[Show abstract][Hide abstract] 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.
The Journal of Immunology 07/2005; 174(12):7853-8. · 5.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Adoptive immunotherapy involving the transfer of autologous tumor or virus-reactive T lymphocytes has demonstrated its effectiveness in the eradication of cancer and virally infected cells. Clinical trails and in vitro studies have focused on CD8+ cytotoxic T-cell receptor (TCR) alphabeta lymphocytes since these cells directly kill virally infected- and tumor cells after antigen-specific recognition via their TCR alphabeta. However, increasing evidence suggests that induction of sustained immunity against cancer and viral infections depends on the presence of tumor- or virus-specific CD4+ T lymphocytes, which are restricted by MHC class II. Here, we show that these MHC class II-restricted CD4+ T lymphocytes can efficiently be redirected to MHC class I-restricted tumor cells by retroviral introduction of an HLA-A1/MAGE-A1-specific chimeric two-chain TCR ValphaCalphazeta/VbetaCbetazeta (tcTCR/zeta). However, TCR-transduced CD4+ T lymphocytes were only able to specifically bind to HLA-A1/MAGE-A1 complexes and respond to HLA-A1+/MAGE-A1+ melanoma cells when the CD8alpha gene was cointroduced. These CD4+/CD8alpha+/TCR(POS) T lymphocytes produce IFN-gamma, TNFalpha and IL-2 when specifically stimulated via the introduced TCR with immobilized HLA-A1/MAGE-A1 complexes or HLA-A1+/MAGE-A1+ melanoma cells. Furthermore, introduction of the CD8alpha gene into TCR(POS) T lymphocytes rendered these T lymphocytes cytotoxic for HLA-A1+/MAGE-A1+ melanoma cells. These results demonstrate that human CD4+ T lymphocytes when genetically grafted with an HLA-A1/MAGE-A1-specific TCR and CD8alpha are induced to kill and produce cytokines upon specific interaction with the relevant melanoma cells. Hence, CD4+ T lymphocytes, in addition to CD8+ T lymphocytes, may be critical effector cells for adoptive immuno-gene therapy to generate a sustained tumor-specific immune response in cancer patients.
[Show abstract][Hide abstract] 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.
Journal of Immunological Methods 10/2003; 280(1-2):13-24. · 2.01 Impact Factor
[Show abstract][Hide abstract] 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.
Human Immunology 02/2003; 64(1):56-68. · 2.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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.
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.
Clinical Cancer Research 02/2003; 9(1):76-83. · 8.19 Impact Factor
[Show abstract][Hide abstract] 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.
Cancer Gene Therapy 08/2002; 9(7):613-23. · 2.55 Impact Factor
[Show abstract][Hide abstract] 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.
The Journal of Immunology 08/2002; 169(2):1110-8. · 5.36 Impact Factor
[Show abstract][Hide abstract] 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.
Annals of Hematology 05/2002; 81(4):187-91. · 2.40 Impact Factor
[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] 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.
British Journal of Haematology 03/2001; 112(2):499-505. · 4.96 Impact Factor
[Show abstract][Hide abstract] 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.
International Journal of Cancer 11/2000; 88(1):115-20. · 5.01 Impact Factor
[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] 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.
Clinical Cancer Research 01/2000; 5(12):3983-9. · 8.19 Impact Factor
[Show abstract][Hide abstract] 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.
Journla of Immunotherapy 08/1999; 22(4):299-307. · 3.35 Impact Factor
[Show abstract][Hide abstract] 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.