153 results on '"Bolhuis RL"'
Search Results
2. Hyperbilirubinaemia in patients treated with recombinant human interleukin-2 (rIL-2)
- Author
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Punt, CJA, primary, Henzen-Logmans, SC, additional, Bolhuis, RL, additional, and Stoter, G, additional
- Published
- 1990
- Full Text
- View/download PDF
3. T lymphocyte repopulation and differentiation after bone marrow transplantation. Early shifts in the ratio between T4+ and T8+ T lymphocytes correlate with the occurrence of acute graft-versus-host disease
- Author
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Gratama, JW, Naipal, A, Oljans, P, Zwaan, FE, Verdonck, LF, de Witte, T, Vossen, JM, Bolhuis, RL, de Gast, GC, and Jansen, J
- Abstract
Acute graft-versus-host disease (GVHD), a major complication of allogeneic bone marrow transplantation (BMT), is probably mediated by T lymphocytes present in the marrow graft. In this study, the repopulation of the peripheral blood with T4+ and T8+ T cells was investigated during the period preceding the occurrence of acute GVHD. Twenty-four allogeneic and 11 autologous BMT recipients were monitored from day 4 post-BMT onward by the use of monoclonal antibodies, indirect immunofluorescence, and flow cytometry. The recipients of allogeneic transplants received methotrexate as GVHD prophylaxis. Similar recovery patterns for T4+ and T8+ T cells were found following autologous and allogeneic BMT. However, lymphoid repopulation occurred at a clearly faster rate after autologous BMT. T4+ T cells were the first to reappear in the peripheral blood, followed by T8+ T cells 4–7 days later. The T8+ T cell reconstitution occurred at an even faster rate in patients who were to develop grade II-IV GVHD, as compared with those with grade O-I GVHD, thus leading to an earlier decrease in the T4/T8 ratio. Of 10 patients with a T4/T8 ratio less than 2.5 at day 19, 9 developed grade II-IV GVHD and 1 showed no GVHD. Of 14 patients with a ratio greater than 2.5 at that time, only 2 developed grade II-IV and 12 grade O-I GVHD (p less than 0.001). In the 11 patients developing grade II-IV GVHD, the T4/T8 ratio decreased to values less than 2.5 before the first clinical symptoms of GVHD in 9; it coincided in one and occurred later in another patient. Thus, early monitoring of the T4/T8 ratio can distinguish patients at risk of developing grade II-IV GVHD.
- Published
- 1984
- Full Text
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4. Enzyme analysis of lymphoproliferative diseases: a useful addition to cell surface phenotyping
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van de Griend, RJ, van der Reijden, HJ, Bolhuis, RL, Melief, CJ, von dem Borne, AE, and Roos, D
- Abstract
Lymphocytes from patients with acute and chronic T-cell malignancy or chronic T gamma lymphocytosis were characterized by studying the activity of three enzymes involved in purine metabolism and by determining the isoenzyme pattern of lactate dehydrogenase (LDH) in addition to analysis of surface marker expression with monoclonal antibodies. Four clinically different types of disease were distinguished on the basis of the enzyme parameters. Lymphocytes from patients with acute lymphocytic leukemia (T-ALL) showed an enzyme profile similar to that of normal thymocytes, i.e., an elevated level of adenosine deaminase (ADA) activity as compared with normal T lymphocytes, reduced activities of purine 5'nucleotidase (5'NT) and purine nucleoside phosphorylase (PNP), and a binomial distribution of the LDH isoenzyme pattern. Cells from “null”-ALL patients had an ADA/PNP ratio that was intermediate between that of normal T cells and that of T-ALL cells or thymocytes, but their 5'NT activity and LDH isoenzyme pattern were thymocyte-like. Patients with chronic T-cell proliferation were subdivided into those with chronic T gamma lymphocytosis and those with proven chronic T malignancy. The lymphocytes from these patients had ADA and PNP activities within the ranges of those of normal T lymphocytes. However, the ADA activity and/or the ADA/PNP ratio were consistently higher in the cells from the patients with chronic T gamma lymphocytosis than in those with chronic T malignancy. The enzyme profile of the cells from the T gamma patients was similar to that of T gamma cells of normal individuals. The cells from patients with chronic T malignancies showed a heterogeneous enzyme pattern as compared with that of normal T lymphocytes. Analysis with monoclonal antibodies enabled us to distinguish null-ALL patients from the other leukemias studied, but a distinction between chronic and acute T-cell proliferation disease, for instance, was not possible with monoclonal antibodies alone. Our data demonstrate that the enzyme profiles studied provide supplementary information for classification and diagnosis of lymphoproliferative diseases to that obtained with cell surface markers alone.
- Published
- 1983
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5. In vitro expansion and analysis of cloned cytotoxic T cells derived from patients with chronic T gamma lymphoproliferative disorders
- Author
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van de Griend, RJ and Bolhuis, RL
- Abstract
Patients with T gamma lymphocytosis are a heterogeneous group, clearly distinguishable from other patients with chronic T lymphoproliferative disorders and usually without proven malignancy. We have attempted in vitro cloning of lymphocytes from three patients with an expansion of phenotypically and functionally different types of T gamma cells. One had T3+ B73.1+ T4 T8+ OKM1+ T gamma cells exerting antibody-dependent cellular cytotoxicity (ADCC) and natural killer (NK) cell cytotoxicity; another had T3+B73.1-T4-T8+OKM1-ADCC+NK- and a third had T3-B73.1+T4-T8- OKM1+ADCC+NK+ cells. On morphological characterization, most of the mononuclear cells of these patients resembled large granular lymphocytes (LGLs). Although lymphocytes of these patients showed almost no proliferative response capacity after stimulation with mitogens, they shared the capacity to proliferate after stimulation with Epstein-Barr virus-transformed lymphoblastoid B (B-LCL) feeder cells. Stable clones were established by this procedure. Clones from patient 3 exerted cytolytic activity against a broad spectrum of tumor cell lines, including fresh biopsy specimens of melanoma tumor target cells. All of these clones (termed activated killer [AK] cells) had the surface phenotype T3-, T4-, T8- or +, HNK1-, OKM1-, Lyt3+, WT1+ and showed ADCC in addition to AK cell cytotoxicity. Most of them were B73.1+ and expressed IgG-Fc receptors. They most likely belong to the T cell lineage, since they express IL2 receptors as recognized by the Tac antibody and did not bind monoclonal antibodies directed against monocytes or granulocytes. Thus lymphocytes with the functional and phenotypical characteristics of T gamma cells can be cloned and expanded in vitro from the peripheral lymphocytes of these patients by using the appropriate stimulus. Our results indicate that, of the heterogeneous population of NK cells, the T3- cells are more rapidly expanded than T3+ subsets. It is discussed whether or not our culture system might selectively induce proliferation in “normal” T cells rather than aberrant ones.
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- 1985
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6. Selectivity of compounds isolated from the leaves of Nerium indicum Mill. on various human cancer cell lines.
- Author
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Mae SH, Sofia M, Bolhuis RL, Nooter K, Oostrum RG, Subagus W, and Ibnu GG
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- Antibiotics, Antineoplastic pharmacology, Antibiotics, Antineoplastic therapeutic use, Cisplatin pharmacology, Cisplatin therapeutic use, Doxorubicin pharmacology, Doxorubicin therapeutic use, Humans, Pilot Projects, Antineoplastic Agents therapeutic use, Neoplasms drug therapy, Nerium, Plant Preparations therapeutic use
- Abstract
The leaves of Nerium indicum Mill. have been utilized traditionally to cure cancer. By Bioassay (BST) guided isolation method, six compounds were isolated from the CHCl3 extract of the leaves. Selectivity of these compounds (in 0.6-12,500 ng/ml) was tested on various human cancer (MCF7, EVSA-T, T47D, H226, IGROV, A498, WIDR, M19, HeLa) and normal (Vero) cells in vitro. Doxorubicin and cysplatin were used as positive controls. The result indicated that NiO2D (5alpha-oleandrin) possessed the best cytotoxic effect on HeLa cells (IC50, 8.38 x10(-6) mM) and NiO2C (16, 17-dehidrodeasetil-5alpha-oleandrin) on A498 cells (IC50, 1.43 x 10(-6) mM). Those two compounds were not cytotoxic to normal cell.
- Published
- 2008
7. T cell re-targeting to EBV antigens following TCR gene transfer: CD28-containing receptors mediate enhanced antigen-specific IFNgamma production.
- Author
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Schaft N, Lankiewicz B, Drexhage J, Berrevoets C, Moss DJ, Levitsky V, Bonneville M, Lee SP, McMichael AJ, Gratama JW, Bolhuis RL, Willemsen R, and Debets R
- Subjects
- CD28 Antigens immunology, Enzyme-Linked Immunosorbent Assay, Flow Cytometry, Genetic Therapy methods, Humans, Jurkat Cells, Receptors, Antigen, T-Cell genetics, Receptors, Antigen, T-Cell immunology, Transduction, Genetic, Epstein-Barr Virus Nuclear Antigens immunology, Gene Transfer Techniques, Genes, T-Cell Receptor, Interferon-gamma biosynthesis, T-Lymphocytes immunology
- Abstract
EBV is associated with a broad range of malignancies. Adoptive immunotherapy of these tumors with EBV-specific CTL proved useful. We generated a panel of primary human T cells specific to various EBV antigens (i.e. Epstein-Barr nuclear antigen 3A, 3B and BamHI-M leftward reading frame) via transfer of modified TCR genes that are either coupled to CD3zeta or Fc(epsilon)RIgamma. TCR-transduced T cells from 20-60% of donors (total number of 25) demonstrated specific lysis of EBV peptide-loaded target cells, whereas lymphoblastoid cell lines expressing native EBV antigens were not killed by any of the EBV-specific T cell populations. This non-responsiveness, confirmed at the level of nuclear factor of activated T cells activation, is not due to receptor configuration since identical receptor formats specific for melanoma antigens successfully re-targeted T cells to native melanoma cells. In an effort to generate a more potent receptor, we introduced a CD28 domain into one of the EBV-specific TCR. This TCR did not affect the cytotoxic response of re-targeted T cells, but dramatically enhanced antigen-specific IFNgamma production. We therefore conclude that these novel CD28-containing EBV-specific TCRs provide a basis for further development of TCR gene transfer to treat EBV-induced diseases.
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- 2006
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8. T cell retargeting with MHC class I-restricted antibodies: the CD28 costimulatory domain enhances antigen-specific cytotoxicity and cytokine production.
- Author
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Willemsen RA, Ronteltap C, Chames P, Debets R, and Bolhuis RL
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- Adjuvants, Immunologic genetics, Adjuvants, Immunologic metabolism, Adjuvants, Immunologic toxicity, Antigens, Neoplasm, CD28 Antigens genetics, CD28 Antigens immunology, Cell Line, Tumor, Epitopes, T-Lymphocyte genetics, Epitopes, T-Lymphocyte immunology, HLA-A1 Antigen genetics, HLA-A1 Antigen metabolism, Humans, Immunoglobulin Fab Fragments genetics, Immunoglobulin Fab Fragments metabolism, Immunoglobulin Fab Fragments toxicity, Immunoglobulin Variable Region genetics, Immunoglobulin Variable Region metabolism, K562 Cells, Melanoma immunology, Melanoma pathology, Melanoma-Specific Antigens, Neoplasm Proteins genetics, Neoplasm Proteins immunology, Neoplasm Proteins metabolism, Protein Structure, Tertiary genetics, Receptors, Antigen, T-Cell biosynthesis, Receptors, Antigen, T-Cell genetics, Receptors, Antigen, T-Cell metabolism, Receptors, IgE genetics, Receptors, IgE metabolism, Receptors, IgE physiology, Signal Transduction genetics, Signal Transduction immunology, T-Lymphocytes, Cytotoxic metabolism, Transduction, Genetic methods, Binding Sites, Antibody genetics, CD28 Antigens physiology, Cytokines biosynthesis, Cytotoxicity, Immunologic genetics, Epitopes, T-Lymphocyte toxicity, HLA-A1 Antigen immunology, Lymphocyte Activation genetics, T-Lymphocytes, Cytotoxic immunology
- Abstract
T cells require both primary and costimulatory signals for optimal activation. The primary Ag-specific signal is delivered by engagement of the TCR. The second Ag-independent costimulatory signal is mediated by engagement of the T cell surface costimulatory molecule CD28 with its target cell ligand B7. However, many tumor cells do not express these costimulatory molecules. We previously constructed phage display derived F(AB), G8, and Hyb3, Ab-based receptors with identical specificity but distinct affinities for HLA-A1/MAGE-A1, i.e., "TCR-like" specificity. These chimeric receptors comprised the FcepsilonRI-gamma signaling element. We analyzed whether linking the CD28 costimulation structure to it (gamma + CD28) could affect the levels of MHC-restricted cytolysis and/or cytokine production. Human scFv-G8(POS) T lymphocytes comprising the gamma + CD28 vs the gamma signaling element alone produced substantially more IL-2, TNF-alpha, and IFN-gamma in response to HLA-A1/MAGE-A1(POS) melanoma cells. Also a drastic increase in cytolytic capacity of scFv-G8(POS) T cells, equipped with gamma + CD28 vs the gamma-chain alone was observed.
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- 2005
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9. Flexible and sensitive method to functionally validate tumor-specific receptors via activation of NFAT.
- Author
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Schaft N, Lankiewicz B, Gratama JW, Bolhuis RL, and Debets R
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- Antibodies, Monoclonal genetics, Antibodies, Monoclonal metabolism, Cytotoxicity, Immunologic, Genes, Reporter, Humans, Jurkat Cells, NFATC Transcription Factors, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Sensitivity and Specificity, T-Lymphocytes immunology, T-Lymphocytes metabolism, Transduction, Genetic, Tumor Cells, Cultured, Tumor Necrosis Factor-alpha biosynthesis, DNA-Binding Proteins genetics, DNA-Binding Proteins metabolism, Genetic Techniques statistics & numerical data, Neoplasms genetics, Neoplasms metabolism, Nuclear Proteins, Receptors, Antigen, T-Cell, alpha-beta genetics, Receptors, Antigen, T-Cell, alpha-beta metabolism, Transcription Factors genetics, Transcription Factors metabolism
- Abstract
Tumor-specific receptors may provide effective tools for anti-tumor immunogene therapy. However, the functional analysis of primary human T cells engrafted with tumor-specific receptors is laborious and emphasizes the need for a fast and sensitive method to validate such receptors. To this end, we have set up a Jurkat T cell-based reporter gene assay, and tested receptors with various formats, i.e., receptors based on either a monoclonal antibody (mAb), a full-length T cell receptor (fl-TCR)alphabeta or a chimeric (ch-)TCRalphabeta, and various antigen specificities for their ability to mediate tumor-specific activation of nuclear factor of activated T cells (NFAT). The mAb-based receptor specifically mediates NFAT activation after stimulation with tumor antigen-positive target cells. The observed receptor-mediated NFAT responses were validated by the use of ligand- and receptor-specific mAbs, as well as cyclosporin A (CsA) and a dominant negative mutant of NFAT. Furthermore, anti-TCR mAbs, peptide-loaded tumor cells and antigen-positive tumor cells all resulted in specific NFAT activation in TCR/CD8 co-transduced Jurkat T cells, irrespective of the TCR format used. Importantly, receptor-mediated NFAT responses parallel tumor-specific cytolysis and TNFalpha production of receptor-transduced primary human T lymphocytes. In fact, inhibition of NFAT activation compromises the immune responses of primary human T lymphocytes, pointing to a central involvement of NFAT in anti-tumor T cell responses. Taken together, receptor-mediated activation of NFAT constitutes a representative measure of anti-tumor T cell responses, and the genetically modified Jurkat T cells provide a flexible and sensitive tool with which to select rapidly tumor-specific (chimeric) receptors for immunogene therapy.
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- 2003
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10. Peptide fine specificity of anti-glycoprotein 100 CTL is preserved following transfer of engineered TCR alpha beta genes into primary human T lymphocytes.
- Author
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Schaft N, Willemsen RA, de Vries J, Lankiewicz B, Essers BW, Gratama JW, Figdor CG, Bolhuis RL, Debets R, and Adema GJ
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- Amino Acid Sequence, Amino Acid Substitution genetics, Amino Acid Substitution immunology, Base Sequence, Cell Line, Cell Line, Transformed, Cells, Cultured, Clone Cells, Cytotoxicity Tests, Immunologic, Epitopes, T-Lymphocyte genetics, HLA-A2 Antigen immunology, Humans, K562 Cells, Melanoma immunology, Melanoma therapy, Membrane Glycoproteins genetics, Molecular Sequence Data, Neoplasm Proteins genetics, Peptide Fragments genetics, Receptors, Antigen, T-Cell, alpha-beta biosynthesis, Receptors, Antigen, T-Cell, alpha-beta therapeutic use, T-Lymphocyte Subsets metabolism, T-Lymphocytes, Cytotoxic metabolism, Transfection, Tumor Cells, Cultured, gp100 Melanoma Antigen, Epitopes, T-Lymphocyte immunology, Membrane Glycoproteins immunology, Neoplasm Proteins immunology, Peptide Fragments immunology, Receptors, Antigen, T-Cell, alpha-beta genetics, T-Lymphocyte Subsets immunology, T-Lymphocytes, Cytotoxic immunology, Transduction, Genetic methods
- Abstract
TCR with known antitumor reactivity can be genetically introduced into primary human T lymphocytes and provide promising tools for immunogene therapy of tumors. We molecularly characterized two distinct TCRs specific for the same HLA-A2-restricted peptide derived from the melanocyte differentiation Ag gp100, yet exhibiting different stringencies in peptide requirements. The existence of these two distinct gp100-specific TCRs allowed us to study the preservation of peptide fine specificity of native TCRalphabeta when engineered for TCR gene transfer into human T lymphocytes. Retroviral transduction of primary human T lymphocytes with either one of the two sets of TCRalphabeta constructs enabled T lymphocytes to specifically kill and produce TNF-alpha when triggered by native gp100(pos)/HLA-A2(pos) tumor target cells as well as gp100 peptide-loaded HLA-A2(pos) tumor cells. Peptide titration studies revealed that the cytolytic efficiencies of the T lymphocyte transductants were in the same range as those of the parental CTL clones. Moreover, primary human T lymphocytes expressing either one of the two engineered gp100-specific TCRs show cytolytic activities in response to a large panel of peptide mutants that are identical with those of the parental CTL. The finding that two gp100-specific TCR, derived from two different CTL, can be functionally introduced into primary human T lymphocytes without loss of the Ag reactivity and peptide fine specificity, holds great promise for the application of TCR gene transfer in cancer immunotherapy.
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- 2003
- Full Text
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11. Genetic engineering of T cell specificity for immunotherapy of cancer.
- Author
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Willemsen RA, Debets R, Chames P, and Bolhuis RL
- Subjects
- Humans, Neoplasms immunology, Antibodies, Neoplasm metabolism, Genetic Engineering, Immunotherapy, Neoplasms therapy, T-Lymphocytes, Cytotoxic immunology
- Abstract
The ultimate goal of immunotherapy of cancer is to make use of the immune system of patients to eliminate malignant cells. Research has mainly focused on the generation of effective antigen specific T-cell responses because of the general belief that T-cell immunity is essential in controlling tumor growth and protection against viral infections. However, the isolation of antigen specific T cells for therapeutic application is a laborious task and it is often impossible to derive autologous tumor specific T cells to be used for adoptive immunotherapy. Therefore, strategies were developed to genetically transfer tumor specific immune receptors into patients T cells. To this end, chimeric receptors were constructed that comprise antibody fragments specific for tumor associated antigens, linked to genes encoding signaling domains of the T-cell receptor (TCR) or Fc receptor. T cells expressing such chimeric antibody receptors recapitulate the immune specific responses mediated by the introduced receptor. Recently, we introduced chimeric TCR genes into primary human T lymphocytes and demonstrated that these T cell transductants acquired the exquisite major histocompatibility complex (MHC) restricted tumor specificity dictated by the introduced TCR. Importantly, the introduction of chimeric TCR bypasses problems associated with the introduction of nonmodified TCR genes, such as pairing of introduced TCR chains with endogenous TCR chains and unstable TCRalpha expression. A novel strategy which is completely independent of available tumor specific T-cell clones for cloning of the TCR genes was recently used to transfer MHC restricted tumor specificity to T cells. Human "TCR-like" Fab fragments obtained by in vitro selection of Fab phages on soluble peptide/MHC complexes were functionally expressed on human T lymphocytes, resulting in MHC restricted, tumor specific lysis and cytokine production. In addition, affinity maturation of the antibody fragment on Fab phages allows improvement of the tumor cell killing capacity of chimeric Fab receptor engrafted T cells. Developments in retroviral transfer technology now enables the generation of large numbers of antigen specific T cells that can be used for adoptive transfer to cancer patients. In this article we summarize the developments in adoptive T cell immunogenetic therapy and discuss the limitations and perspectives to improve this technology toward clinical application.
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- 2003
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12. Repeated administrations of interleukin (IL)-12 are associated with persistently elevated plasma levels of IL-10 and declining IFN-gamma, tumor necrosis factor-alpha, IL-6, and IL-8 responses.
- Author
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Portielje JE, Lamers CH, Kruit WH, Sparreboom A, Bolhuis RL, Stoter G, Huber C, and Gratama JW
- Subjects
- Adult, Aged, B-Lymphocytes metabolism, CD4-Positive T-Lymphocytes metabolism, CD8-Positive T-Lymphocytes metabolism, Carcinoma, Renal Cell blood, Cytokines metabolism, Humans, Immunophenotyping, Interferon-gamma blood, Interleukin-10 blood, Interleukin-6 blood, Interleukin-8 blood, Killer Cells, Natural metabolism, Middle Aged, Recombinant Proteins pharmacology, Time Factors, Tumor Necrosis Factor-alpha biosynthesis, Carcinoma, Renal Cell drug therapy, Interleukin-12 administration & dosage
- Abstract
Purpose: Repeated administrations of recombinant human interleukin-12 (rHuIL-12) to cancer patients are characterized by a reduction of side effects during treatment. Induction of IFN-gamma, considered a key mediator of antitumor effects of IL-12, is known to decline on repeated administrations. We studied whether other immunological effects of rHuIL-12 are tapered in the course of treatment., Experimental Design: In a Phase I study of 26 patients with advanced renal cell cancer, rHuIL-12 was administered s.c. on day 1, followed by 7 days rest and six injections administered over a 2-week time period. Plasma concentrations of various cytokines were monitored, as well as absolute counts of circulating leukocyte and lymphocyte subsets., Results: The first injection of IL-12 was accompanied by rapid, transient, and dose-dependent increments of plasma levels IFN-gamma, tumor necrosis factor-alpha, IL-10, IL-6, IL-8, but not IL-4, as well as rapid, transient, and dose-dependent reductions of lymphocyte, monocyte, and neutrophil counts. The major lymphocyte subsets, i.e., CD4+ and CD8+ T cells, B cells, and natural killer cells, followed this pattern. On repeated rHuIL-12 injections, IL-10 concentrations increased further, whereas the transient increments of IFN-gamma, tumor necrosis factor-alpha, IL-6, and IL-8 concentrations, as well as the fluctuations of the leukocyte subset counts, were tapered. Dose escalation of IL-12 within clinically tolerable margins did not reduce the decline of these immunological effects., Conclusions: Induction of pro-inflammatory cytokines and associated fluctuations in leukocyte subset counts decrease on repeated administrations of rHuIL-12. The steady increment of IL-10 plasma levels may mediate the observed down-regulation of clinical and immunological effects.
- Published
- 2003
13. TCR-like human antibodies expressed on human CTLs mediate antibody affinity-dependent cytolytic activity.
- Author
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Chames P, Willemsen RA, Rojas G, Dieckmann D, Rem L, Schuler G, Bolhuis RL, and Hoogenboom HR
- Subjects
- Antibody Specificity, Antigen Presentation genetics, Antigens, Neoplasm, Cloning, Molecular, Gene Targeting, Genetic Vectors chemical synthesis, HLA-A1 Antigen immunology, Humans, Immunodominant Epitopes immunology, Immunodominant Epitopes metabolism, Immunoglobulin Fab Fragments biosynthesis, Immunoglobulin Fab Fragments genetics, Immunoglobulin Fab Fragments metabolism, Immunoglobulin Heavy Chains biosynthesis, Immunoglobulin Heavy Chains chemistry, Immunoglobulin Heavy Chains metabolism, Immunoglobulin Light Chains biosynthesis, Immunoglobulin Light Chains chemistry, Immunoglobulin Light Chains metabolism, Melanoma-Specific Antigens, Neoplasm Proteins immunology, Protein Binding genetics, Protein Binding immunology, Receptors, Antigen, T-Cell biosynthesis, Receptors, Antigen, T-Cell genetics, Receptors, Antigen, T-Cell metabolism, Receptors, Antigen, T-Cell, gamma-delta biosynthesis, Receptors, Antigen, T-Cell, gamma-delta genetics, Receptors, Antigen, T-Cell, gamma-delta metabolism, Antibody Affinity genetics, Cytotoxicity, Immunologic genetics, Immunoglobulin Fab Fragments physiology, Receptors, Antigen, T-Cell physiology, T-Lymphocytes, Cytotoxic immunology, T-Lymphocytes, Cytotoxic metabolism
- Abstract
The permanent genetic programming via gene transfer of autologous T cells with cell surface receptors directed toward tumor-related Ags holds great promise for the development of more-specific tumor therapies. In this study we have explored the use of Abs directed to MHC-peptide complexes (or TCR-like Abs) to engraft CTLs with exquisite specificity for cancer cells. First, we affinity matured in vitro a previously selected TCR-like Ab, Fab-G8, which is highly specific for the peptide melanoma-associated Ag-A1 presented by the HLA-A1 molecule. A combination of L chain shuffling, H chain-targeted mutagenesis, and in vitro selection of phage display libraries yielded a Fab-G8 Ab derivative, Fab-Hyb3, with an 18-fold improved affinity yet identical peptide fine specificity. Fab-G8 and Fab-Hyb3 were expressed on primary human T lymphocytes as cell surface-anchored Fab, demonstrating that T cells expressing the high-affinity Fab-Hyb3 molecule eradicate tumor cells much more effectively. Furthermore, the gain in ligand-binding affinity resulted in a 2-log improvement in the detection of peptide/MHC complexes on melanoma-associated Ag-A1 peptide-loaded cells. In summary, an affinity-matured Ab specifically recognizing a cancer-related peptide/MHC complex was generated and used to improve the tumor cell killing capacity of human T cells. This strategy, based on engraftment of T cells with in vitro engineered Abs, is an attractive alternative to the laborious, and in many cases unsuccessful, generation of highly potent tumor-specific T lymphocytes.
- Published
- 2002
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14. Protocol for gene transduction and expansion of human T lymphocytes for clinical immunogene therapy of cancer.
- Author
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Lamers CH, Willemsen RA, Luider BA, Debets R, and Bolhuis RL
- Subjects
- Antibodies, Monoclonal, CD28 Antigens genetics, CD3 Complex genetics, CD4 Antigens genetics, Carcinoma, Renal Cell therapy, Cell Division, Culture Media, Serum-Free, Flow Cytometry, Humans, Kidney Neoplasms therapy, Leukocytes, Mononuclear cytology, Retroviridae genetics, Spectrometry, Fluorescence, Time Factors, Transgenes, Tumor Cells, Cultured, Gene Transfer Techniques, Genetic Therapy methods, Immunotherapy methods, Neoplasms therapy, T-Lymphocytes metabolism, Transduction, Genetic
- Abstract
In preparation of a clinical phase I/II study in renal cell carcinoma (RCC) patients, we developed a clinically applicable protocol that meets good clinical practice (GCP) criteria regarding the gene transduction and expansion of primary human T lymphocytes. We previously designed a transgene that encodes a single chain (sc) FvG250 antibody chimeric receptor (ch-Rec), specific for a RCC tumor-associated antigen (TAA), and that genetically programs human T lymphocytes with RCC immune specificity. Here we describe the conditions for activation, gene transduction, and proliferation for primary human T lymphocytes to yield: (a) optimal functional expression of the transgene; (b) ch-Rec-mediated cytokine production, and (c) cytolysis of G250-TAA(POS) RCC by the T-lymphocyte transductants. Moreover, these parameters were tested at clinical scale, i.e., yielding up to 5-10 x 10(9) T-cell transductants, defined as the treatment dose according to our clinical protocol. The following parameters were, for the first time, tested in an interactive way: (1) media compositions for production of virus by the stable PG13 packaging cell; (2) T-lymphocyte activation conditions and reagents (anti-CD3 mAb; anti-CD3+anti-CD28 mAbs; and PHA); (3) kinetics of T-lymphocyte activation prior to gene transduction; (4) (i) T-lymphocyte density, and (ii) volume of virus-containing supernatant per surface unit during gene transduction; and (5) medium composition for T-lymphocyte maintenance (i) in-between gene transduction cycles, and (ii) during in vitro T-lymphocyte expansion. Critical to gene transduction of human T lymphocytes at clinical scale appeared to be the use of the fibronectin fragment CH-296 (Retronectin) as well as Lifecell) X-fold cell culture bags. In order to comply with GCP requirements, we used: (a) bovine serum-free human T-lymphocyte transduction system, i.e., media supplemented with autologous patients' plasma, and (b) a closed cell culture system for all lymphocyte processing. This clinical protocol routinely yields 30-65% scFvG250 ch-Rec(POS) T lymphocytes in both healthy donors and RCC patients.
- Published
- 2002
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15. Increased levels of soluble CD27 in the cerebrospinal fluid are not diagnostic for leptomeningeal involvement by lymphoid malignancies.
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van den Bent MJ, Lamers CH, van 't Veer MB, Sillevis Smitt PA, Bolhuis RL, and Gratama JW
- Subjects
- Biomarkers, Tumor cerebrospinal fluid, Diagnostic Errors prevention & control, Humans, Leukemia, Lymphocytic, Chronic, B-Cell cerebrospinal fluid, Lymphoma, B-Cell cerebrospinal fluid, Meningeal Neoplasms cerebrospinal fluid, Predictive Value of Tests, Solubility, Leukemia, Lymphocytic, Chronic, B-Cell diagnosis, Lymphoma, B-Cell diagnosis, Meningeal Neoplasms diagnosis, Tumor Necrosis Factor Receptor Superfamily, Member 7 cerebrospinal fluid
- Abstract
Soluble CD27 (sCD27) reportedly is a sensitive and specific marker for leptomeningeal involvement (LI) of CD27-expressing lymphoproliferations such as B-cell non-Hodgkin's lymphoma (B-NHL) or chronic B-lymphocytic leukemia (B-CLL). Because morphological analysis of cerebrospinal fluid (CSF) in patients suspected of LI is false negative in one-third of patients, a diagnostic marker for LI by B-NHL or B-CLL would be very valuable. sCD27 was determined in the first CSF sample from each of 102 unselected patients submitted for (immuno)morphologic detection of malignant cells. The patients were considered to have LI if either (immuno)morphologic analyses showed tumor cells or if neuroradiological evaluation showed typical abnormalities consistent with LI. Patients were suspected of having LI if CSF samples revealed atypical lymphocytes and/or if clinical symptoms and signs suggestive of LI were present, but clinical follow-up was shorter than 3 months because of deterioration of the patient. LI was considered absent if (immuno)morphologic analyses of CSF samples were negative without evidence for LI during 3 months of clinical follow-up. In patients with chronic lymphoproliferative disorders [mainly B-non-Hodgkin's lymphoma (NHL)], sCD27 concentrations were significantly higher in the CSF samples of 16 patients with confirmed or suspected LI than in those of 46 patients without LI. However, sCD27 was also increased in a variety of other predominantly inflammatory neurological disorders including herpes simplex and zoster infections. The positive predictive value of sCD27 determination for LI was only 54%, but the negative predictive value was 92%. Normal sCD27 concentrations in CSF samples of patients with chronic lymphoproliferation makes LI unlikely, but the determination of CSF sCD27 is not sufficiently specific to serve as a reliable tumor marker.
- Published
- 2002
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16. A phage display selected fab fragment with MHC class I-restricted specificity for MAGE-A1 allows for retargeting of primary human T lymphocytes.
- Author
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Willemsen RA, Debets R, Hart E, Hoogenboom HR, Bolhuis RL, and Chames P
- Subjects
- Antigens, Neoplasm, Epitopes, Genetic Vectors administration & dosage, HLA-A1 Antigen immunology, Humans, Immunoglobulin Fab Fragments metabolism, Melanoma immunology, Melanoma-Specific Antigens, Neoplasm Proteins immunology, Peptide Library, Retroviridae genetics, T-Lymphocytes, Cytotoxic immunology, Transduction, Genetic, Tumor Cells, Cultured, Genetic Therapy methods, Immunoglobulin Fab Fragments genetics, Immunotherapy, Adoptive methods, Melanoma therapy, Receptors, Immunologic genetics
- Abstract
The clinical benefit of adoptive transfer of MHC-restricted cytotoxic T lymphocytes(CTL) for the treatment of cancer is hampered by the low success rate to generate antitumor CTLs. To bypass the need for tumor-specific CTL, we developed a strategy that allows for grafting of human T lymphocytes with MHC-restricted antigen specificity using in vitro selected human Fab fragments fused to the Fc(epsilon)RI-gamma signaling molecule. Retroviral introduction of a Fab-based chimeric receptor specific for MAGE-A1/HLA-A1 into primary human T lymphocytes resulted in binding of relevant peptide/MHC complexes. Transduced T lymphocytes responded to native MAGE-A1/HLA-A1POS target cells by specific cytokine production and cytolysis. Therefore, peptide/MHC-specific Fab fragments represent new alternatives to TCR to confer human T lymphocytes with tumor specificity, which provides a promising rationale for developing immunogene therapies.
- Published
- 2001
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17. Circumventing tolerance to a human MDM2-derived tumor antigen by TCR gene transfer.
- Author
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Stanislawski T, Voss RH, Lotz C, Sadovnikova E, Willemsen RA, Kuball J, Ruppert T, Bolhuis RL, Melief CJ, Huber C, Stauss HJ, and Theobald M
- Subjects
- Animals, Antigens, Neoplasm immunology, Cell Line, Cytotoxicity Tests, Immunologic, Epitopes, T-Lymphocyte immunology, HLA-A2 Antigen genetics, Humans, Immunotherapy, Adoptive, Leukemia immunology, Leukemia therapy, Mice, Mice, Inbred C57BL, Mice, Transgenic, Neoplasms therapy, Proto-Oncogene Proteins c-mdm2, Transduction, Genetic, Tumor Cells, Cultured, Genes, T-Cell Receptor, Genetic Therapy, Neoplasms immunology, Nuclear Proteins, Proto-Oncogene Proteins immunology, Self Tolerance, T-Lymphocytes, Cytotoxic immunology
- Abstract
We identified a tumor-associated cytotoxic T lymphocyte (CTL) epitope derived from the widely expressed human MDM2 oncoprotein and were able to bypass self-tolerance to this tumor antigen in HLA-A*0201 (A2.1) transgenic mice and by generating A2.1-negative, allo-A2.1-restricted human T lymphocytes. A broad range of malignant, as opposed to nontransformed cells, were killed by high-avidity transgenic mouse and allogeneic human CTLs specific for the A2.1-presented MDM2 epitope. Whereas the self-A2.1-restricted human T cell repertoire gave rise only to low-avidity CTLs unable to recognize the natural MDM2 peptide, human A2.1+ T lymphocytes were turned into efficient MDM2-specific CTLs upon expression of wild-type and partially humanized high-affinity T cell antigen receptor (TCR) genes derived from the transgenic mice. These results demonstrate that TCR gene transfer can be used to circumvent self-tolerance of autologous T lymphocytes to universal tumor antigens and thus provide the basis for a TCR gene transfer-based broad-spectrum immunotherapy of malignant disease.
- Published
- 2001
- Full Text
- View/download PDF
18. Tetramer-based quantification of cytomegalovirus (CMV)-specific CD8+ T lymphocytes in T-cell-depleted stem cell grafts and after transplantation may identify patients at risk for progressive CMV infection.
- Author
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Gratama JW, van Esser JW, Lamers CH, Tournay C, Löwenberg B, Bolhuis RL, and Cornelissen JJ
- Subjects
- Adolescent, Adult, Antibodies, Viral immunology, Antiviral Agents therapeutic use, Biotinylation, Cytomegalovirus Infections diagnosis, Cytomegalovirus Infections drug therapy, Cytomegalovirus Infections etiology, Cytomegalovirus Infections immunology, Disease Progression, Female, Ganciclovir therapeutic use, HLA-A2 Antigen immunology, Humans, Immunity, Cellular, Immunologic Deficiency Syndromes complications, Immunologic Deficiency Syndromes immunology, Male, Middle Aged, Peptide Fragments chemistry, Peptide Fragments immunology, Phosphoproteins chemistry, Risk, Sensitivity and Specificity, Viral Matrix Proteins chemistry, Viremia diagnosis, Viremia drug therapy, Viremia epidemiology, Viremia etiology, Viremia immunology, Antigens, Viral immunology, CD8-Positive T-Lymphocytes immunology, Cytomegalovirus immunology, Cytomegalovirus Infections epidemiology, Hematopoietic Stem Cell Transplantation adverse effects, Hematopoietic Stem Cell Transplantation methods, Immunodominant Epitopes immunology, Phosphoproteins immunology, Viral Matrix Proteins immunology
- Abstract
Recovery of cytomegalovirus (CMV)-specific T-cell-mediated immunity after allogeneic hematopoietic stem cell transplantation (SCT) is critical for protection against CMV disease. The study used fluorochrome-conjugated tetrameric complexes of HLA-A2 molecules loaded with the immunodominant NLVPMVATV (NLV) peptide derived from the CMV protein pp65 to quantify A2-NLV-specific CD8+ T cells in partially T-cell-depleted grafts administered to 27 HLA-A*0201+ patients and to monitor recovery of these T cells during the first 12 months after SCT. None of the 9 CMV-seronegative patients became infected with CMV, whereas 14 of 18 CMV-seropositive patients developed CMV antigenemia after SCT. CMV-seropositive recipients of grafts from CMV-seronegative donors required more preemptive treatment with ganciclovir (GCV) than those of grafts from CMV-seropositive donors (3 [1-6] versus 1 [0-3] courses, respectively; P =.009). The number of A2-NLV-specific CD8+ T cells in the grafts correlated inversely with the number of preemptive GCV courses administered (r = -0.61; P =.01). None of the 9 CMV-seronegative patients mounted a CMV-specific immune response as measured by monitoring A2-NLV-specific CD8+ T cells after SCT. Thirteen of 14 CMV-seropositive patients without CMV disease recovered these T cells. In spite of preemptive GCV treatment, CMV disease developed in 4 patients, who all failed to recover A2-NLV-specific CD8+ T cells after SCT (P =.002). Thus, enumeration of HLA-restricted, CMV-specific CD8+ T cells in the grafts and monitoring of these T cells after SCT may constitute a rapid and sensitive tool to identify SCT recipients at risk for developing CMV disease.
- Published
- 2001
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- View/download PDF
19. Interleukin 12 induces activation of fibrinolysis and coagulation in humans.
- Author
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Portielje JE, Kruit WH, Eerenberg AJ, Schuler M, Sparreboom A, Lamers CH, Bolhuis RL, Stoter G, Huber C, and Hack C
- Subjects
- Adult, Aged, Antithrombin III analysis, Biomarkers blood, Carcinoma, Renal Cell blood, Female, Fibrinolysin analysis, Humans, Interferon-gamma blood, Interleukin-12 blood, Kidney Neoplasms blood, Kidney Neoplasms drug therapy, Male, Middle Aged, Plasminogen Activator Inhibitor 1 analysis, Recombinant Proteins blood, Recombinant Proteins pharmacology, Thrombin analysis, Thrombin metabolism, Tissue Plasminogen Activator analysis, Tumor Necrosis Factor-alpha analysis, alpha-2-Antiplasmin analysis, Blood Coagulation drug effects, Carcinoma, Renal Cell drug therapy, Fibrinolysis drug effects, Interleukin-12 therapeutic use
- Abstract
Interleukin 12 (IL-12) has potential efficacy in malignant, infectious and allergic diseases. Its side-effects include activation of coagulation and fibrinolysis, as documented in chimpanzees. We assessed the coagulative and fibrinolytic response in 18 patients with renal cell carcinoma after subcutaneous injection of 0.5 microg/kg recombinant human IL-12. IL-12 induced a fibrinolytic response in 17 patients (94%): plasmin-alpha2-anti-plasmin complexes (PAPc) increased from 11.8 +/- 6.6 nmol/l (mean +/- SD) to a maximum of 18.8 +/- 7.4 nmol/l at 72 h. Baseline levels of tissue plasminogen activator (tPA) and plasminogen-activator inhibitor-I (PAI) were elevated in eight and 14 patients respectively. tPA increased from 12.6 +/- 5.2 ng/ml to a maximum of 19.0 +/- 6.7 ng/ml at 72 h. PAI decreased from 111 +/- 69 ng/ml to a minimum of 65 +/- 53 ng/ml at 8 h, thereafter remaining below baseline. Elevation of PAPc correlated with elevation of tPA and reduction of PAI. A coagulative response occurred in nine patients (50%): thrombin-anti-thrombin III complexes increased from 29 +/- 53 ng/ml to a maximum of 460 +/- 322 ng/ml at 12 h. Patients with and without a coagulative response had similar levels of recombinant human IL-12, interferon-gamma or tumour necrosis factor-alpha. We conclude that IL-12 can activate both fibrinolysis and coagulation in a significant proportion of patients with cancer. The time-frame and sequence of these activation processes differ from those known for other cytokines.
- Published
- 2001
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20. An entirely humanized CD3 zeta-chain signaling receptor that directs peripheral blood t cells to specific lysis of carcinoembryonic antigen-positive tumor cells.
- Author
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Hombach A, Schneider C, Sent D, Koch D, Willemsen RA, Diehl V, Kruis W, Bolhuis RL, Pohl C, and Abken H
- Subjects
- Antibody Specificity, Carcinoembryonic Antigen biosynthesis, Carrier Proteins biosynthesis, Carrier Proteins genetics, Colonic Neoplasms therapy, Cross Reactions, Cytotoxicity, Immunologic, Epitopes, T-Lymphocyte immunology, Gene Transfer Techniques, Humans, Immunoglobulin Fragments genetics, Immunoglobulin Fragments immunology, Immunomagnetic Separation, Immunotherapy, Adoptive methods, Leukemia Virus, Gibbon Ape genetics, Leukemia Virus, Murine genetics, Lymphocyte Activation, Membrane Proteins biosynthesis, Membrane Proteins genetics, Protein Structure, Tertiary, Receptor-CD3 Complex, Antigen, T-Cell genetics, Receptors, Antigen, T-Cell biosynthesis, Receptors, Antigen, T-Cell genetics, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins immunology, Recombinant Proteins genetics, Recombinant Proteins immunology, Signal Transduction immunology, T-Lymphocytes metabolism, T-Lymphocytes virology, Carcinoembryonic Antigen immunology, Carrier Proteins immunology, Colonic Neoplasms immunology, Membrane Proteins immunology, Receptor-CD3 Complex, Antigen, T-Cell immunology, Receptors, Antigen, T-Cell immunology, Receptors, Cell Surface, T-Lymphocytes immunology
- Abstract
Recombinant T-cell receptors with antibody-like specificity for tumor-associated antigens are successfully used to direct the cytolytic activity of T cells toward tumor cells. Clinical application, however, needs to comply with the low immunogenicity of the recombinant receptor, efficient gene transfer into peripheral blood T cells, and enrichment of receptor-grafted cells. Here, we address these issues and describe an entirely humanized immune receptor for use in adoptive immunotherapy of colorectal carcinoma. The receptor consists of a single-chain antibody (scFv) binding domain specific for carcinoembryonic antigen (CEA), the IgG hinge and CH2/CH3 (Fc) joining region, and the transmembrane and intracellular CD3 zeta signaling chain. To express the receptor in peripheral blood T cells, both GALV envelope and MuLV 4070A pseudotyped retrovirus turned out to be equally efficient, with transduction efficiencies of about 5% to 40%, depending on the lymphocyte donor. Furthermore, receptor-grafted T cells could be 2- to 6-fold enriched by magnetic activated cell sorting, utilizing an antibody directed to the extracellular IgG domain of the receptor. Upon co-culture with CEA(+) tumor cells, receptor-grafted T cells are specifically and efficiently activated to cytolysis and IFN-gamma secretion, demonstrating their feasibility for the adoptive immunotherapy of CEA(+) carcinomas., (Copyright 2000 Wiley-Liss, Inc.)
- Published
- 2000
21. Grafting primary human T lymphocytes with cancer-specific chimeric single chain and two chain TCR.
- Author
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Willemsen RA, Weijtens ME, Ronteltap C, Eshhar Z, Gratama JW, Chames P, and Bolhuis RL
- Subjects
- Chimera, Clone Cells, Genetic Vectors, Granulocyte-Macrophage Colony-Stimulating Factor biosynthesis, Humans, Interferon-gamma biosynthesis, Melanoma immunology, Protein Engineering, Retroviridae, T-Lymphocytes immunology, Tumor Necrosis Factor-alpha biosynthesis, Gene Transfer Techniques, Genetic Therapy methods, Receptors, Antigen, T-Cell, alpha-beta genetics, T-Lymphocytes metabolism
- Abstract
Primary human activated T lymphocytes were genetically grafted with chimeric T cell receptors (TCR). Three domain single chain (sc-) TCR as well as two chain (tc-) TCR gene constructs were derived from the melanoma-specific cytotoxic human T cell (CTL) clone 82/30, and linked to the CD3-zeta signaling element. Chimeric TCR alpha and beta receptor genes were structurally designed to prevent pairing with endogenous TCR alpha and beta chains in order to prevent the generation of unpredictable immune specificities. After transduction of polyclonally activated human peripheral blood lymphocytes with retroviral vectors harboring the chimeric receptor genes, genetically engineered cells specifically recognized and responded to MAGE-A1POS/HLA-A1POS cells. Importantly, each type of transduced T lymphocytes that bound specifically to peptide/MHC complexes also showed specific antitumor reactivity as well as lymphokine production. Genetically engineered primary human T lymphocytes expressing chimeric sc- or tc-TCR therefore hold promise for disease-specific therapies.
- Published
- 2000
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22. Functional balance between T cell chimeric receptor density and tumor associated antigen density: CTL mediated cytolysis and lymphokine production.
- Author
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Weijtens ME, Hart EH, and Bolhuis RL
- Subjects
- Flow Cytometry, Genetic Vectors, Humans, Interferon-gamma metabolism, Tumor Necrosis Factor-alpha metabolism, Antigens, Neoplasm metabolism, Lymphokines metabolism, Receptors, Antigen, T-Cell metabolism, Recombinant Fusion Proteins immunology, T-Lymphocytes, Cytotoxic immunology
- Abstract
Genetically engineered expression of tumor-specific single chain antibody chimeric receptors (ch-Rec) on human T lymphocytes endow these cells with the parental monoclonal antibody (mAb) dictated tumor specificity and may be useful for clinical immuno-genetherapy. Therefore it was of importance to assess how the densities of tumor-specific receptors and tumor associated antigens (TAA), respectively, affect primary human T lymphocyte functions in relation to target cell susceptibilities to lysis. We therefore studied the functional balance between ch-Rec densities on human T lymphocytes and TAA on tumor cells. The gene construct encoding a ch-Rec derived from (1) a renal carcinoma cell (RCC) specific mouse mAb (G250), and (2) the human signal transducing Fc(epsilon)RI gamma-chain was used. To obtain ch-RecHIGH-POS and ch-RecLOW-POS T lymphocytes, two distinct retroviral vectors were used to introduce the gene constructs into primary human T lymphocytes. Levels of ch-Rec-redirected T lymphocyte mediated tumor cell lysis, as well as lymphokine production were determined using RCC lines as target/stimulator cells, which express either no or increasing densities of the TAA. A functional and dynamic balance between ch-Rec densities on cytotoxic T lymphocytes (CTLs) on the one hand and TAA densities on RCCs on the other, was found. In short, ch-RecHIGH-POS CTLs are triggered by TAAHIGH-POS as well as TAALOW-POS RCCs to lyse tumor cells and produce (IFN-gamma and TNF-alpha) lymphokine. In contrast, ch-RecLOW-POS T lymphocytes are only triggered for cytolysis and lymphokine production by relatively TAAHIGH-POS RCCs. In conclusion, (1) the activation of T lymphocyte responses is co-determined by the expression levels of the ch-Rec on T lymphocytes and the TAA on tumor cells and (2) at relatively high T lymphocyte ch-Rec expression levels the CTLs lyse tumor cells with a wide range of TAA densities. Gene Therapy (2000) 7, 35-42.
- Published
- 2000
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23. Phase I study of subcutaneously administered recombinant human interleukin 12 in patients with advanced renal cell cancer.
- Author
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Portielje JE, Kruit WH, Schuler M, Beck J, Lamers CH, Stoter G, Huber C, de Boer-Dennert M, Rakhit A, Bolhuis RL, and Aulitzky WE
- Subjects
- Adjuvants, Immunologic administration & dosage, Adjuvants, Immunologic adverse effects, Adjuvants, Immunologic blood, Adjuvants, Immunologic pharmacokinetics, Adult, Aged, Angiogenesis Inhibitors administration & dosage, Angiogenesis Inhibitors adverse effects, Angiogenesis Inhibitors blood, Angiogenesis Inhibitors pharmacokinetics, Carcinoma, Renal Cell blood, Dose-Response Relationship, Drug, Drug Administration Schedule, Female, Humans, Injections, Subcutaneous, Interleukin-12 administration & dosage, Interleukin-12 blood, Kidney Neoplasms blood, Male, Middle Aged, Recombinant Proteins administration & dosage, Recombinant Proteins adverse effects, Recombinant Proteins blood, Recombinant Proteins pharmacokinetics, Carcinoma, Renal Cell drug therapy, Carcinoma, Renal Cell metabolism, Interleukin-12 adverse effects, Interleukin-12 pharmacokinetics, Kidney Neoplasms drug therapy, Kidney Neoplasms metabolism
- Abstract
A phase I study was conducted to characterize the maximum tolerated dose (MTD), dose-limiting toxicity (DLT), and pharmacokinetics of a single dose followed by three times weekly s.c. injections of recombinant human interleukin 12 (rHuIL-12). The study encompassed 28 patients with advanced renal cell carcinoma. rHuIL-12 was administered on day 1, followed by an observation period of 7 days. Starting on day 8, repeated s.c. injections were administered 3 times a week for 2 weeks. The MTD of the initial injection was evaluated at dose levels of 0.1, 0.5, and 1.0 microg/kg. DLT was observed at 1.0 microg/kg and consisted of fever, perivasculitis of the skin, and leukopenia. The MTD of the subsequent repeated injections after 1 week of rest was studied at dose levels 0.5, 1.0, and 1.25 microg/kg. DLT at 1.25 microg/kg comprised deterioration of performance status, fever, vomiting, mental depression, and leukopenia. Other notable toxicities were oral mucositis and elevation of hepatic enzymes. Fever, leukopenia, and elevation of hepatic enzymes were more severe after the initial injection than after repeated injections at the same dose level. At dose level 0.5 microg/kg, the mean area under the plasma concentration-time curve decreased from 7.4 ng/h/ml after the first injection to 3.3 ng x h/ml (P = 0.034) after repeated administrations, and at dose level 1.0 microg/kg, it ranged from 31.8 ng/h/ml to 6.0 ng x h/ml (P = 0.041). One patient had a partial response and seven had stable disease. The MTD of a single s.c. injection of rHuIL-12 was 0.5 microg/kg, and the MTD of three subsequent administrations per week was 1.0 microg/kg. In comparison with a single administration, the three times weekly administrations at the same dose level was accompanied with a milder pattern of side effects and a reduction of the area under the plasma concentration-time curve.
- Published
- 1999
24. Restoration of expression of signal-transduction molecules in lymphocytes from patients with metastatic renal cell cancer after combination immunotherapy.
- Author
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Gratama JW, Zea AH, Bolhuis RL, and Ochoa AC
- Subjects
- Antineoplastic Agents pharmacology, Biomarkers, Dose-Response Relationship, Immunologic, Gene Expression Regulation, Neoplastic, Humans, Immunoglobulin gamma-Chains immunology, Immunoglobulin gamma-Chains metabolism, Immunophenotyping, Interferon-alpha pharmacology, Interleukin-2 pharmacology, K562 Cells, Killer Cells, Lymphokine-Activated immunology, Leukocytes, Mononuclear immunology, Lymphocyte Specific Protein Tyrosine Kinase p56(lck) metabolism, Membrane Proteins metabolism, Receptors, Antigen, T-Cell metabolism, Retrospective Studies, Time Factors, Carcinoma, Renal Cell drug therapy, Carcinoma, Renal Cell immunology, Immunotherapy, Kidney Neoplasms drug therapy, Kidney Neoplasms immunology, Lymphocyte Specific Protein Tyrosine Kinase p56(lck) immunology, Lymphocytes immunology, Membrane Proteins immunology, Receptors, Antigen, T-Cell immunology, Signal Transduction
- Abstract
A decrease in lymphocyte signal-transduction molecules, described in cancer patients and patients with chronic infectious diseases, has been proposed as a possible mechanism leading to an impaired immune response in cancer patients. Here we report the effects of combination immunotherapy on the levels of T cell receptor zeta chain and p56lck tyrosine kinase in a retrospective study of cryopreserved lymphocytes from 26 metastatic renal cell carcinoma patients treated with high-dose interleukin-2 (IL-2), interferon alpha (IFNalpha) and ex vivo IL-2-activated lymphocytes. Of the 26 patients, 12 were responders (5 complete and 7 partial) and 14 were non-responders (6 stable and 8 with progressive disease). Prior to treatment, 21 of 26 patients (81%) and 13 of 21 patients (62%) respectively expressed zeta chain and p56lck at less than 50% of the levels observed in healthy controls. During therapy, this low zeta chain and p56lck expression increased to at least 50% of normal in 13 of the 21 patients (62%) and in 6 of the 13 patients (46%) respectively; in the remaining patients expression levels remained at 50% of normal or more, or declined. Although, in this limited study, pretreatment levels of and p56lck did not show significant correlation with antitumor response, 4 of 5 patients that achieved a complete response (80%) corrected both zeta chain and p56lck levels to at least 50% of normal, while restoration of both signal-transduction molecules to such levels was only observed in 3 of 7 partial responders (43%), 1 of 5 patients with stable disease (20%) and 2 of 7 patients with progressive disease (29%). Thus, these results suggest that analysis of changes in signal-transduction molecules may a be useful tool for immunological monitoring of patients throughout immunotherapy, and could provide important information for designing new clinical trials that restore impaired signal transduction while activating T cell responses.
- Published
- 1999
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25. Large-scale production of natural cytokines during activation and expansion of human T lymphocytes in hollow fiber bioreactor cultures.
- Author
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Lamers CH, Gratama JW, Luider-Vrieling B, Bolhuis RL, and Bast EJ
- Subjects
- Cells, Cultured, Humans, Leukocytes, Mononuclear metabolism, Models, Theoretical, Reference Values, Reproducibility of Results, Sensitivity and Specificity, Bioreactors, Cytokines biosynthesis, T-Lymphocytes, Cytotoxic metabolism
- Abstract
We studied the large-scale production of a variety of natural cytokines during the activation and expansion of human T lymphocytes in a hollow fiber bioreactor culture system. Peripheral blood mononuclear cells (PBMC) were activated using phytohemagglutinin plus recombinant interleukin-2 (IL-2). Phytohemagglutinin was either present in the hollow fiber bioreactor during the entire 15-16-day culture period or only during the 20-h preactivation of the PBMC in culture bags. The expanding T lymphocytes were mainly CD3+,8+ and exerted maximal natural, activated, bispecific monoclonal antibody-redirected and lectin-dependent cytolytic activities between days 9 and 13 of culture. IL-1 and IL-4 were only produced in low amounts. IL-8 and lymphotoxin were primarily produced during the first week of culture. Harvest of the hollow fiber bioreactor culture supernatant at the time of peak cytokine concentration would have yielded per 10(8) PBMC input between 3.7 and 4.9 micrograms of IL-8 (at days 2 or 3), and between 0.02 and 0.5 microgram of lymphotoxin (at days 6 or 7). Tumor necrosis factor-alpha and IL-6 were produced during the entire culture period of 15 or 16 days: per 10(8) PBMC input, between 0.1 and 0.4 microgram of tumor necrosis factor-alpha (at days 2 or 3) and between 0.03 and 0.5 microgram of IL-6 (at days 15 or 16). Production of interferon-gamma and granulocyte-macrophage colony-stimulating factor started from initiation of cultures onwards to reach peak levels at the end of the 15- or 16-day culture period, yielding at that time between 2.1 and 17.7 micrograms/ml of interferon-gamma and between 0.4 and 4.2 micrograms of granulocyte-macrophage colony-stimulating factor per 10(8) PBMC input. The production of tumor necrosis factor-alpha, IL-6, interferon-gamma, and granulocyte-macrophage colony-stimulating factor was proportional to the extent of lymphocyte multiplication. These results demonstrate the usefulness of hollow fiber bioreactor cultures to produce natural cytokines during the activation and expansion of predominantly CD3+,8+ T lymphocytes.
- Published
- 1999
- Full Text
- View/download PDF
26. Quality control of flow cytometric immunophenotyping of haematological malignancies.
- Author
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Gratama JW, Bolhuis RL, and Van 't Veer MB
- Subjects
- Flow Cytometry standards, Humans, Quality Control, Hematologic Neoplasms immunology, Immunophenotyping
- Abstract
Immunophenotyping of haematological malignancies has developed as a clinically valuable but technically complicated diagnostic procedure. It involves a variety of methodological features, in-process strategic judgements and an extensive knowledge of clinical, morphological and other laboratory features of the disease processes under study. We discuss the various internal quality control steps necessary to guarantee reliable results with respect to instrument set-up and calibration; sample preparation; selection and validation of monoclonal antibody panels; and flow cytometric data acquisition, analysis and interpretation of results. The quality of the entire procedure is documented by the analysis of representative specimens in the setting of an external quality assurance programme.
- Published
- 1999
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- View/download PDF
27. Expression of T cell activation antigen CD134 (OX40) has no predictive value for the occurrence or response to therapy of acute graft-versus-host disease in partial T cell-depleted bone marrow transplantation.
- Author
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Gadisseur AP, Gratama JW, Lamers C, van Esser JW, Bolhuis RL, and Cornelissen JJ
- Subjects
- Acute Disease, Adult, Aged, Biomarkers, Bone Marrow Transplantation immunology, CD4-Positive T-Lymphocytes immunology, CD8-Positive T-Lymphocytes immunology, Female, Graft vs Host Disease therapy, Hematologic Diseases therapy, Humans, Leukemia therapy, Lymphocyte Activation, Lymphocyte Depletion, Lymphoma therapy, Male, Middle Aged, Receptors, OX40, Transplantation, Autologous, Transplantation, Homologous, Bone Marrow Transplantation adverse effects, Graft vs Host Disease etiology, Graft vs Host Disease immunology, Receptors, Tumor Necrosis Factor, T-Lymphocytes immunology, Tumor Necrosis Factor Receptor Superfamily, Member 7 metabolism
- Abstract
CD134 (OX40) is a member of the tumor necrosis factor family which is expressed by activated T lymphocytes. CD134 expression on T cells was monitored during the first 35 days post-transplant in 14 patients, receiving either an HLA-identical sibling bone marrow transplant (BMT), a matched unrelated transplant (MUD-BMT) or an autologous peripheral blood progenitor cell transplant (PBPCT). The sibling and unrelated grafts were partially depleted of T cells. CD134 expression on CD4+ T cells peaked between 7 and 14 days after BMT, with a mean peak value of 45% of CD4+ cells (range 26-70%) over all three patient groups. The observed pattern of CD4+ CD134+ expression, an increase during the first 2 weeks post-BMT followed by a gradual decline towards values of 15-40%, was similar in all groups. No difference in the kinetics of CD134 expression by CD4+ T cells was observed between the patients that did or did not develop graft-versus-host disease (GVHD), nor did the clinical effect of any treatment given for GVHD correlate with alterations in CD134 expression by CD4+ T cells. Absolute CD4+,CD134+ T cell numbers showed a more rapid increment after autologous PBPCT than after sibling or MUD transplants. We conclude that expression of CD134+ by CD4+ T lymphocytes cannot serve as a surrogate marker for allo-reactivity. CD134+ expression may reflect lymphocyte regeneration, rather than alloreactivity.
- Published
- 1999
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28. Level of anti-mouse-antibody response induced by bi-specific monoclonal antibody OC/TR in ovarian-carcinoma patients is associated with longer survival.
- Author
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Miotti S, Negri DR, Valota O, Calabrese M, Bolhuis RL, Gratama JW, Colnaghi MI, and Canevari S
- Subjects
- Animals, Antibodies, Anti-Idiotypic biosynthesis, Antibodies, Bispecific immunology, Antibodies, Heterophile immunology, Antibodies, Monoclonal immunology, Biomarkers, Female, Humans, Mice, Neoplasm Staging, Ovarian Neoplasms immunology, Prognosis, Survival Rate, T-Lymphocytes immunology, Time Factors, Antibodies, Anti-Idiotypic immunology, Antibodies, Bispecific therapeutic use, Antibodies, Monoclonal therapeutic use, Ovarian Neoplasms therapy
- Abstract
More than 60% of cancer patients injected with intact murine monoclonal antibody (MAb) develop a humoral response against the antigen even after a single dose. Analysis of a series of 35 ovarian-cancer patients entered in phase-I and -II clinical studies of T-cells retargeted with the bi-specific F(ab')2 OC/TR revealed: (i) a detectable human anti-mouse antibody (HAMA) response in 31/35 (88%) patients, with high HAMA levels (> or = 150 ng/ml) in 18/31 (58%) cases by the end of the treatment; (ii) no correlation between HAMA levels and the form of delivery of the mAb (OC/TR bound to T cells or bound plus soluble), time schedule or cumulative dose; (iii) an association between high HAMA levels and favorable clinical parameters and response to immunotherapy; and (iv) a significantly longer median survival probability in patients with high HAMA levels than in patients with lower HAMA levels, even when the sub-group of non-responder patients was considered. Evaluation of the anti-idiotypic response in HAMA-positive sera indicated that 11/17 sera showed high-titer (>6000) binding of OC/TR, as evaluated by a specific radioimmunoassay, and 15/18 and 16/16 sera specifically inhibited the binding of the MOv18 and anti-CD3 parental MAbs to ovarian-carcinoma cells and T lymphocytes respectively. Of 7 patients evaluated for duration of the HAMA response, 5 showed stable or even increased HAMA levels. The long-lasting HAMA response maintained an anti-idiotypic component, directed mainly against the alphaCD3 idiotype of bi-MAb OC/TR in 2 out of 3 cases tested.
- Published
- 1999
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29. Genetic re-targeting of T lymphocyte specificity.
- Author
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Bolhuis RL and Gratama JW
- Subjects
- Antibodies, Monoclonal therapeutic use, Autoimmune Diseases therapy, Epitopes, Humans, Recombinant Fusion Proteins genetics, Gene Targeting, Gene Transfer Techniques, Genetic Therapy methods, Lymphocytes, Tumor-Infiltrating immunology, Neoplasms therapy, Receptors, Antigen, T-Cell genetics, Virus Diseases therapy
- Published
- 1998
- Full Text
- View/download PDF
30. A retroviral vector system 'STITCH' in combination with an optimized single chain antibody chimeric receptor gene structure allows efficient gene transduction and expression in human T lymphocytes.
- Author
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Weijtens ME, Willemsen RA, Hart EH, and Bolhuis RL
- Subjects
- Carcinoma, Renal Cell immunology, Carcinoma, Renal Cell therapy, Cell Membrane metabolism, Gene Expression, Humans, Kidney Neoplasms immunology, Kidney Neoplasms therapy, Receptors, Antigen, T-Cell genetics, Recombinant Fusion Proteins genetics, Transduction, Genetic, Tumor Cells, Cultured, Genetic Therapy methods, Genetic Vectors, Lymphocytes, Tumor-Infiltrating metabolism, Receptors, Immunologic genetics, Retroviridae genetics
- Abstract
Genetic engineering of T lymphocytes for adoptive clinical immunotherapy calls for efficient gene transduction methods. Therefore, a transient retroviral gene transduction system 'STITCH' was developed comprising pSTITCH retroviral vector encoding the transgene, plasmids encoding Moloney murine leukemia virus gag/pol and gibbon ape leukemia virus envelope, and the human kidney cell line 293T as a packaging line. Cotransfection of retroviral vector and packaging plasmids in 293T cells results in the production of GALV env pseudotyped viral particles with a titer of 10(7) infectious units per milliliter. The 'STITCH' gene transduction system efficiently transduces genes into activated human T lymphocytes derived from healthy donors and cancer patients. The efficacy of gene transduction is donor-independent. A direct application of the 'STITCH' gene transduction system is the genetic engineering of activated human T lymphocytes to induce expression of antibody based chimeric receptors in their membrane. Introduction of these chimeric receptors into activated human T lymphocytes graft these cells with specificity for, for example, renal cell carcinoma. In order to study the effect of the chimeric receptor gene structure on the processes ultimately leading to functional membrane expression, we designed a number of different chimeric receptor gene structures and subsequently compared their membrane expression on 293T cells and activated human T lymphocytes. Distinct membrane expression densities were observed on 293T cells and human T lymphocytes for the different chimeric receptor gene constructs. Gene transduction of activated human T lymphocytes with four out of five chimeric receptor gene constructs resulted in functional expression of chimeric receptor as demonstrated by specific recognition and cytolysis of renal cell carcinoma.
- Published
- 1998
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31. Chimeric scFv/gamma receptor-mediated T-cell lysis of tumor cells is coregulated by adhesion and accessory molecules.
- Author
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Weijtens ME, Willemsen RA, van Krimpen BA, and Bolhuis RL
- Subjects
- Antibodies, Monoclonal pharmacology, CD11 Antigens immunology, CD18 Antigens immunology, CD2 Antigens immunology, CD3 Complex immunology, CD58 Antigens immunology, Cytotoxicity, Immunologic, Humans, Intercellular Adhesion Molecule-1 immunology, Melanoma immunology, Transfection, Tumor Cells, Cultured, Antigens, CD immunology, Antigens, Neoplasm immunology, Carcinoma, Renal Cell immunology, Immunoglobulin Fragments immunology, Receptors, Antigen, T-Cell, gamma-delta immunology, Recombinant Fusion Proteins immunology, T-Lymphocytes, Cytotoxic immunology
- Abstract
Adhesion and accessory molecules play a critical role in T-cell activation and effector function in general and in tumor cell recognition and lysis in particular. We investigated the contribution of CD2, CD3, CD11a/CD18, CD54 and CD58 molecules in T lymphocyte-tumor cell interactions mediated by chimeric immunoglobulin receptors. The chimeric receptor is composed of a single chain antibody binding site and a gamma-chain signal transducing molecule (scFv/gamma). T lymphocytes expressing such scFv/gamma receptors recognize the G250 Ag on renal cell carcinoma (RCC) in an major histocompatibility complex (MHC)-unrestricted manner and exert RCC selective cytolysis. A coregulatory role for CD2, CD3 and CD11a/CD18 molecules in scFv/gamma-mediated cytolysis was demonstrated using monoclonal antibody (MAb)-induced inhibition of scFv/gamma-mediated cytolysis. The inhibition of lysis was not due to inhibition of cytotoxic T lymphocyte (CTL)-target cell conjugation but rather to a post-conjugate signaling event. Binding of CD54 and CD58 MAbs to the RCC did not inhibit cytolysis of RCC cells that expressed high levels of both CD54 and the G250 antigen (Ag) (A75), whereas cytolysis of RCC expressing intermediate levels of CD54 and G250 Ag (SK-RC-17 cl.4) was partly inhibited by the CD54 MAb. Binding of low concentrations of G250 MAb to RCC (A75) rendered these cells sensitive to CD54 MAb inhibition, demonstrating a direct functional relation between G250 Ag expression level and adhesion molecules. Taken together, our findings indicate a coregulatory role for CD2, CD3 and CD11a/CD18 molecules in the scFv/gamma-mediated cytolysis of tumor cells and show that the requirement of CD11a/CD18-CD54 interactions is dependent on the level of free Ag. This make these gene-transduced T lymphocytes attractive tools for adoptive immunogene therapy of cancer.
- Published
- 1998
- Full Text
- View/download PDF
32. A phase I/II study of multicyclic dose-intensive chemotherapy supported with G-CSF, or G-CSF and haematopoietic progenitor cells in whole blood, in two consecutive cohorts of patients.
- Author
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de Wit R, Kruit WH, Lamers CH, van 't Veer MB, Luyten AA, van Beurden V, Harteveld M, Planting AS, Schmitz PI, Stoter G, Bolhuis RL, and Verweij J
- Subjects
- Adult, Aged, Antineoplastic Combined Chemotherapy Protocols adverse effects, Cisplatin administration & dosage, Dose-Response Relationship, Drug, Drug Administration Schedule, Female, Humans, Ifosfamide administration & dosage, Male, Middle Aged, Neoplasms blood, Antineoplastic Combined Chemotherapy Protocols therapeutic use, Granulocyte Colony-Stimulating Factor therapeutic use, Hematopoietic Stem Cell Transplantation, Neoplasms drug therapy, Neoplasms therapy
- Abstract
We investigated the reconstitutive potential of haematopoietic progenitor cells collected in autologous whole blood during multicycle dose-intensified chemotherapy. Forty patients with metastatic solid tumours were treated with up to six cycles of cisplatin and escalating doses of ifosfamide every 14 days. Cisplatin was administered in 3% sodium chloride over 3 h, followed by ifosfamide over 24 h and mesna over 36 h. The first cohort of patients received granulocyte colony-stimulating factor (G-CSF) days 4-14. Once dose-limiting toxicity was reached in cohort 1, the study continued with a second cohort of patients, in whom, in addition to G-CSF on days 4-14, 500 ml of G-CSF and chemotherapy-'primed' whole blood was collected on day 15, i.e. on day 1 of treatment cycles two to six, before cisplatin administration. This volume of blood was kept unprocessed at 4 degrees C and reinfused 20-24 h after the completion of ifosfamide. In cohort 1, dose-limiting toxicity (DLT) was reached at ifosfamide 6.0 g m(-2) with two out of six of the patients developing neutropenic fever. Although in cohort 2 no neutropenic fever was encountered, neither the frequency nor the duration of grade 4 neutropenia and thrombocytopenia were reduced. Cumulative asthenia resulted in DLT at 7.0 g m(-2). The median number of CD34+ cells in 500 ml of whole blood after the first cycle (i.e. at start of cycle 2) was 1.15 x 10(6) kg(-1). This number was significantly greater after the second cycle (2.06 x 10(6) kg(-1), P = 0.01) and then gradually decreased after cycles three to six. After storing whole blood, the number of CD34+ cells had not decreased (median + 10%). We conclude that the method of combined bone marrow support by G-CSF and haematopoietic progenitor cells in autologous whole blood collected before each cycle of a 2-weekly regimen of cisplatin-ifosfamide does not result in clinically measurable reduced bone marrow toxicity compared with what can be expected by the use of G-CSF alone.
- Published
- 1998
- Full Text
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33. Preparation for a phase I/II study using autologous gene modified T lymphocytes for treatment of metastatic renal cancer patients.
- Author
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Bolhuis RL, Willemsen RA, Lamers CH, Stam K, Gratama JW, and Weijtens ME
- Subjects
- CD4 Antigens biosynthesis, Humans, Immunoglobulin Variable Region biosynthesis, Lymphocyte Activation, Recombinant Fusion Proteins biosynthesis, Transfection methods, Transplantation, Autologous, CD4 Antigens genetics, Carcinoma, Renal Cell immunology, Carcinoma, Renal Cell therapy, Genetic Therapy adverse effects, Immunoglobulin Variable Region genetics, Kidney Neoplasms immunology, Kidney Neoplasms therapy, Lymphocyte Transfusion adverse effects, T-Lymphocytes immunology
- Published
- 1998
- Full Text
- View/download PDF
34. Chimeric bispecific OC/TR monoclonal antibody mediates lysis of tumor cells expressing the folate-binding protein (MOv18) and displays decreased immunogenicity in patients.
- Author
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Luiten RM, Warnaar SO, Sanborn D, Lamers CH, Bolhuis RL, Litvinov SV, Zurawski VR Jr, and Coney LR
- Subjects
- Animals, Antibodies, Anti-Idiotypic blood, Antibodies, Bispecific genetics, Antibodies, Monoclonal genetics, CD3 Complex immunology, Cell Line, Female, Folate Receptors, GPI-Anchored, Gene Expression, Humans, Hybridomas, Immunoglobulin Fab Fragments immunology, Immunotherapy, Mice, Recombinant Fusion Proteins, T-Lymphocytes immunology, Transfection, Antibodies, Bispecific immunology, Antibodies, Monoclonal immunology, Carrier Proteins analysis, Cytotoxicity, Immunologic, Ovarian Neoplasms immunology, Receptors, Cell Surface
- Abstract
The bispecific OC/TR monoclonal antibody (mAb) cross-links the CD3 molecule on T cells with the human folate-binding protein (FBP), which is highly expressed on nonmucinous ovarian carcinomas. Clinical trials of patients with ovarian carcinoma with the OC/TR mAb have shown some complete and partial responses. Most patients developed human anti-murine immunoglobulin antibodies (HAMA), which can inhibit OC/TR mAb-mediated lysis. We generated a chimeric version of the OC/TR mAb to decrease the immunogenicity of the OC/TR mAb and to allow more extended treatment schedules. Sp2/0 myeloma cells were transfected with chimeric heavy- and light-chain genes encoding the anti-CD3 mAb and the MOv18 mAb, respectively, which are reactive with FBP. The resulting cell line produced 80 micrograms/ml of total immunoglobulin G (IgG), of which 11.5% was the functionally active chimeric OC/TR mAb. Chimeric OC/TR F(ab')2 fragments mediated lysis of IGROV-1 ovarian carcinoma cells by human T cells at antibody concentrations of > or = 1 pg/ml. Specific lysis was still detectable at an effector-to-target cell ratio as low as 0.4. Two patients with ovarian carcinoma treated with F(ab')2 fragments of the murine OC/TR developed distinct HAMA titers, which were mainly anti-idiotypic and only partly directed against the murine antibody constant regions. However, of the two patients that were treated with the F(ab')2 fragments of the chimeric OC/TR mAb, only one developed a low transient HAMA response just above background level. In conclusion, the generation of chimeric OC/TR may allow more extended clinical studies of bispecific mAb-mediated immunotherapy of ovarian carcinoma.
- Published
- 1997
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35. Analysis of factors contributing to the formation of mononuclear cell aggregates ("escapees") in flow cytometric immunophenotyping.
- Author
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Gratama JW, van der Linden R, van der Holt B, Bolhuis RL, and van de Winkel JG
- Subjects
- Antibodies, Monoclonal immunology, Antigens, CD analysis, Antigens, CD genetics, Cell Aggregation, Cell Separation methods, Humans, Immunoglobulin Fab Fragments immunology, Immunophenotyping methods, Light, Polymorphism, Genetic, Receptors, IgG analysis, Receptors, IgG genetics, Scattering, Radiation, Flow Cytometry methods, Monocytes cytology
- Abstract
During immunostaining, human lymphocytes may form aggregates with activated platelets and monocytes, resulting in increased forward (FSC) and sideward (SSC) light scatter signals. Consequently, aggregated cells "escape" from the standard FSC-SSC analysis gate, thereby producing erroneous results. We observed that the frequency of aggregate formation in peripheral blood mononuclear cell (PBMC) suspensions depended on cell donor, murine (m) monoclonal antibody (mAb) specificity and IgG subclass, type of fluorochrome conjugated to the mAb, amount of mAb used for immunostaining, time lapse between fixation of PBMC and flow cytometry, and other, as yet unidentified, factors. Platelets, monocytes, and granulocytes express the polymorphic class IIa IgG receptor (Fc gammaRIIa; CD32). A single amino acid difference, either arginine (R) or histidine (H) at amino acid position 131, underlies differential interaction with mIgG1. Because the Fc gammaRIIa-R131 allotypic form binds mIgG1 well in contrast to Fc gammaRIIa-H131, we studied the frequency of aggregate formation in PBMC suspensions from apparently healthy individuals allotyped for Fc gammaRIIa. The Fc gammaRIIa polymorphism contributed significantly to the frequency of mIgG1-induced cell aggregates, which was highest in Fc gammaRIIa-R/R131 individuals, intermediate in Fc gammaRIIa-R/H131 individuals, and lowest in Fc gammaRIIa-H/H131 individuals. The role of mIgG1-Fc gammaRIIa interactions in aggregate formation was confirmed by blocking Fc gammaRIIa by using F(ab')2 fragments of CD32 mAb. These data document the role of mIgG1 mAb binding by human class IIa IgG receptors in the formation of cell aggregates and show that inhibition of this interaction reduces this technical problem in flow cytometric immunophenotyping.
- Published
- 1997
36. Local but no systemic immunomodulation by intraperitoneal treatment of advanced ovarian cancer with autologous T lymphocytes re-targeted by a bi-specific monoclonal antibody.
- Author
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Lamers CH, Bolhuis RL, Warnaar SO, Stoter G, and Gratama JW
- Subjects
- Ascitic Fluid cytology, CD3 Complex immunology, Cytotoxicity, Immunologic, Female, Flow Cytometry, Humans, Immunophenotyping, Infusions, Parenteral, Leukapheresis, Lymphocyte Activation immunology, Lymphocyte Count, Ovarian Neoplasms therapy, T-Lymphocytes, Cytotoxic immunology, Antibodies, Bispecific therapeutic use, Antibodies, Monoclonal therapeutic use, Immunotherapy, Ovarian Neoplasms immunology, T-Lymphocytes immunology
- Abstract
We have reported a 27% overall anti-tumor response using i.p. immunotherapy of advanced ovarian carcinoma with autologous, ex vivo expanded, T lymphocytes re-targeted with bi-specific monoclonal antibody OC/TR, combined with soluble OC/TR and low-dose recombinant interleukin-2 (IL-2). This treatment had no effect on extraperitoneal disease. Therefore we studied in 13 patients whether this immunotherapeutic protocol resulted only in local or also in systemic immunomodulation. The phenotype of the ex vivo expanded lymphocytes was mainly CD3+, 4-, 8+, 16-, 56-. Their OC/TR-re-targeted cytolytic activity against Igrov-1 ovarian-carcinoma cells was approximately as high in responders as in non-responders. Following most therapeutic cycles, the immunophenotype of lymphocytes recovered from the peritoneal fluid was similar to that of the infused T cells (i.e., mainly CD3+, 4-, 8+) and they were coated with OC/TR. However, cytolytic activity of the recovered lymphocytes against Igrov- 1 cells was low in direct assays, and only slightly increased after additional in vitro re-targeting with OC/TR. Systemically, the i.p. immunotherapy resulted in a transient lymphopenia lasting for about 7 days, low (i.e., 5 to 13 ng/ml) serum concentrations of free, functional OC/TR, and very weak coating of circulating T lymphocytes with OC/TR. These peripheral-blood T lymphocytes did not exert OC/TR-re-targeted cytolytic activity. Thus, locoregional OC/TR-re-targeted cellular immunotherapy resulted in substantial local immunomodulation and anti-tumor effects but virtually no systemic immunomodulation.
- Published
- 1997
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37. Sequence analysis of the Epstein-Barr virus (EBV) latent membrane protein-1 gene and promoter region: identification of four variants among wild-type EBV isolates.
- Author
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Sandvej K, Gratama JW, Munch M, Zhou XG, Bolhuis RL, Andresen BS, Gregersen N, and Hamilton-Dutoit S
- Subjects
- Amino Acid Sequence, Base Sequence, Cell Line, Herpesvirus 4, Human isolation & purification, Humans, Lymphocytes virology, Molecular Sequence Data, Sequence Alignment, Sequence Analysis, Genes, Viral, Herpesvirus 4, Human genetics, Promoter Regions, Genetic genetics, Viral Matrix Proteins genetics
- Abstract
Sequence variations in the Epstein-Barr virus (EBV) encoded latent membrane protein-1 (LMP-1) gene have been described in a Chinese nasopharyngeal carcinoma-derived isolate (CAO), and in viral isolates from various EBV-associated tumors. It has been suggested that these genetic changes, which include loss of a Xho I restriction site (position 169425) and a C-terminal 30-base pair (bp) deletion (position 168287-168256), define EBV genotypes associated with increased tumorigenicity or with disease among particular geographic populations. To determine the frequency of LMP-1 variations in European wild-type virus isolates, we sequenced the LMP-1 promoter and gene in EBV from lymphoblastoid cell lines from healthy carriers and patients without EBV-associated disease. Sequence changes were often present, and defined at least four main groups of viral isolates, which we designate Groups A through D. The widespread prevalence of LMP-1 sequence variations, particularly the Xho I polymorphism and the 30-bp deletion, indicate that they cannot be used as simple markers for oncogenic viruses related to particular forms of EBV-associated tumor. Several of the structural changes detected occur, however, at sites where they may affect transcription, translation, or function of LMP-1. Future in vitro studies should aim to establish the functional importance of variations at these sites.
- Published
- 1997
38. High-dose regimen of interleukin-2 and interferon-alpha in combination with lymphokine-activated killer cells in patients with metastatic renal cell cancer.
- Author
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Kruit WH, Goey SH, Lamers CH, Gratama JW, Visser B, Schmitz PI, Eggermont AM, Bolhuis RL, and Stoter G
- Subjects
- Adult, Aged, Carcinoma, Renal Cell immunology, Female, Humans, Interferon-alpha adverse effects, Interleukin-2 adverse effects, Kidney Neoplasms immunology, Male, Middle Aged, Neoplasm Metastasis, Prognosis, Adoptive Transfer, Carcinoma, Renal Cell therapy, Interferon-alpha administration & dosage, Interleukin-2 administration & dosage, Kidney Neoplasms therapy, Killer Cells, Lymphokine-Activated immunology
- Abstract
Seventy-two patients with metastatic renal cell cancer were treated with the combination of high-dose interleukin-2 (IL2), interferon-alpha (IFN alpha), and lymphokine-activated killer cells (LAK). Seventeen patients were entered in a feasibility part of the study (protocol 1) and 55 in an efficacy part (protocol 2). Protocol 2 differed from protocol 1 in the addition of IFN alpha to the first 5 days of IL2 infusion. Each patient was planned to receive two induction cycles. IL2, 18 MIU/m2/day, was administered continuously i.v. on days 1-5, and IFN alpha, 5 MIU/m2/day (protocol 2), was administered i.m. on days 1-5, followed by three daily lymphaphereses on days 7-9. On day 12, treatment was resumed with IL2 and IFN alpha on days 12-15 and LAK reinfusions on days 12-14. In protocol 1, three complete (CR) and one partial (PR) responses were achieved (response rate 24%). The median duration of response and the median survival were 18.1 and 13.9 months, respectively. The 3-year survival was 35%. Of the 51 evaluable patients in protocol 2, 6 achieved a CR and 13 a PR (response rate 37%). The median duration of response was 11.1 months. The median survival was 16.9 months. The 3-year survival was 35%. There were three treatment-related deaths. Other severe toxicities included hypotension, cardiotoxicity, pulmonary edema, renal toxicity, and infectious complications. In the two induction cycles, only 54 and 42% of the planned doses could be administered. We conclude that the use of high-dose regimens of IL2 and IFN alpha is not warranted, unless we can define more accurately which patients may experience long-term survival as a result of treatment.
- Published
- 1997
- Full Text
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39. Tunnelled central venous catheters yield a low incidence of septicaemia in interleukin-2-treated patients.
- Author
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Goey SH, Verweij J, Bolhuis RL, de Gooyer D, Eggermont AM, Schmitz PI, and Stoter G
- Subjects
- Carcinoma, Renal Cell drug therapy, Catheterization, Central Venous instrumentation, Female, Humans, Interleukin-2 therapeutic use, Karnofsky Performance Status, Kidney Neoplasms drug therapy, Male, Middle Aged, Neoplasm Metastasis, Retrospective Studies, Catheterization, Central Venous adverse effects, Catheters, Indwelling adverse effects, Interleukin-2 administration & dosage, Sepsis epidemiology, Staphylococcal Infections epidemiology
- Abstract
A retrospective study on the incidence of catheter-related complications and catheter indwelling time (tCI) during treatment with continuous interleukin-2 (IL-2) infusion in patients with metastatic renal cell cancer, who were equipped with tunnelled central venous catheters (CVC). A group of 72 patients were treated with IL-2-based immunotherapy. Two induction treatment cycles of 35 days each were used. Treatment consisted of IL-2 as a continuous intravenous infusion (c.i.v.) with lymphokine-activated killer cells and interferon alpha intramuscularly. A tunnelled CVC was inserted at the start of treatment and was kept in place for the duration of the therapy or until the occurrence of complications. Out of 72 CVC, 30 (42%) functioned uneventfully for a median tCI of 64 days. In another 12 clinically uncomplicated cases (16%), catheter tips were positive in routine culture after a median tCI of 33 days. In 18 patients (25%), CVC-related infections were noted, including 8 (11%) local tunnel infections and 10 (14%) septic episodes. These complications occurred at a median tCI of 28 and 20 days respectively. In 15 (83%) of these 18 catheter infections, Staphylococcus aureus was isolated, whereas in the remaining 3 (17%) Staphylococcus epidermidis was found. Subclavian vein thrombosis was noted in 12 (17%) CVC at a median tCI of 31 days: 5 (36%) of these were diagnosed in the first 14 patients. This prompted us to administer prophylactic heparin 15,000 i.u. c.i.v. daily during IL-2 treatment. Thereafter the incidence of thrombosis dropped to 7 (12%) in the subsequent 58 CVC inserted (P = 0.03). In conclusion, in contrast to previous reports on the high incidence of CVC-related septicaemia and thrombosis, we observed a relatively low incidence of these complications, which we ascribe to the use of tunnelled catheters and prophylactic heparin.
- Published
- 1997
- Full Text
- View/download PDF
40. Reduction of interlaboratory variability in flow cytometric immunophenotyping by standardization of instrument set-up and calibration, and standard list mode data analysis.
- Author
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Gratama JW, Kraan J, Adriaansen H, Hooibrink B, Levering W, Reinders P, Van den Beemd MW, Van der Holt B, and Bolhuis RL
- Subjects
- Antigens, CD analysis, Calibration, Flow Cytometry instrumentation, Flow Cytometry methods, Humans, Immunophenotyping instrumentation, Immunophenotyping methods, Reference Standards, Reproducibility of Results, Flow Cytometry standards, Immunophenotyping standards, Lymphocytes immunology
- Abstract
Two workshops addressed the question to which degree standardization of instrument set-up and calibration, and standard list mode data analysis would reduce interlaboratory variability of flow cytometric results on prestained peripheral blood mononuclear cells (PBMC). Standard instrument set-up included uniform positioning of the "windows of analysis" for the forward and sideward light scatter and fluorescence (FL) 1 (i.e., fluorescein isothiocyanate [FITC]) and 2 (i.e., phycoerythrin [PE]) parameters. Reference standards and PBMC, double-stained with FITC- and PE-conjugated monoclonal antibodies covering a wide range of FL intensities and coexpression patterns, were sent out to 25 laboratories in Workshop 1 and to 35 laboratories in Workshop 2 with the following requests: a) to set up instruments according to local and standard protocols, b) to acquire list mode data on the PBMC with both instrument settings, and c) to analyze both datasets according to local protocols. Standard analysis of the list mode data acquired with uniform instrument settings was performed centrally using so-called "latent class model" software (Van Putten et al., Cytometry 14:86-96, 1993). This software provides an automated, "no-gating" analytical method of lymphocyte immunophenotypes and employs fixed FL marker settings as defined prior to each analytical run. In Workshop 1, these markers were set in identical histogram channels for all instruments based on results obtained with a reference instrument. Standard analysis of list mode data acquired after uniform instrument set-up led only to a 13% reduction of interlaboratory variability of results as compared to data analysis using local protocols. The standard protocol for instrument set-up led to uniform positioning of relatively strong FL signals but variable positioning of unstained cells on the FL histogram scales. Hence, standard FL marker settings were inappropriate for some instruments. Therefore, instrument responses to FITC and PE signals in Workshop 2 were calibrated using microbeads labeled with FITC or PE in a range of predefined FL intensities expressed in MESF units (molecules of equivalent soluble fluorochrome). That approach allowed the positioning of the FL markers for the standard analysis on the basis of identical FL1 and FL2 intensities, expressed in MESF units, for all instruments. Standard analysis of list mode data acquired after uniform instrument set-up and calibrated FL marker settings led to a 43% reduction of interlaboratory variability as compared to data analysis to local protocols. We conclude that standard list mode data analysis using fixed FL marker settings reduces the interlaboratory variability of flow cytometric results on prestained PBMC, provided that the instruments have been set up in a uniform way and that FL markers have been standardized on the basis of calibration of each instrument's response to the corresponding FL signals.
- Published
- 1997
41. Dose efficacy study of two schedules of high-dose bolus administration of interleukin 2 and interferon alpha in metastatic melanoma.
- Author
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Kruit WH, Punt CJ, Goey SH, de Mulder PH, Gratama JW, Eggermont AM, Bolhuis RL, and Stoter G
- Subjects
- Adult, Aged, Drug Administration Schedule, Female, Humans, Interferon-alpha adverse effects, Interleukin-2 adverse effects, Killer Cells, Natural immunology, Male, Melanoma immunology, Middle Aged, Interferon-alpha administration & dosage, Interleukin-2 administration & dosage, Melanoma secondary, Melanoma therapy
- Abstract
Forty-three patients with metastatic melanoma were treated with a 5 day (18 patients) and a 3 day (25 patients) schedule of high-dose IL-2 11.7 MIU m2 and IFN-alpha 3 MIU m2 i.v. by bolus administration every 8 h, repeated every 21 days for a total of three courses. The 5 day schedule resulted in a high response rate of 41% (CI 18-67%), but was accompanied by severe cardiotoxicity (41%) and central nervous system toxicity (28%). The 3 day schedule was associated with manageable toxicity, but yielded a moderate response rate of 20% (CI 7-43%).
- Published
- 1996
- Full Text
- View/download PDF
42. Single chain Ig/gamma gene-redirected human T lymphocytes produce cytokines, specifically lyse tumor cells, and recycle lytic capacity.
- Author
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Weijtens ME, Willemsen RA, Valerio D, Stam K, and Bolhuis RL
- Subjects
- Base Sequence, Carcinoma, Renal Cell immunology, Cytokines metabolism, Cytotoxicity Tests, Immunologic, Genetic Vectors immunology, Humans, Molecular Sequence Data, Receptors, IgE genetics, Receptors, IgE physiology, Recombinant Fusion Proteins pharmacology, Transfection immunology, Tumor Cells, Cultured, Cytokines biosynthesis, Cytotoxicity, Immunologic genetics, Genes, Immunoglobulin immunology, Immunoglobulin Fragments genetics, Immunoglobulin G genetics, T-Lymphocytes, Cytotoxic immunology, T-Lymphocytes, Cytotoxic metabolism
- Abstract
To enable construction of CTL with known predefined Ab specificity for adoptive immunotherapy, we constructed a chimeric scFv/gamma gene composed of the variable regions of a mAb joined to the Fc(epsilon)RI signaling receptor gamma-chain of mast cells. Introduction of this chimeric receptor into CTL rendered these lymphocytes specific for renal cell carcinoma. This approach combines the specificity of tumor-selective Abs with the efficacy of CTL to destroy tumor cells. We not only demonstrated that the transduced CTL functionally express the scFv/gamma receptor for a prolonged period of time (4.5 mo of in vitro culture), but also showed high levels of Ab-dictated lysis of renal cell carcinoma similar to that of normal CTL, and importantly, we demonstrated that these CTL can recycle their lytic activity. Moreover, these scFv/gamma-expressing T lymphocytes produce cytokines upon stimulation with the relevant target cell. These results together with the donor independence of our gene transduction protocol demonstrate the feasibility of redirecting T lymphocytes for cancer treatment.
- Published
- 1996
43. Prolonged continuous hepatic artery infusion with interleukin-2 in unresectable liver metastases of colorectal cancer: a phase IA-B study.
- Author
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Goey SH, Eggermont AM, Oskam R, Wiggers T, Bolhuis RL, and Stoter G
- Subjects
- Adult, Female, Hepatic Artery, Humans, Infusions, Intra-Arterial, Liver Neoplasms secondary, Male, Middle Aged, Time Factors, Antineoplastic Agents therapeutic use, Colorectal Neoplasms pathology, Interleukin-2 therapeutic use, Liver Neoplasms drug therapy
- Published
- 1996
- Full Text
- View/download PDF
44. Modulation of immune parameters in patients with metastatic renal-cell cancer receiving combination immunotherapy (IL-2, IFN alpha and autologous IL-2-activated lymphocytes).
- Author
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Gratama JW, Schmitz PI, Goey SH, Lamers CH, Stoter G, and Bolhuis RL
- Subjects
- Adult, Aged, Carcinoma, Renal Cell blood, Carcinoma, Renal Cell therapy, Cell Count, Cytokines blood, Female, Humans, Immunophenotyping, Kidney Neoplasms blood, Kidney Neoplasms therapy, Leukocytes, Mononuclear transplantation, Lymphocyte Subsets, Male, Middle Aged, Neoplasm Metastasis, Carcinoma, Renal Cell immunology, Immunotherapy, Adoptive, Interferon-alpha therapeutic use, Interleukin-2 therapeutic use, Kidney Neoplasms immunology, Leukocytes, Mononuclear immunology
- Abstract
We treated 72 patients with metastatic renal-cell cancer according to 2 protocols consisting of two 5-week induction cycles of continuous i.v. high-dose interleukin-2 (IL-2), i.m. interferon-alpha (IFN alpha) and ex vivo IL-2-activated lymphocytes, followed for patients with stable disease (SD), partial response (PR) or complete response (CR) by four 4-week maintenance cycles of IL-2 and IFN alpha. Protocol 2 (55 patients) differed from protocol 1 (17 patients) in (i) the addition of IFN alpha to the first IL-2 infusions in both induction cycles; (ii) the use of Teceleukin IL-2, reconstituted with carrier protein, instead of Proleukin IL-2 without carrier protein. We classified 23 patients with CR and PR as responders (4 in protocol 1 and 19 in protocol 2) and 45 patients with SD and progressive disease as non-responders. Prior to immunotherapy, patients entered into protocol 2 already had higher IFN gamma serum concentrations, higher peripheral blood CD56-,3+ and CD8-,4+ lymphocyte numbers and lower NKK562 activity than those entered into protocol 1. These differences persisted during and after immunotherapy. In line with these observations, ex vivo IL-2-activated lymphocytes had larger proportions of CD56-,3+ and CD8-,4+ lymphocytes and lower NKK562 activity in protocol 2 than in protocol 1. Higher IL-2 serum concentrations were reached during the IL-2 infusion in protocol 2 than in protocol 1. In addition, the immunomodulation in protocol 2 was stronger than in protocol 1 as indicated by higher TNF alpha serum concentrations and a more pronounced eosinophilia. Differences between responders and non-responders treated according to the 2 protocols were not significant, except for the total number of lymphocytes obtained by apheresis, which was higher in responders than in non-responders.
- Published
- 1996
- Full Text
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45. Targeting of peripheral blood T lymphocytes.
- Author
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Bolhuis RL, Hoogenboom HR, and Gratama JW
- Subjects
- Animals, Humans, Epitopes immunology, Immunologic Memory immunology, Leukocytes immunology, T-Lymphocytes, Cytotoxic immunology
- Published
- 1996
- Full Text
- View/download PDF
46. Intrapleural administration of interleukin 2 in pleural mesothelioma: a phase I-II study.
- Author
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Goey SH, Eggermont AM, Punt CJ, Slingerland R, Gratama JW, Oosterom R, Oskam R, Bolhuis RL, and Stoter G
- Subjects
- Aged, Catheters, Indwelling adverse effects, Disease-Free Survival, Dose-Response Relationship, Drug, Fever chemically induced, Humans, Immunologic Factors adverse effects, Immunologic Factors therapeutic use, Infections etiology, Infusions, Parenteral, Interleukin-2 adverse effects, Interleukin-2 therapeutic use, Killer Cells, Lymphokine-Activated immunology, Male, Mesothelioma mortality, Middle Aged, Neoplasm Proteins analysis, Pleura, Pleural Effusion chemistry, Pleural Neoplasms mortality, Survival Analysis, Treatment Outcome, Tumor Necrosis Factor-alpha analysis, Immunologic Factors administration & dosage, Interleukin-2 administration & dosage, Mesothelioma therapy, Pleural Neoplasms therapy
- Abstract
Twenty-three patients with pleural mesothelioma stage I-IIA were entered in a study of continuous daily intrapleural infusion of interleukin 2 (IL-2) for 14 days, repeated every 4 weeks. IL-2 was administered according to a groupwise dose escalation schedule (group A, 3 x 10(4); group B, 3 x 10(5); group C, 3 x 10(6); group D, 6 x 10(6); group E, 18 x 10(6); and group F, 36 x 10(6) IU day-1). Each group consisted of at least three patients. Intrapleural administration of IL-2 was associated with acceptable toxicity. All patients were treated on an outpatient basis except for the patients at dose levels E and F. Dose-limiting toxicity was observed at level F, 36 x 10(6) IU daily, and consisted of catheter infection, fever and flu-like symptoms. Intrapleural IL-2 levels were high (> 20,000 IU ml-1) at levels E and F, while serum levels in most patients were not or barely detectable (< 3-30 IU ml-1). Intrapleural IL-2 levels were up to 6000-fold higher than systemic levels. Intrapleural tumour necrosis factor alpha (TNF-alpha) levels varied greatly and did not correlate with IL-2 dosage. Intrapleural mononuclear cells (MNCs) displayed IL-2-induced lymphokine-activated killer (LAK) activity in all patients. Two patients were not evaluable for response owing to catheter-related problems which precluded the delivery of IL-2. Partial response (PR) occurred in 4 of 21 evaluable patients (19%; 95% confidence interval 5-42%) with a median time to progression of 12 months (range 5-37). Stable disease (SD) occurred in seven patients with a median time to progression of 5 months (range 2-7). There were no complete responses (CRs). The median overall survival was 15.6 months (range 3.0-43). No relationship between the dose of IL-2 and response rate was observed. We conclude that IL-2 given intrapleurally is accompanied with acceptable toxicity and has anti-tumour activity against mesothelioma. In view of the refractory nature of the disease IL-2 may be a treatment option for mesothelioma. A formal phase II study is warranted. Based on the observed toxicity, the lack of dose-response relationship and the immunomodulatory effects seen at relatively low-dose IL-2, the recommended dose for a phase II study is 3 x 10(6) IU day-1 using the present treatment schedule.
- Published
- 1995
- Full Text
- View/download PDF
47. Bispecific antibody targeted T cell therapy of ovarian cancer: clinical results and future directions.
- Author
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Canevari S, Mezzanzanica D, Mazzoni A, Negri DR, Ramakrishna V, Bolhuis RL, Colnaghi MI, and Bolis G
- Subjects
- Antibodies, Bispecific administration & dosage, Antibodies, Bispecific immunology, Antibody Specificity, Combined Modality Therapy, Cytotoxicity, Immunologic, Female, Folate Receptors, GPI-Anchored, Humans, Immunization, Passive, Injections, Intraperitoneal, Injections, Intravenous, Lymphatic Metastasis, Muromonab-CD3 immunology, Ovarian Neoplasms immunology, Ovarian Neoplasms pathology, Ovarian Neoplasms surgery, Peritoneal Neoplasms immunology, Peritoneal Neoplasms therapy, Retroperitoneal Neoplasms secondary, Treatment Outcome, Tumor Cells, Cultured, Antibodies, Bispecific therapeutic use, Carrier Proteins immunology, Lymphocyte Activation, Muromonab-CD3 therapeutic use, Neoplasm Proteins immunology, Ovarian Neoplasms therapy, Peritoneal Neoplasms secondary, Receptors, Cell Surface, T-Lymphocytes immunology
- Abstract
The high frequency of relapse after induction chemotherapy in advanced ovarian carcinoma patients calls for new therapeutic modalities. Retargeted T cell-mediated lysis can be achieved using the bispecific antibody (BsmAb) OCTR, directed to CD3 on T cells and to the folate receptor on ovarian carcinoma cells. Twenty-eight patients with limited intraperitoneal disease after first-line therapy entered a phase II study. They received two i.p. 5 day cycles of activated PBMC retargeted with OCTR. Despite unfavorable tumor characteristics, 7 of 26 patients (27%) showed complete or partial intraperitoneal responses with strict surgicopathologic evaluation. In most cases, the disease relapsed outside the peritoneal cavity, and in 1 case complete intraperitoneal response was accompanied by progression in retroperitoneal lymph nodes. The morbidity was mild to moderate and transient. Combination of i.v. and i.p. administration of OCTR-retargeted lymphocytes will possibly lead to extraperitoneal cure. Ongoing clinical studies indicate that the i.v. infusion of up to 8 x 10(8) OCTR-retargeted T lymphocytes does not induce a higher toxicity than the i.p. treatment. To avoid PBMC preactivation, new approaches for delivering accessory signals are under investigation. Preliminary results indicate that nonactivated PBMC retargeted by OCTR in the presence of an anti-CD28 monoclonal antibody (mAb) are able to significantly inhibit tumor growth.
- Published
- 1995
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48. Inhibition of bispecific monoclonal antibody (bsAb)-targeted cytolysis by human anti-mouse antibodies in ovarian carcinoma patients treated with bsAb-targeted activated T-lymphocytes.
- Author
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Lamers CH, Gratama JW, Warnaar SO, Stoter G, and Bolhuis RL
- Subjects
- Animals, Antibodies, Heterophile biosynthesis, Antibodies, Monoclonal, Antibody Specificity, Antigens, Tumor-Associated, Carbohydrate metabolism, Ascitic Fluid immunology, Binding Sites, Antibody, CD3 Complex immunology, CD3 Complex metabolism, Cytotoxicity, Immunologic, Enzyme-Linked Immunosorbent Assay, Female, Humans, Immunoglobulin Fab Fragments immunology, Immunoglobulin G immunology, Kinetics, Mice, Neoplasm Staging, Ovarian Neoplasms immunology, Ovarian Neoplasms pathology, T-Lymphocytes immunology, Tumor Cells, Cultured immunology, Antibodies, Anti-Idiotypic immunology, Antibodies, Bispecific therapeutic use, Antibodies, Heterophile immunology, Immunotherapy, Adoptive methods, Ovarian Neoplasms therapy
- Abstract
T lymphocytes of 8 patients with ovarian cancer were targeted to the tumor cells using F(ab')2 fragments of a bispecific monoclonal antibody (bsAb), specific for CD3 (a component of the T lymphocyte receptor for antigen) and for the folate receptor MOv18 (overexpressed by ovarian carcinoma cells) as part of a phase I/II study. Phase I (days 0 to 3) consisted of increasing intraperitoneal (i.p.) numbers (10(6)-10(9)) of bsAb-targeted T lymphocytes plus low-dose interleukin-2 (IL-2). Phase II (days 6 to 13, and 27 to 33) consisted of daily i.p. infusions of 10(9) targeted T lymphocytes, 2 mg soluble bsAb, and low-dose IL-2. Using enzyme-linked immunosorbent assays (ELISA), human anti-mouse antibodies (HAMA) were detected in all patients: in the serum from day 13 onwards and in the peritoneal fluid from day 20 onwards. A significant proportion of the HAMA appeared to be directed against the idiotypes of the bsAb specific for CD3 and MOv18, as suggested by (1) the clearly higher ELISA titers against OC/TR bsAb as compared to those against a monoclonal antibody (MAb) with unrelated specificity, and (2) failure to abrogate the capacity of peritoneal fluid containing HAMA to block the binding of OC/TR bsAb to MOv18+ or CD3+ cells by absorption of human anti-mouse IgG-framework antibodies in peritoneal fluid to immobilized mouse IgG. The OC/TR-targeted cytolysis of the MOv18+ ovarian carcinoma cell line Igrov-I by autologous T lymphocytes was inhibited by peritoneal fluid samples containing relatively high HAMA titers. Such inhibitory activity was never detected at the start of phase II, but coincided with the last series of i.p. infusions of targeted T lymphocytes in 2 patients.
- Published
- 1995
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49. Detection of multiple 'Ebnotypes' in individual Epstein-Barr virus carriers following lymphocyte transformation by virus derived from peripheral blood and oropharynx.
- Author
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Gratama JW, Oosterveer MA, Weimar W, Sintnicolaas K, Sizoo W, Bolhuis RL, and Ernberg I
- Subjects
- Acquired Immunodeficiency Syndrome microbiology, Antigens, Viral analysis, Cell Line, DNA-Binding Proteins analysis, Epstein-Barr Virus Nuclear Antigens, Herpesvirus 4, Human isolation & purification, Humans, Infectious Mononucleosis microbiology, Lymphocyte Activation, Transplantation, Tumor Virus Infections microbiology, Antigens, Viral genetics, Blood microbiology, Carrier State microbiology, DNA-Binding Proteins genetics, Herpesviridae Infections microbiology, Herpesvirus 4, Human genetics, Oropharynx microbiology
- Abstract
Transformation of a B lymphocyte into a lymphoblastoid cell line (LCL) by Epstein-Barr virus (EBV) results in the expression of EBV nuclear antigens (EBNAs) of which the size spectrum ('Ebnotype') is characteristic for the transforming virion. Ebnotyping has been used as an epidemiological tool for studies of EBV infection. We compared the occurrence of a single and of multiple Ebnotypes, as defined by EBNAs 1, 2 and 6, in healthy and diseased EBV carriers. Cases from which two or more LCLs could be established from peripheral blood or oropharyngeal cultures were considered informative. The frequency of multiple Ebnotypes was relatively low in healthy individuals and in patients with infectious mononucleosis or with haematological diseases who were awaiting a bone marrow transplant [blood, 11 of 74 patients (15%); oropharynx, 12 of 49 patients (24%)], whereas it was relatively high in recipients of bone marrow or cardiac allografts and one patient with AIDS [blood, 12 of 34 patients (35%); oropharynx, 11 of 16 patients (69%)]. Three patterns of the simultaneous presence of multiple Ebnotypes were distinguished. The first, most frequent, pattern observed predominantly in oropharyngeal cultures of all groups consisted of minority Ebnotypes differing from the majority type by only a single EBNA protein (usually EBNA 1). The second, less frequent, pattern observed in the healthy carriers and the (candidate) transplant recipients consisted of minority Ebnotypes differing from the majority type by two EBNA proteins (mostly EBNAs 1 and 6). The third pattern, characterized by the simultaneous presence of totally different Ebnotypes, was restricted to the (candidate) transplant recipients and the AIDS patient and was more frequently observed in the blood than in the oropharynx. We suggest that the first two patterns result from heterologous recombinations occurring during viral replication at repeat sequences within the EBNA coding regions, whereas the third pattern reflects multiple infections with exogenous viruses.
- Published
- 1994
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50. Characterization of Epstein-Barr viral strains in parotid gland saliva and peripheral blood of patients with primary Sjögren's syndrome and healthy EBV carriers.
- Author
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Oosterveer MA, Markusse HM, Lennette ET, Zou JZ, Bolhuis RL, and Gratama JW
- Subjects
- Aged, Antigens, Viral analysis, B-Lymphocytes microbiology, Base Sequence, Blotting, Western, Carrier State, Cell Line, Transformed, DNA-Binding Proteins analysis, Epithelium microbiology, Epstein-Barr Virus Nuclear Antigens, Female, Herpesviridae Infections complications, Herpesvirus 4, Human genetics, Herpesvirus 4, Human immunology, Herpesvirus 4, Human isolation & purification, Humans, Lymphocyte Activation, Middle Aged, Molecular Sequence Data, Polymerase Chain Reaction, Serotyping, Sjogren's Syndrome complications, Superinfection, Antibodies, Viral blood, Herpesvirus 4, Human classification, Parotid Gland microbiology, Saliva microbiology, Sjogren's Syndrome microbiology
- Abstract
Increased Epstein-Barr virus (EBV) replication has been reported in the salivary and lacrimal glands in Sjögren's syndrome (SS). We studied whether or not certain EBV strains would occur preferentially in the peripheral blood and parotid gland saliva of 18 EBV-seropositive patients with primary Sjögren's syndrome (pSS) and 12 EBV-seropositive control persons. Transforming EBV was detected in the blood of 11 of 18 (61%) pSS patients and 9 of 12 controls (75%). Unexpectedly, neither transforming nor Raji-superinfecting EBV strains were detected in SS parotid saliva, whereas these EBV types were detected in control saliva in 7 and 8 cases, respectively (P < 0.001). Transforming EBV strains were further characterized by 'Ebno-typing,' i.e., analysis of the size spectrum of the viral antigens EBNA 1, 2, 3, and 6 in immunoblots of lymphoblastoid cell lines (LCL). Previous work has shown that a single EBV strain (Ebnotype) dominates the blood and oropharynx of healthy carriers and that unrelated individuals carry different EBV strains, reflecting the vast polymorphism of Ebnotypes in the general population. Two unexpected observations were made. First, an identical Ebnotype was detected in 4 unrelated individuals, i.e., in the blood of 1 pSS patient and in the saliva of 3 control persons. Second, carriage of 2 to 4 different Ebnotypes by a single individual was observed in 4 cases, i.e., in the blood of 1 pSS patient, and in the blood and saliva of 3 control persons.(ABSTRACT TRUNCATED AT 250 WORDS)
- Published
- 1993
- Full Text
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