Biomedical subjects
Publications and source records attributed to J L Schultze.
Explore the source record for details and available documents.
The widely expressed tumour antigens hTERT and CYP1B1 are commonly expressed in multiple myeloma (MM) cells. Several trials targeting these antigens by immunotherapy have been initiated. The aim of this study was to explore whether patients with MM have an endogenous pre-existing immune response against recently identified epitopes from hTERT and CYP1B1. Peripheral blood T cells from 27 HLA-A*0201+ multiple myeloma patients at different stages of disease and 20 healthy HLA-A*0201+ donors were enriched and studied for the presence of hTERT- and CYP1B1-specific cytotoxic T cells using MHC tetramer detection and short-term ex vivo expansion. No significant expansion of tetramer-positive cells was detected in the peripheral blood of either MM patients or healthy controls when cells were stained with tetramers containing the dominant hTERT-derived epitope or two peptides derived from CYP1B1. A single ex vivo peptide stimulation led to the detection of a small population (0.3-0.5%) of hTERT-specific cells in two of 27 patients with MM. None of the patients or controls showed significant expansion of CYP1B1-specific cells after a single peptide stimulation. Thus, endogenous in vivo priming of T cells against hTERT and CYP1B1 is a rare event in MM patients. These results suggest that strategies targeting hTERT and CYP1B1 may have to utilize techniques to induce T cell responses from a naive precursor frequency.
Owing to its clinical accessibility, peripheral blood is probably the best source for the assessment of differences or changes in gene expression associated with disease or drug response and therapy. Gene expression patterns in peripheral blood cells greatly depend on temporal and interindividual variations. However, technical aspects of blood sampling, isolation of cellular components, RNA isolation techniques and clinical aspects such as time to analysis and temperature during processing have been suggested to affect gene expression patterns. We therefore assessed gene expression patterns in peripheral blood from 29 healthy individuals by using Affymetrix microarrays. When RNA isolation was delayed for 20-24 h-a typical situation in clinical studies-gene signatures related to hypoxia were observed, and downregulation of genes associated with metabolism, cell cycle or apoptosis became dominant preventing the assessment of gene signatures of interindividual variation. Similarly, gene expression patterns were strongly dependent on choice of cell and RNA isolation and preparation techniques. We conclude that for large clinical studies, it is crucial to reduce maximally the time to RNA isolation. Furthermore, prior to study initiation, the cell type of interest should already be defined. Our data therefore will help to optimize clinical studies applying gene expression analysis of peripheral blood to exploit drug responses and to better understand changes associated with disease.
Explore the source record for details and available documents.
To target the neural cell adhesion molecule (NCAM, CD56) on neuroblastoma by T cell-based immunotherapy we have generated a bi-specific CD3 x NCAM antibody (OE-1). This antibody can be used to redirect T cells to NCAM+ cells. Expectedly, the antibody binds specifically to NCAM+ neuroblastoma cells and CD3+ T cells. OE-1 induces T cell activation, expansion and effector function in peripheral blood mononuclear cell (PBMC)-derived CD4+ and CD8+ T cells. T cell activation was shown to depend on the presence of normal natural killer (NK) cells in the culture. Interestingly, while PBMC- derived T cells were activated by OE-1, NK cells were almost completely depleted, suggesting that T cells activated by OE-1 deleted the NK cells. Activated CD4+ and CD8+ T cells differentiate into a larger CCR7+ central memory and a smaller CCR7- effector memory cell population. Most importantly, preactivated T cells were highly cytotoxic for neuroblastoma cells. In eight of 11 experiments tumour-directed cytotoxicity was enhanced when NK cells were present during preactivation with OE-1. These data strongly support a bi-phasic therapeutic concept of primarily stimulating T cells with the bi-specific antibody in the presence of normal NCAM+ cells to induce T cell activation, migratory capacity and finally tumour cell lysis.
Although high frequencies of T lymphocytes specific for certain tumor-associated antigens have been detected in some cancer patients, increasing evidence suggests that these T cells may be functionally defective in vivo and fail to induce meaningful clinical responses. One strategy to overcome this limitation is to target novel antigens that are ignored during the natural antitumor immune response but are nevertheless capable of triggering effector T-cell responses against tumors after optimal presentation by antigen-presenting cells. Here, we show that the telomerase catalytic subunit (hTERT)-a nearly universal tumor antigen identified by epitope deduction rather than from patient immune responses-is immunologically ignored by patients despite progressive tumor burden. Nevertheless, HLA-A2-restricted CTLs against hTERT are equivalently induced ex vivo from patients and healthy individuals and efficiently kill human tumor cell lines and primary tumors. Thus, telomerase-specific T cells from cancer patients are spared functional inactivation because of immunological ignorance. These findings support clinical efforts to target the hTERT as a tumor antigen with broad therapeutic potential.
A growing number of human tumor antigens have been described that can be recognized by CTLs in a MHC class I restricted fashion. The epithelial cell adhesion molecule (Ep-CAM) is expressed in a variety of human tumors and has attracted attention as a therapeutic target for monoclonal antibody serotherapy. We have identified immunogenic peptides derived from Ep-CAM, that bind to human leukocyte antigen-A*0201 and elicit strong peptide-specific human CTL responses, demonstrating that there is an effective T-cell repertoire against these Ep-CAM-derived peptides that can be recruited. Alterations to these peptides were made to increase their binding affinity to MHC class I molecules. The use of such "heteroclitic" peptides allowed generation of cytotoxic T cells that demonstrated increased killing of target cells pulsed not only with the heteroclitic but also with the native peptide. Most important, CTL cell lines that are generated against these peptides specifically lyse epithelial tumor cells expressing Ep-CAM but not normal hematopoietic or bronchial epithelial cells.
Clinically successful specific cancer immunotherapy depends on the identification of tumor-rejection antigens (Ags). Historically, tumor Ags have been identified by analyzing either T-cell or antibody responses of cancer patients against the autologous cancer cells. The unveiling of the sequence of the human genome, improved bioinformatics tools and optimized immunological analytical tools have made it possible to screen any given protein for immunogenic epitopes. Overexpressed genes in cancer can be identified by gene-expression profiling; immunogenic epitopes can be predicted based on HLA-binding motifs; candidate peptides can be identified by mass spectrometry of tumor-cell-derived HLA molecules; and peptide-specific T cells can be qualitatively and quantitatively analyzed at the single-cell level using ELISPOT and tetramer technologies. Here, we suggest that, based on these advancements, a new class of tumor Ags can be identified by directly linking cancer genomics to cancer immunology and immunotherapy.
Most B-cell malignancies are incurable diseases and therefore warrant new therapeutic approaches. In a pilot study, we tested the feasibility and safety of combined immunotherapy consisting of adoptive transfer of autologous tumour-specific T cells, low-dose interleukin 2 (IL-2) and a cellular vaccine of CD40-activated plasma cell leukaemia (PCL) cells in a patient who failed tandem repeat stem cell transplantation and idiotype vaccination. Autologous tumour-specific T cells for adoptive T-cell transfer were propagated in vitro by repetitive stimulation with autologous ex vivo CD40-activated PCL cells. CD40-activated PCL cells for vaccination were similarly generated ex vivo by co-culture with CD40 ligand transfectants. Autologous T cells (5 x 108 and 2.5 x 109 for two separate treatment cycles) generated ex vivo and cytotoxic against autologous tumours were infused and well tolerated by the patient. Fever and myalgias were closely related to IL-2 injections and no other adverse effects were observed. A temporary decrease of PCL cells in peripheral blood was seen after the first cycle of adoptive T-cell therapy, tumour cell vaccination and low-dose IL-2. Tumour progression was associated with tumour cells that (1) expressed a complex karyotype, (2) demonstrated loss of MHC class II, and (3) did not induce autologous tumour-specific T-cell lines ex vivo. We demonstrated the safety and feasibility in combining autologous tumour-specific T-cell therapy with low-dose IL-2 and that clinical trials based on the use of CD40-activated autologous tumour cell vaccines are warranted in patients with CD40-activated autologous tumour cells, either as a vaccine or for ex vivo stimulation of autologous T cells.
Tumour immunology has seen many exciting developments in the last few years. In addition to tumour antigens that are defined by antitumour T- and B-cell responses in patients, the human telomerase reverse transcriptase has been identified by 'reverse immunology' as the first truly universal tumour antigen. Molecular remission has been associated with a cancer vaccine that targets the clonal idiotype of B-cell malignancies, and sophisticated cellular vaccines (including fusions of tumour cells and antigen-presenting cells) have demonstrated promising results. Moreover, our capabilities of measuring immunity have been significantly enhanced by novel technology, such as major histocompatibility complex (MHC)-peptide tetramers and ELISPOT analysis. We are now capable of tracking antigen-specific T cells at a single cell level. This review will analyse recent developments and highlight some important issues that need to be addressed in the future.
The disclosure of the human genome sequence and rapid advances in genomic expression profiling have revolutionized our knowledge about molecular changes in malignant diseases. Rapidly growing gene expression databases and improvements in bioinformatics tools set the stage for new approaches using large-scale molecular information to develop specific therapeutics in cancer. On one hand, the ability to detect clusters of genes differentially expressed in normal and malignant tissue may lead to widely applicable targeting of defined molecular structures. On the other hand, analyzing the 'molecular fingerprint' of an individual tumor raises the possibility of developing customized therapeutics. One approach to use the emerging new datasets for the development of novel therapeutics is to identify genes that are specifically expressed in tumors as targets for immune intervention. This review will focus on the process from in silico analysis of expression databases and screening of potential candidate genes by bioinformatics to the in vitro and in vivo analysis to determine the immunogenicity of candidate tumor antigens. Basic biological principles of 'reverse immunology' as well as technical advantages and difficulties will be addressed.
A major obstacle for the development of cancer immunotherapy is the poor capacity of most tumor cells to present antigen. It has previously been shown that ligation of CD40 on the surface of malignant B cells results in the induction of efficient antigen presentation primarily because of upregulated expression of MHC, costimulatory, and adhesion molecules. Ongoing clinical trials are testing the impact of CD40 ligation as immunotherapy for B cell malignancies. Because CD40 is also widely expressed in carcinomas, we studied whether CD40 activation of these cells using soluble recombinant trimeric human CD40 ligand (srhCD40L) can also induce T cell responses. Here, we show that carcinoma cells upregulate expression of CD54 and MHC molecules following in vitro exposure to srhCD40L but do not upregulate CD80 or CD86. CD40-activated carcinoma cells failed to trigger mixed lymphocyte reactions, in sharp contrast to CD40-activated lymphoma cells for which CD40 activation, as expected, resulted in increased expression of MHC, adhesion, and costimulatory molecules, and generated brisk allogeneic lymphocyte reactions. Retroviral-mediated expression of CD80 in carcinoma cells, with or without CD40 activation, triggered mixed lymphocyte reactions, provided cells were treated with IFN-gamma. Thus, the cell surface phenotype induced on carcinoma cells following CD40 activation is not fully capable of inducing T cell proliferation; however, these results support ongoing efforts to exploit costimulation in clinical efforts aimed at increasing carcinoma immunogenicity.
PURPOSE: We have reported previously that the telomerase catalytic subunit, human telomerase reverse transcriptase (hTERT), is a widely expressed tumor-associated antigen recognized by CTLs. A nine-amino acid peptide derived from hTERT binds strongly to HLA-A2 antigen and elicits CTL responses against a broad panel of hTERT+ tumors (but not hTERT+ hematopoietic progenitor cells). The applicability of hTERT as a potential target for anticancer immunotherapy would be widened by the identification of epitopes restricted to other common HLA alleles, such as HLA-A3 antigen. EXPERIMENTAL DESIGN: Using a method of epitope deduction, HLA-A3-restricted peptide epitopes were screened from hTERT and tested for immunogenicity in a human in vitro T-cell system. RESULTS: The hTERT peptide K973 was used to generate specific CD8+ CTLs from HLA-A3+ cancer patients and healthy individuals. These CTLs lysed hTERT+ tumors from multiple histologies in an MHC-restricted fashion, suggesting that the epitope is naturally processed and presented by tumors. In contrast, highly enriched HLA-A3+ CD34+ peripheral blood progenitor cells or activated T cells were not lysed. CONCLUSION: Given the expression of HLA-A2 and HLA-A3 antigen in the general population, these findings extend the potential applicability of hTERT as a therapeutic target to >60% of all cancer patients. The characterization of hTERT as a polyepitope, polyallelic tumor-associated antigen may provide an approach for circumventing therapy-induced resistance potentially mediated by antigenic- and allelic-loss tumor escape mutants.
Although the idiotypic structures of immunoglobulin from malignant B cells were the first tumor-specific determinants recognized, and clinical vaccination trials have demonstrated induction of tumor-specific immunity, the function of immunoglobulin-specific CD8+ cytotoxic T lymphocytes in tumor rejection remains elusive. Here, we combined bioinformatics and a T cell-expansion system to identify human immunoglobulin-derived peptides capable of inducing cytotoxic T-lymphocyte responses. Immunogenic peptides were derived from framework regions of the variable regions of the immunoglobulin that were shared among patients. Human-leukocyte-antigen-matched and autologous cytotoxic T lymphocytes specific for these peptides killed primary malignant B cells, demonstrating that malignant B cells are capable of processing and presenting such peptides. Targeting shared peptides to induce T-cell responses might further improve current vaccination strategies in B-cell malignancies.
Interleukin (IL)-12 is expressed mainly in antigen-presenting cells after challenge with microbial material or after CD40 activation. Although IL-12 was cloned from human Epstein-Barr virus (EBV)-transformed B cell lines, surprisingly, CD40 ligation on murine B cells did not lead to IL-12 production, suggesting that murine B cells do not produce IL-12. Here we demonstrate that a subset of human tonsillar B cells can be induced to express and secrete bioactive IL-12. The major stimulus to produce IL-12 in human B cells was CD40 ligation. In contrast, B cell receptor cross-linking did not induce IL-12. Expression of IL-12 after CD40 activation was restricted to CD38(-)IgD+/- non-germinal center (non-GC) B cells. CD40 ligation and interferon (IFN)-gamma exhibited synergistic effects on IL-12 production, whereas IL-10 abrogated and IL-4 significantly inhibited IL-12 production by these B cells. In contrast to IL-12, production of IL-6 is conversely regulated, leading to significant increase after CD40 ligation in the presence of the T helper type 2 (Th2) cytokine IL-4. Cord blood T cells skewed towards either a Th1 or a Th2 phenotype maintained their cytokine expression pattern when restimulated with allogeneic resting B cells. Blockade of CD40 and/or IL-12 during T-B interaction significantly reduced IFN-gamma production by the T cells. This suggests a model whereby B cells produce either IL-12 or IL-6 after contact with T cells previously differentiated towards Th1 or Th2. Furthermore, IL-12 and IL-6 might provide a positive feedback during cognate T-B interactions, thereby maintaining T cells' differentiation pattern during amplification of the immune response.
Detection of clonal tumor cells in leukemias and lymphomas by PCR in minimal residual disease (MRD) has been shown to be a valuable parameter for identifying patients who may require further treatment. Here we introduce the studies underway in our own and other institutions addressing the value of PCR technology in detecting residual CLL cells either in the autologous stem cell product or after induction of MRD in patients after autologous or allogeneic stem cell transplant. The PCR technology used for these questions and the results are discussed.
Increasing evidence suggests that chemotherapy does not cure the majority of patients with B cell non-Hodgkin's lymphoma (NHL). Therefore new treatment modalities are necessary. Immunotherapy of B cell lymphomas using monoclonal antibodies has been shown to be efficacious in murine model systems and also in patients. With the identification of tumor-specific antigens as targets for autologous T cells, T cell mediated immunity has been revived as an immunotherapeutic modality in B cell lymphomas. For B cell lymphomas the lymphoma-specific idiotype can be used as a tumor-specific antigen to stimulate T cells. Alternatively, the malignant B cells can be modified to become efficient antigen-presenting cells and present peptides from their own tumor-specific antigens to the autologous T cells. Here we discuss previous and currently explored immunotherapeutic strategies for B cell lymphoma.
The discovery of tumor-associated antigens (TAA) in certain human malignancies has prompted renewed efforts to develop antigen-specific immunotherapy of cancer. However, most TAA described thus far are expressed in one or a few tumor types, and, among patients with these types of tumors, TAA expression is not universal. Here, we characterize the telomerase catalytic subunit (hTERT) as a widely expressed TAA capable of triggering antitumor cytotoxic T lymphocyte (CTL) responses. More than 85% of human cancers exhibit strong telomerase activity, but normal adult tissues, with few exceptions, do not. In a human system, CD8+ CTL specific for an hTERT peptide and restricted to MHC HLA-A2 lysed hTERT+ tumors from multiple histologies. These findings identify hTERT as a potentially important and widely applicable target for anticancer immunotherapeutic strategies.