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Background: The high frequency of relapse after induction chemotherapy of advanced ovarian carcinoma calls for new therapeutic approaches. Lysis of ovarian carcinoma cells can be achieved by retargeting of T lymphocytes using F(ab')2 fragments of the bispecific monoclonal antibody (MAb) OC/TR, which is directed to the CD3 molecule on T lymphocytes and to the folate receptor on ovarian carcinoma cells.Purpose: Our purpose was to assess in ovarian carcinoma patients the antitumor activity of in vitro-activated autologous peripheral blood T lymphocytes retargeted with OC/TR.Methods: Patients with epithelial ovarian cancer (International Federation of Gynecology and Obstetrics stages III and IV) meeting specific criteria were eligible to enter a phase II immunotherapy trial. Before immunotherapy, the 28 patients who entered the trial underwent laparotomy to reduce their tumor load and to allow measurement of all indicator lesions. They then received two cycles of five daily intraperitoneal infusions of autologous in vitro activated peripheral blood T lymphocytes retargeted with OC/TR plus recombinant interleukin 2 (IL-2) with (n = 11) or without (n = 17) a second daily infusion of OC/TR F(ab')2 and IL-2. Response to treatment could be assessed in 26 patients following explorative laparotomy; time to progression could be assessed in 27 patients.Results: Seven patients had clinical evidence of progressive disease after treatment and therefore did not undergo laparotomy. Of the 19 patients evaluated by surgery and histology, three showed complete response, one showed complete intraperitoneal response with progressive disease in retroperitoneal lymph nodes, three showed partial response, seven had stable disease, and five had progressive disease. The overall intraperitoneal response rate was 27% (95% confidence interval [CI] = 10%-44%). The complete responses seen in three patients lasted 26 months in one patient, 23 months in the second, and 18 months in the third. Two patients were not assessable for response. One of these patients had bowel perforation during catheter removal, which precluded further evaluation. The second patient was positive only by cytologic examination before immunotherapy, was tumor free at laparotomy after immunotherapy, and remained so for the entire 21 months of follow-up, as determined by cytologic examination of random biopsy specimens. The median time to disease progression in the 15 assessable patients plus those who had stable disease was 11 months (95% CI = 6-18 months). Immunotherapy-related toxic effects included mild to moderate fever, nausea, emesis, and fatigue. Anti-mouse antibodies were detectable by the end of the treatment in 21 of 25 patients tested.Conclusions: Locoregional immunotherapy of ovarian cancer with bispecific MAb-retargeted T lymphocytes can result in tumor regression. Toxicity was mild to moderate and only transient.Implications: Improvement in systemic antitumor responses is needed before this approach can prove useful as adjunctive treatment following induction chemotherapy in patients with minimal residual disease. [ABSTRACT FROM AUTHOR]

Umbilical cord blood stem cell transplantation (UCBT) is associated with retarded hematopoietic recovery and immune reconstitution and a high infection-related morbidity and mortality, especially after conditioning including anti-thymocyte globulin (ATG). However, data on immune recovery, incidence of infections, and outcome in double UCBT (dUCBT) recipients receiving an ATG-free reduced intensity conditioning (RIC) are lacking. In this study, recovery of lymphocyte subsets, thymopoiesis, and its association with severe infections and clinical outcome was assessed in a group of 55 recipients of a dUCBT ATG-free RIC regimen. T cell recovery was severely protracted in the majority of patients. However, T cell receptor excision circle TREC + T cells were detectable in 62% of patients at 3 months post-transplantation. A total of 128 common toxicity criteria grade 3-4 infections were observed in the first year post-transplantation. Non-relapse mortality at 12 months post-transplant was 16%, of which 78% infectious mortality. One-year overall survival was 73%. Patients who failed to recover thymopoiesis at 3 months post-transplantation were at a 3.3-fold higher risk of subsequent severe grade 3-4 infections.

Autologous T cells were genetically modified to express a chimeric antigen receptor (CAR) directed toward carboxy-anhydrase-IX (CAIX) and used to treat patients with CAIX-positive metastatic renal cell carcinoma. In this study, we questioned whether the T cell maturation stage in the pre-infusion product affected CAIX CAR expression and function in vitro as well as in vivo CAR T cell numbers and expansion. During the 14 days expansion of CAR T cells prior to administration, we observed shifts from a predominant CD4 to a CD8 T cell phenotype and from a significant fraction of naïve to central effector T cells. Surface expression of the CAR was equally distributed among different T cell subsets and T cell maturation stages. During T cell culture days 14-18 (which covered patient treatment days 1-5), T cells demonstrated a decline in CAR expression level per cell irrespective of T cell maturation stage, although the proportion of CAR-positive T cells and CAR-mediated T cell effector functions remained similar for both CD4 and CD8 T cell populations. Notably, patients with a higher fraction of naïve CD8 T cells at baseline (prior to genetic modification) or central effector CD8 T cells at 2 weeks of CAR T cell culture demonstrated a higher fold expansion and absolute numbers of circulating CAR T cells at 1 month after start of therapy. We conclude that the T cell maturation stage prior to and during CAR T cell expansion culture is related to in vivo CAR T cell expansion.

Although double umbilical cord blood transplantation (dUCBT) in adult patients may be associated with less graft failure compared with single UCBT, hematopoietic recovery generally originates from a single cord blood unit (CBU). CBU predominance is still incompletely understood. We recently showed that blood CD4 + T-cell numbers rapidly increase after dUCBT, and early CD4 + T-cell chimerism predicts for graft predominance. Given the frequent HLA class II allele mismatches between CBUs in dUCBT, we hypothesized that alloreactive HLA class II-specific CD4 + T cells from the "winning" CBU may contribute to rejection of the "loser" CBU. We evaluated whether CD4 + T cells originating from the predominant (PD)-CBU would recognize HLA class II allele mismatches, expressed by the nonengrafting (NE)-CBU. Alloreactive effector CD4 + T cells toward 1 or more mismatched HLA class II alleles of the NE-CBU were detected in 11 of 11 patients, with reactivity toward 29 of 33 (88%) tested mismatches, and the strongest reactivity toward DR and DQ alleles early after dUCBT. Mismatched HLA class II allele-specific CD4 + T cells recognized primary leukemic cells when the mismatched HLA class II allele was shared between NE-CBU and patient. Our results suggest that cytotoxicity exerted by CD4 + T cells from the PD-CBU drives the rapid rejection of the NE-CBU, whose alloreactive effect might also contribute to graft-versus-leukemia., (© 2016 by The American Society of Hematology.)

We studied safety and proof of concept of a phase I/II trial with chimeric antigen receptor (CAR) T-cells in patients with metastatic renal cell carcinoma (mRCC). The CAR was based on the G250 mAb that recognized an epitope of carboxy-anhydrase-IX (CAIX). Twelve patients with CAIX+ mRCC were treated in three cohorts with a maximum of 10 daily infusions of 2×10(7) to 2×10(9) CAR T-cells. Circulating CAR T-cells were transiently detectable in all patients and maintained antigen-specific immune functions following their isolation post-treatment. Blood cytokine profiles mirrored CAR T-cell presence and in vivo activity. Unfortunately, patients developed anti-CAR T-cell antibodies and cellular immune responses. Moreover, CAR T-cell infusions induced liver enzyme disturbances reaching CTC grades 2-4, which necessitated cessation of treatment in four out of eight patients (cohort 1+2). Examination of liver biopsies revealed T-cell infiltration around bile ducts and CAIX expression on bile duct epithelium, adding to the notion of on-target toxicity. No such toxicities were observed in four patients that were pretreated with G250 mAb (cohort 3). The study was stopped due to the advent of competing treatments before reaching therapeutic or maximum tolerated dose in cohort 3. No clinical responses have been recorded. Despite that, from this trial numerous recommendations for future trials and their immune monitoring could be formulated, such as choice of the target antigen, format and immunogenicity of receptor and how the latter relates to peripheral T-cell persistence., (© 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.)

Cancer immune therapy, in particular the use of checkpoint inhibitors and adoptive transfer of T cells has recently demonstrated significant clinical responses against several tumor types. Unfortunately, these therapies are frequently accompanied by severe toxicities, underscoring the need for markers that provide information on therapy response. Monitoring immune responses in the tumor microenvironment and peripheral blood prior to and during these therapies will provide better insight into the mechanisms underlying clinical activities, and will potentially enable the identification of such markers. In this review, we present an overview of adoptive T-cell trials conducted with a special focus on immune monitoring, and argue that accurate monitoring of T cells is pivotal to further development of immune therapies to treat cancer.

Therapy with autologous T cells that have been gene-engineered to express chimeric antigen receptors (CAR) or T cell receptors (TCR) provides a feasible and broadly applicable treatment for cancer patients. In a clinical study in advanced renal cell carcinoma (RCC) patients with CAR T cells specific for carbonic anhydrase IX (CAIX), we observed toxicities that (most likely) indicated in vivo function of CAR T cells as well as low T cell persistence and clinical response rates. The latter observations were confirmed by later clinical trials in other solid tumor types and other gene-modified T cells. To improve the efficacy of T cell therapy, we have redefined in vitro conditions to generate T cells with young phenotype, a key correlate with clinical outcome. For their impact on gene-modified T cell phenotype and function, we have tested various anti-CD3/CD28 mAb-based T cell activation and expansion conditions as well as several cytokines prior to and/or after gene transfer using two different receptors: CAIX CAR and MAGE-C2(ALK)/HLA-A2 TCR. In a total set of 16 healthy donors, we observed that T cell activation with soluble anti-CD3/CD28 mAbs in the presence of both IL15 and IL21 prior to TCR gene transfer resulted in enhanced proportions of gene-modified T cells with a preferred in vitro phenotype and better function. T cells generated according to these processing methods demonstrated enhanced binding of pMHC, and an enhanced proportion of CD8+, CD27+, CD62L+, CD45RA+T cells. These new conditions will be translated into a GMP protocol in preparation of a clinical adoptive therapy trial to treat patients with MAGE-C2-positive tumors.

Purpose: RGB-286638 is a multitargeted inhibitor with targets comprising the family of cyclin-dependent kinases (CDK) and a range of other cancer-relevant tyrosine and serine/threonine kinases. The objectives of this first in human trial of RGB-286638, given i.v. on days 1 to 5 every 28 days, were to determine the maximum tolerated dose (MTD) and to evaluate the pharmacokinetic (PK) and pharmacodynamic (PD) profiles of this new drug., Experimental Design: Sequential cohorts of 3 to 6 patients were treated per dose level. Blood, urine samples, and skin biopsies for full PK and/or PD analyses were collected., Results: Twenty-six patients were enrolled in 6-dose levels from 10 to 160 mg/d. Four dose-limiting toxicities were observed in 2 of the 6 patients enrolled at the highest dose level. These toxicities were AST/ALT elevations in 1 patient, paroxysmal supraventricular tachycardias (SVTs), hypotension, and an increase in troponin T in another patient. The plasma PK of RGB-286638 was shown to be linear over the studied doses. The interpatient variability in clearance was moderate (variation coefficient 7%-36%). The PD analyses in peripheral blood mononuclear cells, serum (apoptosis induction) and skin biopsies (Rb, p-Rb, Ki-67, and p27(KIP1) expression) did not demonstrate a consistent modulation of mechanism-related biomarkers with the exception of lowered Ki-67 levels at the MTD level. The recommended MTD for phase II studies is 120 mg/d., Conclusions: RGB-286638 is tolerated when administered at 120 mg/d for 5 days every 28 days. Prolonged disease stabilization (range, 2-14 months) was seen across different dose levels., (©2014 American Association for Cancer Research.)

Evaluation of: Russo V, Pilla L, Lunghi F et al. Clinical and immunologic responses in melanoma patients vaccinated with MAGE-A3-genetically modified lymphocytes. Int. J. Cancer 132(11), 2557-2566 (2012). When one mentions T lymphocytes, one easily recognizes the effective and antigen-specific manner by which T lymphocytes execute cellular immune responses towards pathogen-infected or cancerous cells. Russo and coworkers recognized the other side of the coin and exploited the potency of T cells to act as a cellular vaccine, to which end they used T cells transduced with the cancer-testis antigen MAGE-A3. Twenty-three patients with MAGE-A3-expressing melanoma were treated and six patients developed MAGE-A3-specific immune responses and showed clinical benefit, whereas patients without a MAGE-A3-specific immune response did not show clinical benefit. This report includes and extends on results from a pilot study including ten patients, of which three developed MAGE-A3-specific immune responses. The present study further explores a potential beneficial application of the observed immunogenicity of genetically modified T cells.

Background Aims: The generation of gene-modified T cells for clinical adoptive T-cell therapy is challenged by the potential instability and concomitant high financial costs of critical T-cell activation and transduction components. As part of a clinical trial to treat patients with metastatic renal cell cancer with autologous T cells engineered with a chimeric antigen receptor (CAR) recognizing carboxy-anhydrase-IX (CAIX), we evaluated functional stability of the retroviral vector, T-cell activation agent Orthoclone OKT3 (Janssen-Cilag, Beerse, Belgium) monoclonal antibody (mAb) and the transduction promoting agent RetroNectin (Takara, Otsu, Japan)., Methods: Carboxy-anhydrase-IX chimeric antigen receptor retrovirus-containing culture supernatants (RTVsups) were generated from two packaging cell lines, Phoenix-Ampho (BioReliance, Sterling, UK) and PG13, and stored at -80°C over 10 years and 14 years. For Orthoclone OKT3 and RetroNectin, aliquots for single use were prepared and stored at -80°C. Transduction efficiencies of both batches of RTVsups were analyzed using the same lots of cryopreserved donor peripheral blood mononuclear cells, Orthoclone OKT3 and RetroNectin over time., Results: We revisit here an earlier report on the long-term functional stability of the RTVsup, observed to be 9 years, and demonstrate that this stability is at least 14 years. Also, we now demonstrate that Orthoclone OKT3 and RetroNectin are functionally stable for periods of at least 6 years and 10 years., Conclusions: High-cost critical components for adoptive T-cell therapy can be preserved for ≥10 years when prepared in aliquots for single use and stored at -80°C. These findings may significantly facilitate, and decrease the financial risks of, clinical application of gene-modified T cells in multicenter studies., (Copyright © 2013 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Autologous T cells genetically modified to express a chimeric antibody receptor (CAR) against carboxy-anhydrase-IX (CAIX) were administered to 12 patients with CAIX-expressing metastatic renal cell carcinoma (RCC). Patients were treated in three cohorts with a maximum of 10 infusions of a total of 0.2 to 2.1 × 10(9) CAR T cells. CTC grade 2-4 liver enzyme disturbances occurred at the lowest CAR T cell doses, necessitating cessation of treatment in four out of eight patients in cohorts 1 and 2. Examination of liver biopsies revealed CAIX expression on bile duct epithelium with infiltration of T cells, including CAR T cells. Subsequently four patients were pre-treated with CAIX monoclonal antibody (mAb) G250 to prevent CAR-specific toxicity and showed no liver toxicities and indications for enhanced peripheral T cell persistence. No clinical responses were recorded. This report shows that CAIX-targeting CAR T cells exerted antigen-specific effects in vivo and induced liver toxicity at the lowest dose of 0.2 × 10(9) T cells applied, illustrating the potency of receptor-modified T cells. We provide in-patient proof that the observed "on-target" toxicity is antigen-directed and can be prevented by blocking antigenic sites in off-tumor organs and allowing higher T cell doses.

Background: Inflammation may underlie cancer-related fatigue; however, there are no studies that assess the relation between fatigue and cytokines in patients with advanced disease versus patients without disease activity. Furthermore, the relation between cytokines and the separate dimensions of fatigue is unknown. Here, association of plasma levels of inflammatory markers with physical fatigue and mental fatigue was explored in advanced cancer patients and cancer survivors., Methods: A total of 45 advanced cancer patients and 47 cancer survivors completed the subscales Physical Fatigue and Mental Fatigue of the Multidimensional Fatigue Inventory. Plasma concentrations of C-reactive protein (CRP), interleukin-1 receptor antagonist (IL-1-ra), interleukin-6 (IL-6), interleukin-8 (IL-8), and neopterin were measured. Nonparametric tests were used to assess differences in fatigue intensity and levels of inflammatory markers and to determine correlation coefficients between the fatigue dimensions and inflammatory markers., Results: Compared with cancer survivors, patients with advanced cancer had higher levels of physical fatigue (median 16 vs 9, P < .001) and mental fatigue (median 11 vs 6, P = .01). They also had higher levels of all cytokines (P < .01). In advanced cancer, CRP (r = 0.49, P = .001), IL-6 (r = 0.43, P = .003), IL-1-ra (r = 0.32, P = .03), and neopterin (r = 0.25, P = .10) were correlated with physical but not with mental fatigue. In cancer survivors, only IL-1-ra was related to both physical fatigue (r = 0.24, P = .10) and mental fatigue (r = 0.35, P = .02)., Conclusions: In advanced cancer, inflammation seems to be associated with physical fatigue, but not to mental fatigue. In cancer survivors, there was no convincing evidence that inflammation plays a major role in fatigue., (Copyright © 2012 American Cancer Society.)

Anti-Hu antibody-associated paraneoplastic neurological syndromes (Hu-PNSs) are severe and often precede the detection of a malignancy, usually small-cell lung cancer. In Hu-PNS, it is hypothesized that neuronal cells are destroyed by T cells targeted against HuD, a protein expressed by small-cell lung cancer cells and neurons. There is only limited evidence for the existence of HuD-specific T cells. To detect these T cells in the blood of Hu-PNS patients, we employed 3 highly sensitive assays that included T cell stimulation with dendritic cells (DCs) to specifically expand the number of any HuD-specific T cells. A total of 17 Hu-PNS patients were tested with 1 or more of the following 3 assays: (1) tetramer staining after stimulation of T cells with conventionally generated DCs (n = 9), (2) interleukin (IL)-13 enzyme-linked immunosorbent spot (ELISpot; n = 3), IL-4 and IL-5 and interferon (IFN)-γ multiplex cytokine bead array (n = 2) to assay cytokine production by T cells after stimulation with conventionally generated DCs, and (iii) IFN-γ ELISpot and tetramer staining after T cell stimulation with accelerated co-cultured DCs (n = 11). No circulating HuD-specific T cells were found. We suggest that either autoaggressive T cells in Hu-PNS are not targeted against HuD or that their numbers in the blood are too low for detection by highly sensitive techniques.

Adoptive transfer of immune effector cells that are gene modified by retroviral transduction to express tumor-specific receptors constitutes an attractive approach to treat cancer. In patients with metastatic renal cell carcinoma, we performed a study with autologous T cells genetically retargeted with a chimeric antibody receptor (CAR) directed toward carbonic anhydrase IX (CAIX), an antigen highly expressed in renal cell carcinoma. In the majority of patients, we observed distinct humoral and/or cellular anti-CAIX-CAR T-cell immune responses in combination with a limited peripheral persistence of transferred CAIX-CAR T cells in the majority of patients. Humoral immune responses were anti-idiotypic in nature and neutralized CAIX-CAR-mediated T-cell function. Cellular anti-CAIX-CAR immune responses were directed to the complementarity-determining and framework regions of the CAR variable domains. In addition, 2 patients developed immunity directed against presumed retroviral vector epitopes. Here, we document the novel feature that therapeutic cells, which were ex vivo engineered by means of transduction with a minimal γ-retroviral vector, do express immunogenic vector-encoded epitopes, which might compromise persistence of these cells. These observations may constitute a critical concern for clinical ex vivo γ-retroviral gene transduction in general and CAR-retargeted T-cell therapy in particular, and underscore the need to attenuate the immunogenicity of both transgene and vector.

Purpose: Immune responses to gene-modified cells are a concern in the field of human gene therapy, as they may impede effective treatment. We conducted 2 clinical trials in which cancer patients were treated with lymphocytes genetically engineered to express murine T-cell receptors (mTCR) specific for tumor-associated antigens p53 and gp100., Experimental Design: Twenty-six patients treated with autologous lymphocytes expressing mTCR had blood and serum samples available for analysis. Patient sera were assayed for the development of a humoral immune response. Adoptive cell transfer characteristics were analyzed to identify correlates to immune response., Results: Six of 26 (23%) patients' posttreatment sera exhibited specific binding of human anti-mTCR antibodies to lymphocytes transduced with the mTCR. Antibody development was found in both responding and nonresponding patients. The posttreatment sera of 3 of these 6 patients mediated a 60% to 99% inhibition of mTCR activity as measured by a reduction in antigen-specific interferon-γ release. Detailed analysis of posttreatment serum revealed that antibody binding was β-chain specific in 1 patient whereas it was α-chain specific in another., Conclusions: A subset of patients treated with mTCR-engineered T cells developed antibodies directed to the mTCR variable regions and not to the constant region domains common to all mTCR. Overall, the development of a host immune response was not associated with the level of transduced cell persistence or response to therapy. In summary, patients treated with mTCR can develop an immune response to gene-modified cells in a minority of cases, but this may not affect clinical outcome., (©2010 AACR.)

Background: Preeclampsia is a disease hypothesized to originate from widespread endothelial dysfunction or damage. This study investigated whether circulating endothelial cells (CEC) can serve as a surrogate marker for disease severity in patients with preeclampsia, and if their number correlates to serum endothelial biomarkers for activation, dysfunction, or damage of those cells., Methods: Blood was drawn consecutively from 30 patients admitted with a diagnosis of severe preeclampsia. Thirty healthy, normotensive, patients matched for age, body mass index, and gestational age served as a control group. We determined the number of CEC and serum concentrations of biomarkers indicative of endothelial damage (thrombomodulin) and activation (E-selectin), and the antiangiogenic protein (endoglin), which reflects endothelial dysfunction., Results: Median CEC counts did not differ significantly between preeclamptic patients and the control group (median 5.3 vs. 3.5 CEC/mL, respectively) and were mostly within the normal range (i.e., <20 CEC/mL). However, serum concentrations of thrombomodulin (median 3.6 vs. 5.2 ng/mL; P = 0.006), E-selectin (median 32.0 vs. 42.9 ng/mL; P = 0.02), and especially endoglin (median 5.0 vs. 76.2 ng/mL; P < 0.0001) were significantly increased in severe preeclamptic patients. CEC counts did not correlate with any of the clinical parameters or routinely determined laboratory indices., Conclusion: Preeclampsia is characterized by endothelial dysfunction and activation rather than actual endothelial damage as characterized by increased CEC counts., (© 2010 International Clinical Cytometry Society.)

Circulating endothelial cells (CEC) are considered a promising marker to determine the extent of vascular damage. However, currently available and validated CEC enumeration assays are laborious, time consuming and costly, which limits their clinical utility. Here, we evaluated the feasibility of quantifying mRNA levels of the endothelium-associated markers CD31, CD144, CD146 and von Willebrand factor (vWf) in peripheral blood (PB) of healthy donors, patients, and human umbilical veins by real-time reverse transcriptase polymerase chain reaction (RT-PCR) and their use as surrogate markers for CEC. Whole blood samples and CD146+ cell-enriched fractions were assessed for mRNA and protein expression of CD31, CD144, CD146 and vWf by RT-PCR and flow cytometry, respectively. We showed the feasibility to detect endothelial mRNA isolated from HUVEC numbers as low as 10. However, no endothelial mRNA could be measure in whole blood samples, and only low levels of CD31 and CD146 mRNA were detected in suspensions of isolated CEC with numbers up to 4,450 CEC per sample. We conclude that mRNA levels of CD31, CD144, CD146 and vWf in whole blood as detected by real time RT-PCR cannot be used as biomarkers for end-stage endothelial cells such as CEC.

Resistance of cancer cells to cytotoxic therapy can be caused by the activation of strong anti-apoptotic effectors, for example NF-kappaB. Therefore, compounds that inhibit NF-kappaB stimulation might overcome chemotherapy resistance. F60008, a semi-synthetic derivate of triptolide, is converted to triptolide in vivo and activates apoptosis in human tumour cells. We performed a phase I and pharmacological study of F60008 given intravenously as a weekly infusion for 2 weeks every 3 weeks in patients with advanced solid tumours. Twenty patients were enrolled, and a total of 35 cycles were administered. The most frequent haematological side-effect was mild grade 1-2 anaemia. Non-haematological toxicities included fatigue, nausea, vomiting, diarrhoea and constipation, all grade 1-2. Two lethal events were observed in which an increase in caspase-3 activity and overt apoptosis in monocytes and neutrophils could be seen. Pharmacokinetic studies showed high inter-individual variability and rendered F60008 a far from optimal derivate of triptolide.

The Food and Drug Administration/Center for Biologics Evaluation and Research has defined that for retroviral gene therapy, the vector-producing cell, the vector preparation, and the ex vivo gene-transduced cells have to be tested for absence of replication-competent retrovirus (RCR) if the transduced cells are cultured for >4 days. We assessed the sensitivity of the "extended PG4(S+L-) assay" to detect gibbon ape leukemia virus (GALV) RCR, and applied this assay to measure GALV RCR spread in retrovirally transduced T cells. To this end, T cells were expanded for 12 days after transduction with a GALV-envelope pseudotyped retroviral vector expressing single chain variable fragment (anticarbonic anhydrase IX) in presence or absence of GALV RCR. Results showed that: (1) the "extended PG4(S+L-) assay" detects 1 focus-forming unit (ffu) GALV RCR and thus is applicable and sufficiently sensitive to screen human T-cell cultures for absence of infectious GALV RCR; (2) although GALV RCR infect human T cells, it very poorly replicate in T cells; (3) GALV RCR, when present at low levels immediately upon gene transduction (ie, 100 ffu/20x10 T cells in 100 mL), did not spread during a 12-day T-cell culture at clinical scale. Our observation that GALV RCR poorly spreads in primary human T-cell cultures questions the relevance of testing T-cell transductants for RCR on top of testing the vector-producing cells and the clinical vector batch for RCR and warrants evaluation of the current policy for safety testing of ex vivo retrovirally transduced T lymphocytes for GALV RCR.

Increased numbers of circulating endothelial cells (CEC) in peripheral blood have been observed in diseases with vascular involvement, and are considered a promising surrogate marker for vascular damage. It was the objective of this study to evaluate the correlation between putative soluble markers of endothelial injury, activation, and endothelial proliferation, and absolute numbers of CEC. CEC were evaluated in 125 healthy donors and 40 patients with metastatic carcinoma by automated CD146 driven immunomagnetic isolation. Plasma concentrations of E-selectin, endoglin, and thrombomodulin were assessed by ELISA in plasma obtained from 40 healthy donors and 40 patients. CEC numbers in blood were positively correlated with plasma thrombomodulin levels, but not with levels of E-selectin and endoglin. Multivariate analysis demonstrated a significant increase in CEC numbers with age. The levels of plasma biomarkers were not influenced by age. Higher levels of thrombomodulin and E-selectin were observed in males when compared to females. In conclusion, CEC numbers correlate positively with plasma levels of thrombomodulin.

Background: Given the presumed key role for autoreactive lymphocytes in multiple sclerosis (MS), treatment strategies have been developed to ablate lymphocyte activity. Intrathecal lymphocyte activation can be measured by CSF-soluble(s)CD27., Objective: To determine the effect of maximum whole-body immune ablation on two different markers that detect lymphocyte activation in CSF-oligoclonal IgG bands and levels of CSF-sCD27., Design, Setting and Patients: The study quantified sCD27 levels and assessed the presence of oligoclonal IgG bands in CSF samples of secondary progressive patients with MS treated by autologous bone-marrow transplantation. In eight individuals, CSF was taken before and 6-9 months after conditioning. CSF-sCD27 levels were compared with other MS and non-inflammatory neurological disease controls. Regarding the effect of stem-cell transplantation on CSF oligoclonal bands, the study analysed pooled data of this and four other international studies on stem-cell transplantation in MS., Results: CSF-sCD27 was significantly lower after the extremely immunoablative protocol. However, levels remained elevated compared with non-inflammatory controls and stayed within the range observed in other MS controls. The joint analysis of CSF oligoclonal bands demonstrated persistence of this immune abnormality in 88% of the reported cases (n = 34)., Conclusions: The persistence of CSF lymphocyte activation markers sCD27 and intrathecal oligoclonal IgG bands after maximum immunoablative treatment indicates that complete eradication of activated lymphocytes from the CNS has not been established. This is paralleled by disease progression observed in several studies on the effect of stem-cell transplantation in MS.

Adequate blood supply is a prerequisite in the pathogenesis of solid malignancies. As a result, depriving a tumour from its oxygen and nutrients, either by preventing the formation of new vessels, or by disrupting vessels already present in the tumour, appears to be an effective treatment modality in oncology. Given the mechanism by which these agents exert their anti-tumour activity together with the crucial role of tumour vasculature in the pathogenesis of tumours, there is a great need for markers properly reflecting its impact. Circulating endothelial cells (CEC), which are thought to derive from damaged vasculature, may be such a marker. Appropriate enumeration of these cells appears to be a technical challenge. Nevertheless, first studies using validated CEC assays have shown that CEC numbers in patients with advanced malignancies are elevated compared to healthy controls making CEC a potential tool for among other establishing prognosis and therapy-induced effects. In this review, we will address the possible clinical applications of CEC detection in oncology, as well as the pitfalls encountered in this process.

Recombinant retroviruses are one of the most commonly used gene transfer vehicles for therapeutic gene delivery. The stability of viral vectors upon long-term storage, anticipated to be short lived, is expected to impact timeline and financial course of clinical immunogene therapy. However, to date little is known about vector stability. Therefore, we analyzed the stability of retroviral vectors produced in culture supernatants (RTVsup) for ex vivo gene therapy upon long-term storage. We have generated RTVsups derived from two packaging cell lines, PG13 and Phoenix(Ampho). Both lines produced murine leukemia virus-derived SFG-scFv(G250)-CD4gamma vector, which were pseudotyped with the gibbon ape leukemia virus envelope and amphotropic envelope, respectively. The supernatants were stored at -80 or -196 degrees C. To date, the PG13-derived RTVsups have been evaluated over a period of 9 years (1998-2007). In addition, a clinical batch of Phoenix(Ampho)-derived RTVsup has been evaluated over a period of 5 years (2002-2007). Here, we show that both RTVsups, when stored up to 9 and 5 years, respectively, do not show any sign of decay in their capacity to functionally transduce primary human T cells. These data provide evidence that in terms of 'life expectancy' the production and storage of clinical batches of RTVsup for gene therapy warrants the corresponding professional and financial risks.

Background: For our clinical immunogene therapy study for the treatment of renal cell carcinoma (RCC) patients, we had developed a protocol for gene transduction and expansion of human T cells in compliance with good manufacturing practice (GMP) criteria. Critical to our successful clinical-scale transductions of patient T cells was the use of Retronectin in combination with Lifecell X-foldtrade mark cell culture bags., Methods: In our current study, we evaluated two alternative types of bags for the Retronectin-mediated retroviral transduction of human T cells: the Miltenyi DC-generation bag and the Takara CultiLife Spin bag., Results: In static transductions, but not in spinoculation, the DC-generation bags and CultiLife Spin bags performed as well as Lifecell X-foldtrade mark bags in Retronectin-assisted retroviral transduction of human T cells with respect to transduction efficiency, lymphocyte subset composition and lymphocyte function. However, both types of bags performed less well than Lifecell X-foldtrade mark cell culture bags in terms of cell yield., Discussion: Adjusted numbers of cells at the start of transduction should be used when using the Miltenyi or Takara bags in order to compensate for the lower cell yield following transduction.

Background: We have treated three patients with carboxy-anhydrase-IX (CAIX) positive metastatic renal cell cancer (RCC) by adoptive transfer of autologous T-cells that had been gene-transduced to express a single-chain antibody-G250 chimeric receptor [scFv(G250)], and encountered liver toxicity necessitating adaptation of the treatment protocol. Here, we investigate whether or not the in vivo activity of the infused scFv(G250)(+) T cells is reflected by changes of selected immune parameters measured in peripheral blood., Methods: ScFv(G250)-chimeric receptor-mediated functions of peripheral blood mononuclear cells (PBMC) obtained from three patients during and after treatment were compared to the same functions of scFv(G250)(+) T lymphocytes prior to infusion, and were correlated with plasma cytokine levels., Results: Prior to infusion, scFv(G250)(+) T lymphocytes showed in vitro high levels of scFv(G250)-chimeric receptor-mediated functions such as killing of CAIX(+) RCC cell lines and cytokine production upon exposure to these cells. High levels of IFN-gamma were produced, whilst production of TNF-alpha, interleukin-4 (IL-4), IL-5 and IL-10 was variable and to lower levels, and that of IL-2 virtually absent. PBMC taken from patients during therapy showed lower levels of in vitro scFv(G250)-receptor-mediated functions as compared to pre-infusion, whilst IFN-gamma was the only detectable cytokine upon in vitro PBMC exposure to CAIX. During treatment, plasma levels of IFN-gamma increased only in the patient with the most prominent liver toxicity. IL-5 plasma levels increased transiently during treatment in all patients, which may have been triggered by the co-administration of IL-2., Conclusion: ScFv(G250)-receptor-mediated functions of the scFv(G250)(+) T lymphocytes are, by and large, preserved in vivo upon administration, and may be reflected by fluctuations in plasma IFN-gamma levels.

We have designed a transgene that encodes a scFv(G250) chimeric receptor, which is specific for carboxyanhydrase IX (G250-ligand, G250L), a molecule overexpressed by renal cell cancer (RCC). Retroviral transduction of this transgene into primary human T lymphocytes confers these cells with specific functional responses towards G250L-positive RCC cells. In preparation of a clinical phase (I/II) study in RCC patients, we set up a protocol for gene transduction and expansion of primary human T cells. For this purpose, we directly compared two packaging cell lines, that is, the GALV-pseudotyped MLV producing cell line PG13, and the MLV-A-producing cell line Phi-NX-Ampho (a.k.a. Phoenix-A). We generated and characterized stable scFv(G250)-positive clones of both PG13 and Phoenix cells and optimized the retrovirus production conditions. Transductions of primary human T cells yielded 30-60% scFv(G250)+ T cells using PG13-derived retrovirus versus up to 90% scFv(G250)+ T cells using Phoenix-derived retrovirus. The median number of transgene integrations per scFv(G250)+ T cell differed only 1.5-fold as determined by real-time PCR (mean number of integrations per T cell 2.6 and 3.7 for PG13 and Phoenix-based transductions, respectively). In addition, T cells transduced with Phoenix-derived retrovirus showed, on a per cell basis, 10-30% higher levels of scFv(G250)-mediated TNFalpha production and cytolysis of G250L+ RCC cells than T cells transduced with PG13-derived retrovirus. The improved functional transduction efficiency together with a limited increase in the number of integrations per recipient cell, made us select Phoenix clone 58 for our clinical immunogene therapy study.

We compared the kinetics of T-cell recovery after extensive ex vivo and in vivo T-cell depleted autologous stem cell transplantation (SCT) for multiple sclerosis (MS; n=8) with unmodified SCT for hematological malignancies (HM; n=39). Both patient group showed a very protracted recovery of 'naive' CD4(+), 45R0(-) ( approximately CD45RA(+)) T-cells. Within the 'primed' CD4(+), 45R0(+) T-cells, the 'central memory' cells expressing the CD62L and CD27 markers were the slowest to recover. The repopulating T-cells were highly activated, as shown by increased expression of HLA-DR and the apoptosis marker CD95. The capability of CD4(+) and CD8(+) T-cells to produce IFN-gamma, IL-2 and TNF-alpha had reached normal ranges from 2 months post SCT onwards. Unexpectedly, the kinetics of T-cell recovery between 3 and 12 months post transplant was similar in T-depleted and unmodified SCT. Before SCT, the HM patients showed lymphopenia of all T-cell subsets, upregulated HLA-DR and CD95 expression and increased cytokine responses. We suggest that the similar kinetics of T-cell recovery in the two patient groups may be explained by the susceptibility to apoptosis of the activated CD4(+) T-cells in the autografts of the HM patients. This susceptibility to apoptosis would interfere with a swift and sustained CD4(+) T-cell regeneration post SCT.

Background: Adoptive transfer of autologous T cells that are gene-transduced to express Ag-specific receptors represents an experimental strategy to provide tumor-specific immunity to cancer patients. We studied this concept in patients with metastatic renal cell cancer (RCC) using retroviral transduction of T cells with a single-chain Ab-G250 chimeric receptor [scFv(G250)]. We describe the validation of our clinical protocol for gene transduction and expansion of human T lymphocytes., Methods: A batch of scFv(G250) transgene-containing retrovirus was produced under conditions of good manufacturing practice (GMP). In addition to quality control and safety testing of the virus batch, extensive potency testing was performed, i.e. assessment of its functional transduction efficiency in primary human T cells. Subsequently, the clinical gene transduction and cell-expansion protocol was subjected to a series of process validations and a clinical evaluation using T cells obtained from healthy donors and three RCC patients., Results: The clinical batch of scFv(G250) transgene-containing retrovirus met the quality and safety control criteria. Small-scale transductions yielded 62-92% scFv(G250)+ T cells and, at a clinical scale, 50-84% transduction efficiencies were obtained. Patient and healthy donor T cells showed similar expansion potencies, and also yielded similar levels of scFv(G250)-mediated immune functions, i.e. specific cytolysis of G250-ligand expressing RCC cells and production of IFN-gamma upon stimulation with such cells. All T cell cultures were free of replication competent retroviruses., Discussion: We have shown that the validated batch of scFv(G250) transgene-containing retrovirus in combination with our GMP T-cell transduction and expansion protocol successfully generates clinically relevant numbers of functional scFv(G250) gene-modified T cells for patient treatment.

We have started a phase I/II immunogene therapy study of metastatic renal cell cancer (RCC), using autologous T lymphocytes transduced ex vivo with a gene encoding a single-chain receptor based on the monoclonal antibody (mAb) G250 [scFv(G250)]. G250 recognizes carbonic anhydrase IX, which is overexpressed by RCC cells. We have developed and validated flow cytometric and real-time polymerase chain reaction (PCR) assays to quantitatively detect transduced T cells in patient blood. The flow assay was based on staining with the anti-G250 idiotype mAb NuH82 and showed a sensitivity of 0.06% scFv(G250)(1) cells within CD3(1) T cells. The real-time PCR method showed a sensitivity of 14 copies of scFv(G250) DNA per 100 ng of total DNA, which enabled detection of 0.008% scFv(G250)(1) T cells within leukocytes. Both assays were further validated for their specificity and reproducibility. When applied to blood samples from three RCC patients treated with intravenous infusions of scFv(G250)(1) T cells, the kinetics of scFv(G250)(1) T cell counts as detected by flow cytometry were similar to those detected by real-time PCR, although PCR allowed detection of transduced T cells over a longer period of time (i.e., for patient 3, 7 versus 32 days, respectively). Interestingly, follow-up studies of patient 3 demonstrated that the number of circulating scFv(G250)(1) T cells remained fairly constant during the first 7 days posttreatment, whereas the number of gene copies increased during the same period of time. These results suggest loss of scFv(G250) membrane expression on adoptive transfer, which would have important implications for the antitumor efficacy of this form of immunogene therapy.

Interleukin 12 (IL-12) is a cytokine with important regulatory functions bridging innate and adaptive immunity. It has been proposed as an immune adjuvant for vaccination therapy of infectious diseases and malignancies. The inflammatory properties of IL-12 play an important role in the adjuvant effect. We studied the effect of s.c. injections of recombinant human IL-12 (rHuIL-12) in 26 patients with renal cell cancer and demonstrated dose-dependent systemic activation of multiple inflammatory mediator systems in humans. rHuIL-12 at a dose of 0.5 microg/kg induced degranulation of neutrophils with a significant increase in the plasma levels of elastase (p < 0.05) and lactoferrin (p = 0.01) at 24 h. Additionally, rHuIL-12 injection mediated the release of lipid mediators, as demonstrated by a sharp increase in the plasma secretory phospholipase A2 (sPLA2) level (p = 0.003). rHuIL-12, when administered at a dose of 0.1 microg/kg, showed minimal systemic effects. In conclusion, when IL-12 is used as an adjuvant, doses should not exceed 0.1 microg/kg, in order to avoid severe systemic inflammatory responses.

Adoptive transfer of antigen-specific T cells has recently shown therapeutic successes in the treatment of viral infections and tumors. T cells specific for the antigen of interest can be generated in vitro, and adoptively transferred back to provide patients with large numbers of immune-competent T cells. Adoptive T cell therapy, however, is a patient-tailored treatment that unfortunately is not universally applicable to treat viral infections and tumors. We and others have demonstrated that the transfer of genes encoding antigen-specific receptors into T cells (i.e., genetic retargeting) represents an attractive alternative to induce antigen-specific immunity. Currently, we evaluate this concept in a clinical protocol to treat patients with metastatic renal cell cancer (RCC) using autologous RCC-specific gene-modified T lymphocytes.

In a phase I/II study, patients with solid metastatic MAGE-3-positive tumors, mainly melanoma, were vaccinated with recombinant MAGE-3 protein combined with the immunologic adjuvant AS02B comprised of MPL and QS21 in an oil-in-water emulsion. The recombinant MAGE-3 protein was made up of a partial sequence of the protein D (ProtD) antigen of Haemophilus influenzae fused to the MAGE-3 sequence. The vaccine was given intramuscularly at 3-week intervals. Patients whose tumors stabilized or regressed after 4 vaccinations received 2 additional vaccinations at 6-week intervals. MAGE-3 and ProtD antibody and cellular immune responses were monitored after vaccination. Ninety-six percent (23/24) of the patients vaccinated with MAGE-3 protein in AS02B adjuvant elicited a significant anti-MAGE-3 IgG antibody response after 4 vaccinations, and all developed anti-ProtD IgG antibodies. For the detection of T-cell activity, total peripheral blood mononuclear cells were restimulated in vitro with MAGE-3- or ProtD-loaded autologous mature dendritic cells. In 30% of the evaluable patients vaccinated with the adjuvanted recombinant protein, IFNgamma production was increased in response to MAGE-3, and 2 patients (14% of evaluable patients) had a concomitant increase in IL-5 production. In 37% and 43% of the patients, respectively, IFNgamma or IL-5 production was increased in response to ProtD. It is concluded that vaccination of advanced cancer patients with MAGE-3 self-antigen in AS02B adjuvant is able to elicit MAGE-3-specific antibody and a T-cell response.

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.

Background and Aim: The aim of this study was to unravel the mechanisms responsible for the increased risk of gall stone disease in hypertriglyceridaemia (HTG) and to compare the effects of triglyceride lowering therapy by bezafibrate and fish oil on determinants of cholelithiasis (biliary lipid composition and gall bladder motility) in HTG patients., Patients and Methods: Gall bladder motility (ultrasonography) was studied postprandially and during infusion of cholecystokinin (CCK). Determinants of cholelithiasis and serum lipids were compared between nine HTG patients and 10 age, sex, and body mass index matched normolipidaemic controls. The effects of bezafibrate and fish oil in HTG patients were studied in a randomised cross over trial., Results: HTG patients showed 14-fold higher serum triglyceride (TG) levels than controls. Biliary lipid composition, fasting gall bladder volumes, and CCK levels did not differ between HTG patients and controls. Gall bladder emptying was reduced in HTG patients compared with controls during CCK infusion (-22%) as well as in response to a meal (-37%; both p<0.001). Postprandial CCK levels were significantly higher in HTG patients. Both bezafibrate and fish oil reduced serum TG levels (-68% and -51% v baseline, respectively; both p<0.01). Fasting CCK levels were not affected whereas CCK induced gall bladder emptying increased during bezafibrate (+29%; p<0.001) and tended to increase on fish oil therapy (+13%; p=0.07). Postprandial gall bladder motility improved on bezafibrate and fish oil (+47 and +25% v baseline, respectively; both p<0.02) at least partly due to increased gall bladder sensitivity to CCK (both p<0.05 v baseline). Bezafibrate but not fish oil increased the molar ratio of cholesterol to bile acids (+40%; p

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Background/aims: Following truncal vagotomy, a heightened contractile response of the gallbladder to cholecystokinin (CCK) has been reported in patients. We investigated whether the gallbladder responsiveness to the CCK analog cerulein is also affected in patients with a highly selective vagotomy (HSV) and in duodenal ulcer patients, since most patients had truncal vagotomy for recurrent peptic ulcer disease., Materials and Methods: Gallbladder emptying (cholescintigraphy) and plasma cholecystokinin like immunoreactivity (CCK-LI) levels were studied during infusion of graded doses of the CCK analog cerulein., Results: In duodenal ulcer patients (n = 9), patients with HSV (n = 9), patients with truncal vagotomy (n = 9) and control subjects (n = 9), infusion of stepwise increasing doses of cerulein (1-16 ng.kg-1.h-1) induced dose related changes in plasma CCK-LI. In patients with truncal vagotomy, the gallbladder contraction in response to 1, 2 and 4 ng.kg-1.h-1 of cerulein was significantly increased over controls; whereas the gallbladder contraction to cerulein in duodenal ulcer patients and patients with HSV was not significantly different from controls., Conclusions: Thus, in patients with truncal vagotomy, gallbladder contractile response to CCK is significantly enhanced, possibly due to denervation of hepatic vagal branches since gallbladder contraction after CCK infusion shows no difference between post HSV or duodenal ulcer patients and the controls.

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.