Esther Tzehoval

Analysis of endogenous peptides bound by soluble MHC class I molecules: a novel approach for identifying tumor‐specific antigens

The Human MHC Project aims at comprehensive cataloging of peptides presented within the context of different human leukocyte antigens (HLA) expressed by cells of various tissue origins, both in health and in disease. Of major interest are peptides presented on cancer cells, which include peptides derived from tumor antigens that are of interest for immunotherapy. Here, HLA‐restricted tumor‐specific antigens were identified by transfecting human breast, ovarian and prostate tumor cell lines with truncated genes of HLA‐A2 and HLA‐B7. Soluble HLA secreted by these cell lines were purified by affinity chromatography and analyzed by nano‐capillary electrospray ionization‐tandem mass spectrometry. Typically, a large peptide pool was recovered and sequenced including peptides derived from MAGE‐B2 and mucin and other new tumor‐derived antigens that may serve as potential candidates for immunotherapy.

Combined Dendritic Cell Cryotherapy of Tumor Induces Systemic Antimetastatic Immunity

Purpose: Cryotherapy of localized prostate, renal, and hepatic primary tumors and metastases is considered a minimally invasive treatment demonstrating a low complication rate in comparison with conventional surgery. The main drawback of cryotherapy is that it has no systemic effect on distant metastases. We investigated whether intratumoral injections of dendritic cells following cryotherapy of local tumors (cryoimmunotherapy) provides an improved approach to cancer treatment, combining local tumor destruction and systemic anticancer immunity. Experimental Designs: The 3LL murine Lewis lung carcinoma clone D122 and the ovalbumin-transfected B16 melanoma clone MO5 served as models for spontaneous metastasis. The antimetastatic effect of cryoimmunotherapy was assessed in the lung carcinoma model by monitoring mouse survival, lung weight, and induction of tumor-specific CTLs. The mechanism of cryoimmunotherapy was elucidated in the melanoma model using adoptive transfer of T cell receptor transgenic OT-I CTLs into the tumor-bearing mice, and analysis of Th1/Th2 responses by intracellular cytokine staining in CD4 and CD8 cells. Results: Cryoimmunotherapy caused robust and tumor-specific CTL responses, increased Th1 responses, significantly prolonged survival and dramatically reduced lung metastasis. Although intratumor administration of dendritic cells alone increased the proliferation rate of CD8 cells, only cryoimmunotherapy resulted in the generation of effector memory cells. Furthermore, cryoimmunotherapyprotected mice that had survived primary MO5 tumors from rechallenge with parental tumors. Conclusions: These results present cryoimmunotherapy as a novel approach for systemic treatment of cancer. We envisage that cryotherapy of tumors combined with subsequent in situ immunotherapy by autologous unmodified immature dendritic cells can be applied in practice.

Human CTL Epitopes Prostatic Acid Phosphatase-3 and Six-Transmembrane Epithelial Antigen of Prostate-3 as Candidates for Prostate Cancer Immunotherapy

Specific immunotherapy of prostate cancer may be an alternative or be complementary to other approaches for treatment of recurrent or metastasized disease. This study aims at identifying and characterizing prostate cancer-associated peptides capable of eliciting specific CTL responses in vivo. Evaluation of peptide-induced CTL activity in vitro was done following immunization of HLA-A2 transgenic (HHD) mice. An in vivo tumor rejection was tested by adoptive transfer of HHD immune lymphocytes to nude mice bearing human tumors. To confirm the existence of peptide-specific CTL precursors in human, lymphocytes from healthy and prostate cancer individuals were stimulated in vitro in the presence of these peptides and CTL activities were assayed. Two novel immunogenic peptides derived from overexpressed prostate antigens, prostatic acid phosphatase (PAP) and six-transmembrane epithelial antigen of prostate (STEAP), were identified; these peptides were designated PAP-3 and STEAP-3. Peptide-specific CTLs lysed HLA-A2.1+ LNCaP cells and inhibited tumor growth on adoptive immunotherapy. Furthermore, peptide-primed human lymphocytes derived from healthy and prostate cancer individuals lysed peptide-pulsed T2 cells and HLA-A2.1+ LNCaP cells. Based on the results presented herein, PAP-3 and STEAP-3 are naturally processed CTL epitopes possessing anti-prostate cancer reactivity in vivo and therefore may constitute vaccine candidates to be investigated in clinical trials.

Novel breast‐tumor‐associated MUC1‐derived peptides: Characterization in Db−/− × β2 microglobulin (β2m) null mice transgenic for a chimeric HLA‐A2.1/Db‐β2 microglobulin single chain

The MUC1 protein was found to be up‐regulated in a spectrum of malignant tumors. T‐cell responses to the MUC1 extracellular tandem repeat array (TRA) were observed in murine models as well as in breast‐carcinoma patients. In the present study, we evaluated the anti‐tumor potential of HLA‐A2.1‐motif‐selected peptides from non‐TRA domains of the molecule. Peptide immunogenicity was examined in the Db−/− × β2 microglobulin (β2m) null mice transgenic for a modified HLA‐A2.1/Db‐β2 microglobulin single chain (HHD mice). Our results show the existence of 3 novel HLA‐A2.1‐restricted MUC1‐derived cytotoxic T‐lymphocyte (CTL) epitopes. These peptides are processed and presented by the HHD‐transfected breast‐tumor cell line MDA‐MB‐157. Moreover, CTL induced by these 3 peptides show higher lysis of target cells pulsed with breast‐carcinoma‐derived peptides than of targets pulsed with normal breast‐tissue‐derived peptides. These data suggest an important role for non‐TRA MUC1‐derived peptides as inducers of a MHC‐restricted CTL reaction to a breast‐carcinoma cell line and patient‐derived tumor extracts. Int. J. Cancer 85:391–397, 2000. ©2000 Wiley‐Liss, Inc.

Thymosins α1 and β4 potentiate the antigen-presenting capacity of macrophages

Abstract The immunomodulatory effects of thymosins on the Ag-presenting capacity of macrophages were investigated. Using an in vitro antigen (Ag)-specific macrophage-dependent T-cell proliferation system, we found that both thymosin alpha 1 (T α 1 ) and thymosin beta 4 (T β 4 ) augment the Ag-presenting capacity of macrophages. Macrophage monolayers were pulsed with keyhole limpet hemocyanin (KLH) in the absence or presence of thymosins, washed and overlaid with spleen cells. Splenocytes were collected, mitomycin C-treated and injected into syngeneic mice. Draining lymph mode cells were tested for Ag-specific response by measuring proliferation, interleukin 2 (IL-2) secretion and expression of IL-2 receptors (IL-2R) on their cell surface. We found that the presence of thymosins during the pulsing of macrophages with KLH led to significantly enhanced lymph node cell proliferation when challenged with KLH. No proliferation was observed in response to bovine serum albumin (BSA). The augmentation in the proliferative responses to KLH was correlated to increased IL-2 production and IL-2R expression. The concentrations of T α 1 and T β 4 required for amplification were 10 −8 to 10 −10 M, well within the physiological range of activity of most peptide hormones. The observed enhancement of IL-2 secretion was not accompanied by interleukin 4 (IL-4) production. This study is the first to demonstrate that thymic hormones have the ability to increase the efficiency of antigen presentation by macrophages. The results suggest that an initial step in the regulation of the immune function by T α 1 and T β 4 may involve activation of the macrophages at the time of antigen presentation.

Characterization of novel breast carcinoma–associated BA46-derived peptides in HLA-A2.1/Db-β2mtransgenic mice

The human milk fat globule membrane protein BA46 (lactadherin) is highly overexpressed in human breast tumors, making it a potential target for tumor immunotherapy. We have identified BA46-derived peptides that contain the motif recognized by the MHC class I molecule HLA-A2.1 and that are processed and presented by human breast carcinoma cells. In mice lacking normal class I molecules but expressing an HLA-A2.1/D(b)-beta2 microglobulin single chain (HHD mice), three peptides elicited specific CTL activity. Two of these peptides also stimulated cytotoxic activity in peripheral blood lymphocytes from HLA-A2.1-positive breast carcinoma patients. Adoptive transfer of HHD-derived bulk CTLs to nude mice bearing human breast carcinoma transplants reduced tumor growth. These peptides therefore represent naturally processed BA46-derived CTL epitopes that can be used in peptide-based antitumor vaccines.

O-glycosylated versus non-glycosylated MUC1-derived peptides as potential targets for cytotoxic immunotherapy of carcinoma

Due to the fact that many cellular proteins are extensively glycosylated, processing and presentation mechanisms are expected to produce a pool of major histocompatibility complex (MHC) class I‐bound protein‐derived peptides, part of which retain sugar moieties. The immunogenic properties of the presented glycosylated peptides in comparison to their non‐glycosylated counterparts have not been determined clearly. We assessed the cellular immunogenicity of MUC1 (mucin)‐derived peptides O‐glycosylated with a Tn epitope (GalNAc) using HLA‐A*0201 single chain (HHD)‐transfected cell lines and transgenic mice. For part of the compounds Tn moiety did not interfere with the HLA‐A*0201 binding. Moreover, part of the glycopeptides elicited effective cytotoxic responses, indicating recognition of the glycopeptide‐HLA‐A*0201 complex by the T cell receptor (TCR) and subsequent cytotoxic T lymphocyte (CTL) activation. The CTLs exhibited a substantial degree of cross‐reactivity against target cells loaded with glycosylated and non‐glycosylated forms of the same peptide. The studied (glyco)peptides showed cellular immunogenicity in both MUC1‐HHD and HHD mice and induced effective lysis of (glyco)peptide‐loaded target cells in CTL assays. However, the elicited CTLs did not induce selective lysis of human MUC1‐expressing murine cell lines. Moreover, immunization with (glyco)peptide‐loaded dendritic cells (DCs) did not induce significant immunotherapeutic effects. We conclude that Tn glycosylated MUC1‐derived peptides can be presented by MHC class I molecules, and may be recognized by specific TCR molecules resulting in cytotoxic immune responses. However, the studied glycopeptides did not offer significant benefit as targets for cytotoxic immune response due apparently to (a) cross‐reactivity of the elicited CTLs against the glycosylated and non‐glycosylated forms of the same peptide and (b) low abundance of glycopeptides on tumour target cells.

Immunotherapy Via Gene Therapy: Comparison of the Effects of Tumor Cells Transduced with the Interleukin-2, Interleukin-6, or Interferon-γ Genes

We have comparatively analyzed the immune mechanisms induced by and the immunotherapeutic potentials of a highly metastatic clone of the Lewis lung carcinoma, D122, transduced with the interleukin-2 (IL-2), IL-6, or interferon-gamma (IFN-gamma) genes. All of the D122 cytokine gene-transduced cells induced antitumor CD8+ cytotoxic T lymphocytes (CTLs), as can be judged from in vivo depletion of CD8+ cells and in vitro CTL assays. In vivo depletion of CD4+ cells did not affect the malignant phenotypes of the different D122 gene-modified cells, but in vivo depletion of natural killer (NK) cells resulted in increased malignancy of both D122 cells and D122 gene-modified cells. In accordance with the effects of in vivo NK depletion, D122 as well as D122 derivative cells were sensitive to lysis by polyinosinic-polycytidylic acid (poly I:C)-induced activity. We discuss the immune responses generated by the different D122 gene-modified cells in view of their in vivo behavior in syngeneic and nude mice. We also performed comparative analysis of the capacity of vaccinations with irradiated D122 gene-modified cells to cure established micrometastases of parental D122 cells in tumor-operated mice. Vaccinations with D122-IL-2 or -IL-6 secretors did not generate a significant effect. Also, vaccinations with D122-IFN-gamma cells, which showed increased major histocompatibility complex class I expression but did not secrete detectable levels of IFN-gamma, did not cure established micrometastases. Only vaccination with D122-IFN-gamma high secretors efficiently cured postoperated mice carrying established micrometastases. We discuss the relevance of these results to the application of immunotherapy via cytokine gene therapy of human malignancy.

Induction of Antitumor Immunity with Modified Autologous Cells Expressing Membrane-Bound Murine Cytokines

Development of cytokine gene-modified autologous tumor vaccines must take into account the strictly paracrine physiology of cytokines whose expression at the tumor microenvironment is important for the successful induction of tumor-specific immunity. In this study, we investigated the efficacy of a tumor vaccine composed of inactivated autologous cells transfected with two plasmid vectors encoding a mutant membrane-bound murine granulocyte-macrophage colony-stimulating factor (MuGM-CSF) and murine interferon-gamma (MuIFN-gamma). Expression of both cytokines as cell surface ligands on the highly metastatic D122 clone of Lewis lung carcinoma led to abrogation of their tumorigenicity and metastatic phenotype. More importantly, vaccination with irradiated tumor cells expressing the membrane-bound GM-CSF and IFN-gamma induced a cytotoxic T lymphocyte (CTL) response that protected syngeneic mice against a subsequent challenge with D122 cells as a primary tumor in preimmunized mice as well as against lung metastasis developing after surgical removal of the primary tumor in naive mice. Autologous cells expressing the membrane-bound GM-CSF and IFN-gamma exhibited comparable efficacy as an antimetastatic vaccine to a vaccine composed of transfectants expressing wild-type secreted cytokine molecules. These results indicate that membrane-bound cytokines can cause enhanced immunogenicity when transfected into tumor cells for the induction of antitumor immunity.

The human 1-8D gene (IFITM2) is a novel p53 independent pro-apoptotic gene

The human 1‐8 interferon inducible gene family consists of at least 3 functional genes; 9‐27, 1‐8D and 1‐8U, which are all linked on an 18‐kb fragment of chromosome 11 and are highly homologous. It has recently been shown by us and others that the 1‐8D gene is overexpressed in colon carcinoma. Here, we show, by sequence comparison of the 1‐8D in pairs of tumor/normal colon tissues, the existence of 6 different alleles, containing single‐nucleotide polymorphisms with no mutations. Transformation assays revealed a possible role for the 1‐8D gene as a transformation inhibitor. Further, transient expression of the human 1‐8D gene in multiple mammalian cell lines showed accumulation of cells in the G1 phase followed by elevation in the subG1 phase. SubG1 elevation was confirmed as apoptosis by Annexin‐V binding assays and transferase‐mediated dUTP nick end labeling assays. Moreover, knock‐down of 1‐8D provided partial protection from Etoposide and UV‐induced apoptosis. The induction of apoptosis by 1‐8D is dependent on caspase activities but not on p53 expression. Although 1‐8D induces apoptosis independently of p53, p53 expression downregulates 1‐8D protein expression. Our data suggest a role for the 1‐8D gene as a novel pro‐apoptotic gene that will provide new insights into the regulated cellular pathways to death.