with low-dose subcutaneous human recombinant interleukin-2 (rIL-2). In all patients, administration of rIL-2 resulted in a significant increase in IL-6 serum levels to peak values within 4 to 6 hours as measured by enzyme-linked immunosorbent assays (ELISA). Repetitive administration of rIL-2 induced significantly lower IL-6 serum peaks when compared to the initial administration of rIL-2. Cumulative IL-6 release, as expressed by the area under the concentration curve (AUC), appeared to be independent of rIL-2 dose distribution (10 million IU rIL-2/m2 versus 20 million IU rIL-2/m2), and IL-6 serum peaks showed no direct dose dependency. Prior rIL-2 immunotherapy had no measurable effect on rIL-2 induced IL-6 release, while steroids resulted in a significant suppression of secondary IL-6 did not correlate with response to rIL-2 therapy or survival of rIL-2 treated renal cell carcinoma patients.
Tumor Biology, 1993
Natural killer (NK) cells are believed to contribute to the clinical efficacy of cancer immunothe... more Natural killer (NK) cells are believed to contribute to the clinical efficacy of cancer immunotherapy using recombinant interleukin-2 (rIL-2) in humans. In previous trials of high-dose i.v. rIL-2, however, no correlation has been established between circulating NK cells and treatment response. Between January 1989 and October 1990, we treated a total of 47 outpatients with advanced tumors using low-dose s.c. rIL-2 and interferon-alpha-2 (rIFN-alpha). Therapy consisted of a 2-day rIL-2 pulse at 18 million IU/m2/day, followed by 6 weeks of rIL-2 (3.6 x 10(6)-4.8 x 10(6) IU/m2/day x 5 days/week) and rIFN-alpha (5 x 10(6)-6 x 10(6) U/m2 x 3/week). Before and after therapy, we phenotypically evaluated circulating lymphocytes and correlated them with clinical response. During 6-week therapy, peripheral blood lymphocytes bearing the CD56 (NK-cell-associated) surface antigen were increased significantly (p < or = 0.005) in treatment responders [complete response (CR) and partial response (PR), n = 10; 3.8-fold] and stable disease (SD) patients (n = 20; 2.1-fold), while patients with progressive disease (PD, n = 17) exhibited no significant expansion of circulating NK cells (p > 0.1). After one 6-week treatment cycle, CR/PR patients had significantly more peripheral NK cells, when compared with patients in SD (1.6-fold) and PD (1.9-fold) (p < 0.04). The overall number of circulating lymphocytes was also increased upon therapy (1.6-fold; p < or = 0.001), but remained independent of response (p > 0.4). These data demonstrate that s.c. rIL-2 and s.c. rIFN-alpha produce a significant increase in peripheral blood NK cells; this expansion correlates significantly with treatment response in advanced tumor patients receiving long-term combination immunotherapy at outpatient doses.
Protein Engineering Design and Selection, 2007
Chemical conjugates of monoclonal antibodies with fluorophores or enzymes have long been used for... more Chemical conjugates of monoclonal antibodies with fluorophores or enzymes have long been used for diagnostic purposes and experimental therapeutic approaches. Recombinant technology allows for the design and expression of tailored genuine fusion proteins, providing defined molecules as to size, molar ratios of the functional components and stability. The production of functional protein, however, is often limited or impossible due to refolding and solubility problems. Here, we report on the production of a soluble recombinant fusion construct, A33scFv-green fluorescent protein (A33scFv::GFP) in Pichia pastoris. A33scFv is a single-chain antibody recognizing the A33 antigen, which is expressed by approximately 95% of colorectal carcinomas and has become a focus of pre-clinical and clinical investigation. The fusion partner GFP was selected both as an experimental tool for functional studies of the A33 antigen and as a potential diagnostic for colon cancer detection and therapy planning. Pichia pastoris yeast strains were transformed with A33scFv::GFP cDNA under the methanol-inducible AOX1 promotor. The construct was properly expressed and secreted into culture supernatants as a soluble protein, which was bifunctional without additional renaturation or solubilization steps. The crude protein solution was purified by affinity chromatography. Surface plasmon resonance, flow cytometry and fluorescence microscopy on sections of normal and cancerous colon tissue revealed specific binding and the applicability of this fusion protein for diagnostic purposes. In addition, the biodistribution of A33scFv::GFP was analyzed in mice bearing A33-positive tumor xenografts, confirming specific tumor targeting.