http://repub.eur.nl/res/aut/8706/ List of Publications en http://repub.eur.nl/static-eur/img/logo.png http://repub.eur.nl http://repub.eur.nl/res/pub/33281/ 2011-10-01T00:00:00Z Fragile X-associated tremor/ataxia syndrome (FXTAS) is an adult-onset neurodegenerative disorder generally presenting with intention tremor and gait ataxia, but with a growing list of co-morbid medical conditions including hypothyroidism, hypertension, peripheral neuropathy, and cognitive decline. The pathological hallmark of FXTAS is the presence of intranuclear inclusions in both neurons and astroglia. However, it is unknown to what extent such inclusions are present outside the central nervous system (CNS). To address this issue, we surveyed non-CNS organs in ten human cases with FXTAS and in a CGG repeat knock-in (CGG KI) mouse model known to possess neuronal and astroglial inclusions. We find inclusions in multiple tissues from FXTAS cases and CGG KI mice, including pancreas, thyroid, adrenal gland, gastrointestinal, pituitary gland, pineal gland, heart, and mitral valve, as well as throughout the associated autonomic ganglia. Inclusions were observed in the testes, epididymis, and kidney of FXTAS cases, but were not observed in mice. These observations demonstrate extensive involvement of the peripheral nervous system and systemic organs. The finding of intranuclear inclusions in non-CNS somatic organ systems, throughout the PNS, and in the enteric nervous system of both FXTAS cases as well as CGG KI mice suggests that these tissues may serve as potential sites to evaluate early intervention strategies or be used as diagnostic factors. http://repub.eur.nl/res/pub/33314/ 2011-09-01T00:00:00Z The FMR1 gene contains a CGG repeat present in the 5'-untranslated region which can be unstable upon transmission to the next generation. The repeat is up to 55 CGGs long in the normal population. In patients with fragile X syndrome (FXS), a repeat length exceeding 200 CGGs (full mutation: FM) generally leads to methylation of the repeat and the promoter region, which is accompanied by silencing of the FMR1 gene. The absence of FMR1 protein, FMRP, seen in FM is the cause of the mental retardation in patients with FXS. The premutation (PM) is defined as 55-200 CGGs. Female PM carriers are at risk of developing primary ovarian insufficiency. Elderly PM carriers might develop a progressive neurodegenerative disorder called fragile X-associated tremor/ataxia syndrome (FXTAS). Although arising from the mutations in the same gene, distinct mechanisms lead to FXS (absence of FMRP), FXTAS (toxic RNA gain-of-function) and FXPOI. The pathogenic mechanisms thought to underlie these disorders are discussed. This review gives insight on the implications of all possible repeat length categories seen in fragile X families. http://repub.eur.nl/res/pub/33842/ 2011-08-01T00:00:00Z 5′-and 3′-untranslated regions (UTRs) are important regulators of gene expression and play key roles in disease progression and susceptibility. The 5′-UTR of the fragile X mental retardation 1 (FMR1) gene contains a CGG repeat element that is expanded (>200 CGG repeats; full mutation) and methylated in fragile X syndrome (FXS), the most common form of inherited intellectual disability (ID) and known cause of autism. Significant phenotypic involvement has also emerged in some individuals with the premutation (55-200 CGG repeats), including fragile X-associated premature ovarian insufficiency (FXPOI) in females, and the neurodegenerative disorder, fragile X-associated tremor/ataxia syndrome (FXTAS), in older adult carriers. Here, we show that FMR1 mRNA in human and mouse brain is expressed as a combination of multiple isoforms that use alternative transcriptional start sites and different polyadenylation sites. Furthermore, we have identified a novel human transcription start site used in brain but not in lymphoblastoid cells, and have detected FMR1 isoforms generated through the use of both canonical and non-canonical polyadenylation signals. Importantly, in both human and mouse, a specific regulation of the UTRs is observed in brain of FMR1 premutation alleles, suggesting that the transcript variants may play a role in premutation-related pathologies. http://repub.eur.nl/res/pub/34211/ 2011-05-01T00:00:00Z Fragile X syndrome (FXS) is the most common inherited form of mental retardation and is caused by the lack of fragile X mental retardation protein (FMRP). In the brain, spine abnormalities have been reported in both patients with FXS and Fmr1 knockout mice. This altered spine morphology has been linked to disturbed synaptic transmission related to altered signaling in the excitatory metabotropic glutamate receptor 5 (mGluR5) pathway. We investigated hippocampal protrusion morphology in adult Fmr1 knockout mice. Our results show a hippocampal CA1-specific altered protrusion phenotype, which was absent in the CA3 region of the hippocampus. This suggests a subregion-specific function of FMRP in synaptic plasticity in the brain. http://repub.eur.nl/res/pub/33545/ 2011-01-06T00:00:00Z 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. http://repub.eur.nl/res/pub/21835/ 2010-11-01T00:00:00Z Purpose: A new universal tool for specific, non-covalent and non-destructive attachment of a recombinant antibody fragment to a polymer-modified adenovirus has been utilised to regulate the tropism of adenoviral gene delivery vector. Methods: We have prepared a multivalent reactive N-(2-hydroxypropyl)methacrylamide-based copolymer (PHPMA) bearing an α-bungarotoxin-binding peptide (BTXbp). The copolymer was used for covalent surface modification of adenoviral vectors (Ad). The α-bungarotoxin protein (BTX) has a nanomolar binding affinity for BTXbp, allowing non-covalent linkage of BTX fusion proteins. A single chain variable fragment of anti-PSMA antibody bearing BTX (scFv-BTX) binding to the prostate-specific membrane antigen (PSMA) was conjugated with the copolymer-coated adenovirus to enable specific infection of prostate cancer cells via PSMA receptors. Results: As shown by ELISA, the copolymer-coated virus exhibited much reduced binding to anti-Ad antibodies. Infection of PC-3 and LNCaP prostate cancer cells was ∼100-fold less efficient with copolymer-coated Ad than with un-modified Ad. Conjugation of scFv-BTX with Ad-PHPMA-BTXbp led to 5-10-fold restoration of infection in PSMA-positive LNCaP cells. In PSMA-negative PC-3 cells, the conjugation of scFv-BTX with Ad-PHPMA-BTXbp gave no enhancement of infection. Conclusions: We have shown that the presented Ad-PHPMA-BTXbp/scFv-BTX system can be used as a universal tool for a receptor-specific virotherapy. http://repub.eur.nl/res/pub/20683/ 2010-07-01T00:00:00Z Gene-engineered T cell therapy represents a promising strategy to treat cancers. To enable pre-selection of patients sensitive to this type of treatment we have setup and validated a T cell activation assay to test antigen expression on patient-derived tumor tissues. Chimeric antibody-based receptor (CAR) directed against CAIX, currently used in a clinical trial to treat RCC patients, was used as a model receptor. Primary human T cells expressing CAIX CAR were able to respond to CAIX-positive but not CAIX-negative tumor tissue and showed an increased production of IFNγ, TNFα, IL-10 and IL-4, but not IL-2 or IL-5. Tumor tissue driven responses of primary T cells were paralleled by NFAT activation measured in CAR-transduced Jurkat T cells, which was shown to be triggered in a CAR and antigen-specific manner. Next, the reporter gene assay was applied to two independent PSMA CARs, which both mediated NFAT activation in response to tumor tissue. Taken together, a sensitive and donor-independent assay was established to measure T cell activation upon exposure to patient-derived tumor tissue, which may facilitate pre-selection of patients for clinical adoptive T cell therapy. http://repub.eur.nl/res/pub/28446/ 2010-02-01T00:00:00Z T cell receptor (TCR) gene therapy provides patients with autologous T cells that are genetically engineered with TCRαβ chains and constitutes a promising approach for the treatment of tumors and virus infections. Among the current challenges of TCR gene therapy is the optimization of TCRα and β transgene pairing to enhance the functional avidity of therapeutic T cells. Recently, various genetically modified TCRs have been developed that enhance TCR pairing and minimize mispairing, i.e. pairing between transgenic and endogenous TCR chains. Here, we classify such receptors according to their CD3-dependence for surface expression and review their abilities to address functional T cell avidity. In addition, we discuss the anticipated clinical value of these and other strategies to generate high-avidity T cells. http://repub.eur.nl/res/pub/25263/ 2009-12-15T00:00:00Z T-cell receptor (TCR) gene transfer for cancer immunotherapy is limited by the availability of large numbers of tumorspecific T cells. TCR α and β chains were isolated from a highly lytic HLA-A2-restricted cytotoxic T lymphocyte (CTL) clone recognizing the melanoma-associated Melan-A/MART-1 antigen and inserted into a lentiviral vector carrying a bidirectional promoter capable of robust and coordinated expression of the two transgenes. Lentiviral vector-based gene delivery systems have shown increased transfer efficiency and transgene expression compared with the widely used γ-retroviral vectors. This vector performed more efficiently than a γ-retrovirus-based vector containing the same expression cassette, resulting in a T-cell population with 60% to 80% of transgenic TCR expression with mainly CD8+intermediate effector phenotype. Transgenic T cells specifically produced cytokine in response to and killed antigen-expressing melanoma cells, retained an overlapping functional avidity in comparison with the TCR donor CTL clone, and exerted significant therapeutic effects in vivo upon adoptive transfer in melanoma-bearing severe combined immunodeficient mice. Optical imaging showed their accumulation in the tumor site. Overall, our results indicate that lentiviral vectors represent a valid tool for stable and high-intensity expression of transgenic TCR and support clinical exploitation of this approach for therapeutic application. http://repub.eur.nl/res/pub/26906/ 2009-09-05T00:00:00Z The CGG-repeat present in the 5′UTR of the FMR1 gene is unstable upon transmission to the next generation. The repeat is up to 55 CGGs long in the normal population. In fragile X patients, a repeat length exceeding 200 CGGs (full mutation: FM) generally leads to methylation of the repeat and the promoter region, which is accompanied by silencing of the FMR1 gene. The gene product FMRP is involved in regulation of transport and translation of certain mRNA in the dendrite, thereby affecting synaptic plasticity. This is central to learning and memory processes. The absence of FMRP seen in FM is the cause of the mental retardation seen in fragile X patients. The premutation (PM) is defined as 55-200CGGs. Female PM carriers are at risk of developing primary ovarian insufficiency. Recently it was discovered that elderly PM carriers might develop a progressive neurodegenerative disorder called fragile X-associated tremor/ ataxia syndrome. Although arising from the mutations in the same gene, distinct mechanisms lead to fragile X syndrome (absence of FMRP) and FXTAS (toxic RNA gain of function). The pathogenic mechanisms thought to underlie these disorders are discussed, with a specific emphasis on FXTAS. This review gives insight on the implications of all possible repeat length categories seen in fragile X families. http://repub.eur.nl/res/pub/21377/ 2009-04-01T00:00:00Z Abstract 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. http://repub.eur.nl/res/pub/30476/ 2008-11-01T00:00:00Z Adoptive transfer of antigen-specific T-cells has shown therapeutic successes in the treatment of tumors in patients with metastatic melanoma. Tumor antigen-specific T-lymphocytes, however, occur only at low frequencies in a small proportion of patients. This low T-lymphocyte frequency together with the difficulties associated with in vitro generation of T-lymphocytes specific for cancers other than melanoma hampers adoptive T cell therapy. To make adoptive T-cell therapy more uniformly applicable, strategies were developed at transferring tumor-specificity to primary human T-lymphocytes via antibody (Ig) or T-cell receptor (TCR) molecules. We exploited the selection power of phage display that allows for the testing of tens of billions of individual clones with a high-throughput selection of Fabs with peptide/MHC complex binding capacity. Following in vitro selection, human "TCR-like" Fab fragments have been functionally expressed on human T-lymphocytes, resulting in MHC-restricted, tumor-specific lysis and cytokine production. Currently, we have extended our selections to a panel of class I and II MHC-restricted MAGE and other tumor-specific epitopes, and would like to propose that phage display represents a technology able to expand T-cell therapy to numerous tumor types. http://repub.eur.nl/res/pub/29921/ 2008-07-01T00:00:00Z Adenovirus vectors have great potential in cancer gene therapy. Targeting of cancer-testis (CT) antigens, which are specifically presented at the surface of tumor cells by human leukocyte antigen (HLA) class I molecules, is an attractive option. In this study, a single-chain T-cell receptor (scTCR) directed against the CT antigen melanoma-associated antigen (MAGE)-A1 in complex with the HLA class I molecule of haplotype HLA-A1 is fused with the C terminus of the adenovirus minor capsid protein IX. Propagation of a protein-IX (pIX)-gene-deleted human adenovirus 5 (HAdV-5) vector on cells that constitutively express the pIXscTCR fusion protein yielded viral particles with the pIXscTCR fusion protein incorporated in their capsid. Generated particles specifically transduced melanoma cell lines expressing the HLA-A1/MAGE-A1 target complex with at least 10-fold higher efficiency than control viruses. Whereas loading of HLA-A1-positive cells with MAGE-A1 peptides leads to enhanced transduction of the cells, the efficiency of virus transduction is strongly reduced if the HLA-A1 molecules are not accessible at the target cell. Taken together, these data provide proof of principle that pIXscTCR fusions can be used to target HAdV-5 vectors to tumor cells expressing intracellular CT antigens. http://repub.eur.nl/res/pub/35067/ 2007-12-01T00:00:00Z To improve safety and specificity of oncolytic adenoviruses, we introduced T-cell receptors (TCR) specific for a unique class of truly tumor-specific antigens into the adenoviral fiber protein. The adenoviral fiber knob responsible for attachment to the coxsackie-adenoviral receptor (CAR) on target cells was replaced by a single-chain TCR (scTCR) molecule with specificity for the melanoma-associated cancer-testis antigen MAGE-A1, presented by HLA-A1, and an extrinsic trimerization motif in a replicating Ad5 vector (Ad5.R1-scTCR). The production of the recombinant virus was initiated in a novel producer cell line that expressed an antibody-based hexonspecific receptor (293T-AdR) in the cell membrane. This new production system allowed CAR-independent and target antigen-independent propagation of Ad5.R1-scTCR. Infection with adenovirus bearing the scTCR-based fiber resulted in an efficient killing of target tumor cells. The infection was cell type specific because only HLA-A1+/MAGE-A1+melanoma cells were killed, and thus, this retargeting strategy provides a versatile tool for future clinical application. http://repub.eur.nl/res/pub/36410/ 2007-09-01T00:00:00Z Progressive multifocal leukoencephalopathy (PML) is a deadly brain disease caused by the polyomavirus JC (JCV). The aim of this study is to develop 'designer T cells' armed with anti-JCV TCR-based chimeric immune receptors (CIRs) by gene modification for PML immunotherapy. Two T cell lines specific to two dominant CTL epitopes derived from JCV VP1 protein (termed p36 and p100) from an HLA-A0201+PML survivor were generated for TCR cloning. Two distinct dominant TCR alpha chains (Vα6 and Vα12) and a unique TCR beta chain (Vβ5.1) were cloned from the p36-specific cell line, while only one alpha (Vα8.6) and one beta (Vβ2) chains were dominant in the p100-specific line. Retroviral constructs encoding CIRs were created with the extracellular domains of TCR α and β chains fused to the transmembrane and cytoplasmic portions of CD3ζ (VαCαCD3ζ or VβCβCD3ζ). Cellular expression and screening for binding specific peptide-HLA-A0201 tetramer confirmed the reactivity of the p100 TCRαβ and of one of the two pairs of p36 TCRαβ (Vα12 and Vβ5.1). Functional tests confirmed CIR-expressing T cells secreted cytokines and expressed potent cytotoxicity on contact with A0201+B-lymphoblastoid line loaded with peptides and/or with HLA-A0201+cells expressing native JCV VP1 protein. In conclusion, anti-JCV designer T cells were generated. http://repub.eur.nl/res/pub/36625/ 2007-07-01T00:00:00Z Background: T cell receptor (TCR) gene therapy represents an attractive anti-cancer treatment but requires further optimization of its efficacy and safety in clinically relevant models, such as those using a tumor antigen and TCR of human origin. Currently, however, there is no consensus as to what protocol is most optimal for retroviral human TCR gene transfer into primary murine T cells, most notably with respect to virus pseudo-type. Methods: Primary murine T cells were transduced, expanded and subsequently tested for transgene expression, proliferation and antigen-specific function. To this end, murine leukemia virus (MLV) retroviruses were produced upon transfection of various packaging cells with genes encoding either green fluorescent protein (GFP) or TCRαβ specific for human melanoma antigen gp100280-288and the helper elements GAG/POL and ENV. Next to viral pseudotyping, the following parameters were studied: T cell densities; T cell activation; the amounts of IL-2 and the source of serum used to supplement medium. Results: The pseudo-type of virus produced by packaging cells critically determines T cell transduction efficiencies. In fact, MLV-A and MLV-E pseudo-typed viruses derived from a co-culture of Phoenix-A and 293T cells resulted in T cell transduction efficiencies that were two-fold higher than those based on retroviruses expressing either VSV-G, GALV, MLV-A or MLV-E envelopes. In addition, T cell densities during transduction were inversely related to transduction efficiencies. Further optimization resulted in transduction efficiencies of over 90% for GFP, and 68% for both a murine and a human (i.e. murinized) TCR. Importantly, TCR-transduced T cells proliferate (i.e. showing a log increase in cell number in a few days) and show antigen-specific function. Conclusions: We set up a quick and versatile method to genetically modify primary murine T cells based on transient production of TCR-positive retroviruses, and show that retroviral gene transfer of a human TCR into primary murine T cells is critically improved by viral pseudo-typing with both MLV-A and MLV-E envelopes. Copyright http://repub.eur.nl/res/pub/21783/ 2006-04-01T00:00:00Z Abstract EBV is associated with a broad range of malignancies. Adoptive immunotherapy of these tumors with EBV-specific CTL proved useful. We generated a panel of primary human T cells specific to various EBV antigens (i.e. Epstein-Barr nuclear antigen 3A, 3B and BamHI-M leftward reading frame) via transfer of modified TCR genes that are either coupled to CD3zeta or Fc(epsilon)RIgamma. TCR-transduced T cells from 20-60% of donors (total number of 25) demonstrated specific lysis of EBV peptide-loaded target cells, whereas lymphoblastoid cell lines expressing native EBV antigens were not killed by any of the EBV-specific T cell populations. This non-responsiveness, confirmed at the level of nuclear factor of activated T cells activation, is not due to receptor configuration since identical receptor formats specific for melanoma antigens successfully re-targeted T cells to native melanoma cells. In an effort to generate a more potent receptor, we introduced a CD28 domain into one of the EBV-specific TCR. This TCR did not affect the cytotoxic response of re-targeted T cells, but dramatically enhanced antigen-specific IFNgamma production. We therefore conclude that these novel CD28-containing EBV-specific TCRs provide a basis for further development of TCR gene transfer to treat EBV-induced diseases. http://repub.eur.nl/res/pub/9350/ 2006-01-01T00:00:00Z Therapeutic success of TCR gene transfer to treat tumors depends on the ability of redirected T cells to become activated upon tumor recognition in vivo. Help provided by tumor-specific Th1 cells is reported to relieve T cells from an anergized state and to induce tumor regression. We recently demonstrated the ability to generate melanoma-specific Th1 cells by genetic introduction of both a CD8-dependent TCR and the CD8α coreceptor into CD4⁺ T cells. In this study, we analyzed a TCR that binds Ag independently of CD8, a property generally preferred to induce tumor-specific T cell responses, and addressed the contribution of CD8α following introduction into TCR-transduced CD4⁺ T cells. To this end, primary human CD4⁺ T cells were gene transferred with a high-avidity TCR, and were shown not only to bind peptide/MHC class I, but also to effectively kill Ag-positive tumor cells in the absence of CD8α. The introduction of CD8α up-regulates the tumor-specific production of TNF-α and IL-2 to some extent, but significantly down-regulates production of IL-4, IL-5, and IL-10 in CD4⁺ T cells. The introduction of a mutated cysteine motif in CD8α, which prevents its binding to LCK and linker for activation of T cells, did not adversely affect expression and T cell cytotoxicity, but counteracted the CD8α-mediated down-regulation of IL-4 and IL-5, but not IL-10. In conclusion, CD8α down-regulates the production of major Th2-type cytokines, in part mediated by LCK and/or linker for activation of T cells, and may induce differentiation of tumor-specific Th1 cells, which makes this coreceptor an interesting candidate to improve the clinical potential of TCR gene transfer to treat cancer. http://repub.eur.nl/res/pub/21351/ 2005-11-01T00:00:00Z 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) þ T cells using PG13-derived retrovirus versus up to 90% scFv(G250) þ T cells using Phoenix-derived retrovirus. The median number of transgene integrations per scFv(G250) þ 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 TNFa production and cytolysis of G250L þ RCC cells than T cells ransduced 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. http://repub.eur.nl/res/pub/10389/ 2005-01-01T00:00:00Z 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. http://repub.eur.nl/res/pub/21366/ 2004-04-01T00:00:00Z Abstract 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 http://repub.eur.nl/res/pub/10098/ 2003-01-01T00:00:00Z TCR with known antitumor reactivity can be genetically introduced into primary human T lymphocytes and provide promising tools for immunogene therapy of tumors. We molecularly characterized two distinct TCRs specific for the same HLA-A2-restricted peptide derived from the melanocyte differentiation Ag gp100, yet exhibiting different stringencies in peptide requirements. The existence of these two distinct gp100-specific TCRs allowed us to study the preservation of peptide fine specificity of native TCRalphabeta when engineered for TCR gene transfer into human T lymphocytes. Retroviral transduction of primary human T lymphocytes with either one of the two sets of TCRalphabeta constructs enabled T lymphocytes to specifically kill and produce TNF-alpha when triggered by native gp100(pos)/HLA-A2(pos) tumor target cells as well as gp100 peptide-loaded HLA-A2(pos) tumor cells. Peptide titration studies revealed that the cytolytic efficiencies of the T lymphocyte transductants were in the same range as those of the parental CTL clones. Moreover, primary human T lymphocytes expressing either one of the two engineered gp100-specific TCRs show cytolytic activities in response to a large panel of peptide mutants that are identical with those of the parental CTL. The finding that two gp100-specific TCR, derived from two different CTL, can be functionally introduced into primary human T lymphocytes without loss of the Ag reactivity and peptide fine specificity, holds great promise for the application of TCR gene transfer in cancer immunotherapy. http://repub.eur.nl/res/pub/21294/ 2002-07-01T00:00:00Z 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