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1.
J Immunol ; 174(12): 7853-8, 2005 Jun 15.
Article in English | MEDLINE | ID: mdl-15944290

ABSTRACT

T cells require both primary and costimulatory signals for optimal activation. The primary Ag-specific signal is delivered by engagement of the TCR. The second Ag-independent costimulatory signal is mediated by engagement of the T cell surface costimulatory molecule CD28 with its target cell ligand B7. However, many tumor cells do not express these costimulatory molecules. We previously constructed phage display derived F(AB), G8, and Hyb3, Ab-based receptors with identical specificity but distinct affinities for HLA-A1/MAGE-A1, i.e., "TCR-like" specificity. These chimeric receptors comprised the FcepsilonRI-gamma signaling element. We analyzed whether linking the CD28 costimulation structure to it (gamma + CD28) could affect the levels of MHC-restricted cytolysis and/or cytokine production. Human scFv-G8(POS) T lymphocytes comprising the gamma + CD28 vs the gamma signaling element alone produced substantially more IL-2, TNF-alpha, and IFN-gamma in response to HLA-A1/MAGE-A1(POS) melanoma cells. Also a drastic increase in cytolytic capacity of scFv-G8(POS) T cells, equipped with gamma + CD28 vs the gamma-chain alone was observed.


Subject(s)
Binding Sites, Antibody , CD28 Antigens/physiology , Cytokines/biosynthesis , Cytotoxicity, Immunologic , Epitopes, T-Lymphocyte/toxicity , HLA-A1 Antigen/immunology , Lymphocyte Activation , T-Lymphocytes, Cytotoxic/immunology , Adjuvants, Immunologic/genetics , Adjuvants, Immunologic/metabolism , Adjuvants, Immunologic/toxicity , Antigens, Neoplasm , Binding Sites, Antibody/genetics , CD28 Antigens/genetics , CD28 Antigens/immunology , Cell Line, Tumor , Cytotoxicity, Immunologic/genetics , Epitopes, T-Lymphocyte/genetics , Epitopes, T-Lymphocyte/immunology , HLA-A1 Antigen/genetics , HLA-A1 Antigen/metabolism , Humans , Immunoglobulin Fab Fragments/genetics , Immunoglobulin Fab Fragments/metabolism , Immunoglobulin Fab Fragments/toxicity , Immunoglobulin Variable Region/genetics , Immunoglobulin Variable Region/metabolism , K562 Cells , Lymphocyte Activation/genetics , Melanoma/immunology , Melanoma/pathology , Melanoma-Specific Antigens , Neoplasm Proteins/genetics , Neoplasm Proteins/immunology , Neoplasm Proteins/metabolism , Protein Structure, Tertiary/genetics , Receptors, Antigen, T-Cell/biosynthesis , Receptors, Antigen, T-Cell/genetics , Receptors, Antigen, T-Cell/metabolism , Receptors, IgE/genetics , Receptors, IgE/metabolism , Receptors, IgE/physiology , Signal Transduction/genetics , Signal Transduction/immunology , T-Lymphocytes, Cytotoxic/metabolism , Transduction, Genetic/methods
2.
J Immunol Methods ; 280(1-2): 13-24, 2003 Sep.
Article in English | MEDLINE | ID: mdl-12972184

ABSTRACT

Tumor-specific receptors may provide effective tools for anti-tumor immunogene therapy. However, the functional analysis of primary human T cells engrafted with tumor-specific receptors is laborious and emphasizes the need for a fast and sensitive method to validate such receptors. To this end, we have set up a Jurkat T cell-based reporter gene assay, and tested receptors with various formats, i.e., receptors based on either a monoclonal antibody (mAb), a full-length T cell receptor (fl-TCR)alphabeta or a chimeric (ch-)TCRalphabeta, and various antigen specificities for their ability to mediate tumor-specific activation of nuclear factor of activated T cells (NFAT). The mAb-based receptor specifically mediates NFAT activation after stimulation with tumor antigen-positive target cells. The observed receptor-mediated NFAT responses were validated by the use of ligand- and receptor-specific mAbs, as well as cyclosporin A (CsA) and a dominant negative mutant of NFAT. Furthermore, anti-TCR mAbs, peptide-loaded tumor cells and antigen-positive tumor cells all resulted in specific NFAT activation in TCR/CD8 co-transduced Jurkat T cells, irrespective of the TCR format used. Importantly, receptor-mediated NFAT responses parallel tumor-specific cytolysis and TNFalpha production of receptor-transduced primary human T lymphocytes. In fact, inhibition of NFAT activation compromises the immune responses of primary human T lymphocytes, pointing to a central involvement of NFAT in anti-tumor T cell responses. Taken together, receptor-mediated activation of NFAT constitutes a representative measure of anti-tumor T cell responses, and the genetically modified Jurkat T cells provide a flexible and sensitive tool with which to select rapidly tumor-specific (chimeric) receptors for immunogene therapy.


Subject(s)
DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Genetic Techniques , Neoplasms/genetics , Neoplasms/metabolism , Nuclear Proteins , Receptors, Antigen, T-Cell, alpha-beta/genetics , Receptors, Antigen, T-Cell, alpha-beta/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/metabolism , Cytotoxicity, Immunologic , Genes, Reporter , Genetic Techniques/statistics & numerical data , Humans , Jurkat Cells , NFATC Transcription Factors , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sensitivity and Specificity , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Transduction, Genetic , Tumor Cells, Cultured , Tumor Necrosis Factor-alpha/biosynthesis
3.
Hum Immunol ; 64(1): 56-68, 2003 Jan.
Article in English | MEDLINE | ID: mdl-12507815

ABSTRACT

The ultimate goal of immunotherapy of cancer is to make use of the immune system of patients to eliminate malignant cells. Research has mainly focused on the generation of effective antigen specific T-cell responses because of the general belief that T-cell immunity is essential in controlling tumor growth and protection against viral infections. However, the isolation of antigen specific T cells for therapeutic application is a laborious task and it is often impossible to derive autologous tumor specific T cells to be used for adoptive immunotherapy. Therefore, strategies were developed to genetically transfer tumor specific immune receptors into patients T cells. To this end, chimeric receptors were constructed that comprise antibody fragments specific for tumor associated antigens, linked to genes encoding signaling domains of the T-cell receptor (TCR) or Fc receptor. T cells expressing such chimeric antibody receptors recapitulate the immune specific responses mediated by the introduced receptor. Recently, we introduced chimeric TCR genes into primary human T lymphocytes and demonstrated that these T cell transductants acquired the exquisite major histocompatibility complex (MHC) restricted tumor specificity dictated by the introduced TCR. Importantly, the introduction of chimeric TCR bypasses problems associated with the introduction of nonmodified TCR genes, such as pairing of introduced TCR chains with endogenous TCR chains and unstable TCRalpha expression. A novel strategy which is completely independent of available tumor specific T-cell clones for cloning of the TCR genes was recently used to transfer MHC restricted tumor specificity to T cells. Human "TCR-like" Fab fragments obtained by in vitro selection of Fab phages on soluble peptide/MHC complexes were functionally expressed on human T lymphocytes, resulting in MHC restricted, tumor specific lysis and cytokine production. In addition, affinity maturation of the antibody fragment on Fab phages allows improvement of the tumor cell killing capacity of chimeric Fab receptor engrafted T cells. Developments in retroviral transfer technology now enables the generation of large numbers of antigen specific T cells that can be used for adoptive transfer to cancer patients. In this article we summarize the developments in adoptive T cell immunogenetic therapy and discuss the limitations and perspectives to improve this technology toward clinical application.


Subject(s)
Antibodies, Neoplasm/metabolism , Genetic Engineering , Immunotherapy , Neoplasms/therapy , T-Lymphocytes, Cytotoxic/immunology , Humans , Neoplasms/immunology
4.
Clin Cancer Res ; 9(1): 76-83, 2003 Jan.
Article in English | MEDLINE | ID: mdl-12538454

ABSTRACT

PURPOSE: Repeated administrations of recombinant human interleukin-12 (rHuIL-12) to cancer patients are characterized by a reduction of side effects during treatment. Induction of IFN-gamma, considered a key mediator of antitumor effects of IL-12, is known to decline on repeated administrations. We studied whether other immunological effects of rHuIL-12 are tapered in the course of treatment. EXPERIMENTAL DESIGN: In a Phase I study of 26 patients with advanced renal cell cancer, rHuIL-12 was administered s.c. on day 1, followed by 7 days rest and six injections administered over a 2-week time period. Plasma concentrations of various cytokines were monitored, as well as absolute counts of circulating leukocyte and lymphocyte subsets. RESULTS: The first injection of IL-12 was accompanied by rapid, transient, and dose-dependent increments of plasma levels IFN-gamma, tumor necrosis factor-alpha, IL-10, IL-6, IL-8, but not IL-4, as well as rapid, transient, and dose-dependent reductions of lymphocyte, monocyte, and neutrophil counts. The major lymphocyte subsets, i.e., CD4+ and CD8+ T cells, B cells, and natural killer cells, followed this pattern. On repeated rHuIL-12 injections, IL-10 concentrations increased further, whereas the transient increments of IFN-gamma, tumor necrosis factor-alpha, IL-6, and IL-8 concentrations, as well as the fluctuations of the leukocyte subset counts, were tapered. Dose escalation of IL-12 within clinically tolerable margins did not reduce the decline of these immunological effects. CONCLUSIONS: Induction of pro-inflammatory cytokines and associated fluctuations in leukocyte subset counts decrease on repeated administrations of rHuIL-12. The steady increment of IL-10 plasma levels may mediate the observed down-regulation of clinical and immunological effects.


Subject(s)
Carcinoma, Renal Cell/drug therapy , Interleukin-12/administration & dosage , Adult , Aged , B-Lymphocytes/metabolism , CD4-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/metabolism , Carcinoma, Renal Cell/blood , Cytokines/metabolism , Humans , Immunophenotyping , Interferon-gamma/blood , Interleukin-10/blood , Interleukin-6/blood , Interleukin-8/blood , Killer Cells, Natural/metabolism , Middle Aged , Recombinant Proteins/pharmacology , Time Factors , Tumor Necrosis Factor-alpha/biosynthesis
7.
J Immunol ; 169(2): 1110-8, 2002 Jul 15.
Article in English | MEDLINE | ID: mdl-12097420

ABSTRACT

The permanent genetic programming via gene transfer of autologous T cells with cell surface receptors directed toward tumor-related Ags holds great promise for the development of more-specific tumor therapies. In this study we have explored the use of Abs directed to MHC-peptide complexes (or TCR-like Abs) to engraft CTLs with exquisite specificity for cancer cells. First, we affinity matured in vitro a previously selected TCR-like Ab, Fab-G8, which is highly specific for the peptide melanoma-associated Ag-A1 presented by the HLA-A1 molecule. A combination of L chain shuffling, H chain-targeted mutagenesis, and in vitro selection of phage display libraries yielded a Fab-G8 Ab derivative, Fab-Hyb3, with an 18-fold improved affinity yet identical peptide fine specificity. Fab-G8 and Fab-Hyb3 were expressed on primary human T lymphocytes as cell surface-anchored Fab, demonstrating that T cells expressing the high-affinity Fab-Hyb3 molecule eradicate tumor cells much more effectively. Furthermore, the gain in ligand-binding affinity resulted in a 2-log improvement in the detection of peptide/MHC complexes on melanoma-associated Ag-A1 peptide-loaded cells. In summary, an affinity-matured Ab specifically recognizing a cancer-related peptide/MHC complex was generated and used to improve the tumor cell killing capacity of human T cells. This strategy, based on engraftment of T cells with in vitro engineered Abs, is an attractive alternative to the laborious, and in many cases unsuccessful, generation of highly potent tumor-specific T lymphocytes.


Subject(s)
Antibody Affinity , Cytotoxicity, Immunologic , Immunoglobulin Fab Fragments/physiology , Receptors, Antigen, T-Cell/physiology , T-Lymphocytes, Cytotoxic/immunology , T-Lymphocytes, Cytotoxic/metabolism , Antibody Affinity/genetics , Antibody Specificity , Antigen Presentation/genetics , Antigens, Neoplasm , Cloning, Molecular , Cytotoxicity, Immunologic/genetics , Gene Targeting , Genetic Vectors/chemical synthesis , HLA-A1 Antigen/immunology , Humans , Immunodominant Epitopes/immunology , Immunodominant Epitopes/metabolism , Immunoglobulin Fab Fragments/biosynthesis , Immunoglobulin Fab Fragments/genetics , Immunoglobulin Fab Fragments/metabolism , Immunoglobulin Heavy Chains/biosynthesis , Immunoglobulin Heavy Chains/chemistry , Immunoglobulin Heavy Chains/metabolism , Immunoglobulin Light Chains/biosynthesis , Immunoglobulin Light Chains/chemistry , Immunoglobulin Light Chains/metabolism , Melanoma-Specific Antigens , Neoplasm Proteins/immunology , Protein Binding/genetics , Protein Binding/immunology , Receptors, Antigen, T-Cell/biosynthesis , Receptors, Antigen, T-Cell/genetics , Receptors, Antigen, T-Cell/metabolism , Receptors, Antigen, T-Cell, gamma-delta/biosynthesis , Receptors, Antigen, T-Cell, gamma-delta/genetics , Receptors, Antigen, T-Cell, gamma-delta/metabolism
8.
Cancer Gene Ther ; 9(7): 613-23, 2002 Jul.
Article in English | MEDLINE | ID: mdl-12082462

ABSTRACT

In preparation of a clinical phase I/II study in renal cell carcinoma (RCC) patients, we developed a clinically applicable protocol that meets good clinical practice (GCP) criteria regarding the gene transduction and expansion of primary human T lymphocytes. We previously designed a transgene that encodes a single chain (sc) FvG250 antibody chimeric receptor (ch-Rec), specific for a RCC tumor-associated antigen (TAA), and that genetically programs human T lymphocytes with RCC immune specificity. Here we describe the conditions for activation, gene transduction, and proliferation for primary human T lymphocytes to yield: (a) optimal functional expression of the transgene; (b) ch-Rec-mediated cytokine production, and (c) cytolysis of G250-TAA(POS) RCC by the T-lymphocyte transductants. Moreover, these parameters were tested at clinical scale, i.e., yielding up to 5-10 x 10(9) T-cell transductants, defined as the treatment dose according to our clinical protocol. The following parameters were, for the first time, tested in an interactive way: (1) media compositions for production of virus by the stable PG13 packaging cell; (2) T-lymphocyte activation conditions and reagents (anti-CD3 mAb; anti-CD3+anti-CD28 mAbs; and PHA); (3) kinetics of T-lymphocyte activation prior to gene transduction; (4) (i) T-lymphocyte density, and (ii) volume of virus-containing supernatant per surface unit during gene transduction; and (5) medium composition for T-lymphocyte maintenance (i) in-between gene transduction cycles, and (ii) during in vitro T-lymphocyte expansion. Critical to gene transduction of human T lymphocytes at clinical scale appeared to be the use of the fibronectin fragment CH-296 (Retronectin) as well as Lifecell) X-fold cell culture bags. In order to comply with GCP requirements, we used: (a) bovine serum-free human T-lymphocyte transduction system, i.e., media supplemented with autologous patients' plasma, and (b) a closed cell culture system for all lymphocyte processing. This clinical protocol routinely yields 30-65% scFvG250 ch-Rec(POS) T lymphocytes in both healthy donors and RCC patients.


Subject(s)
Gene Transfer Techniques , Genetic Therapy/methods , Immunotherapy/methods , Neoplasms/therapy , T-Lymphocytes/metabolism , Transduction, Genetic , Antibodies, Monoclonal , CD28 Antigens/genetics , CD3 Complex/genetics , CD4 Antigens/genetics , Carcinoma, Renal Cell/therapy , Cell Division , Culture Media, Serum-Free , Flow Cytometry , Humans , Kidney Neoplasms/therapy , Leukocytes, Mononuclear/cytology , Retroviridae/genetics , Spectrometry, Fluorescence , Time Factors , Transgenes , Tumor Cells, Cultured
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