Jehad Charo

Small interfering RNA (siRNA) inhibits the expression of the Her2/neu gene, upregulates HLA class I and induces apoptosis of Her2/neu positive tumor cell lines.

Silencing of a specific mRNA using double stranded RNA oligonucleotides represents one of the newest technologies for suppressing a specific gene product. Small interfering RNA (siRNA) are 21 nucleotides long, double stranded RNA fragments that are identical in sequence to the target mRNA. We designed 3 such siRNA against the Her2/neu (HER2) gene. The HER2 gene is known to play an important role in the oncogenesis of several types of cancers, such as breast, ovarian, colon and gastric cancers. Introduction of the siRNA into HER2 positive tumor lines in vitro greatly reduced the cell surface expression of the HER2 protein. Concurrently, a range of effects on cell physiology, such as growth inhibition or apoptosis, was observed. The expression of HLA class I was observed to be upregulated when HER2 was silenced with siRNA. Treatment of SKBr3 and MCF7/HER2 tumor cell lines with the HER2 siRNA resulted in growth arrest of cells in the late G1/S‐phase. Our results suggest that siRNA may be an effective method of abrogating the effect of HER2 in tumorigenesis.

Identification of HER2/neu-derived peptide epitopes recognized by gastric cancer-specific cytotoxic T lymphocytes

We have derived HLA‐A2.1‐restricted, gastric cancer‐specific cytotoxic T lymphocyte (CTL) lines by repetitive in vitro stimulation of tumor‐associated lymphocytes (TAL) with autologous tumor cells. The HER2/neu specificity of these gastric cancer‐specific CTLs was demonstrated using HER2/neu‐transfected cell lines and HER2/neu‐expressing tumors, and with a set of HER2/neu‐derived peptide epitopes. Gastric cancer‐specific CTLs specifically lysed autologous and allogeneic HLA‐A2.1+, HER2/neu+ gastric cancer cells, HER2/neu‐transfected C1R/A2 cell lines (HLA‐A2.1+, HER2+) and HLA‐A2.1‐transfected SW626 tumor cell lines (HLA‐A2.1+, HER2+). This recognition could be inhibited by anti‐HLA‐A2 antibody or by cold target HER2/neu‐transfected C1R/A2 cells. Our results demonstrate that the HER2/neu‐encoded HLA‐A2.1‐associated epitopes recognized by CTLs are presented as naturally processed peptides on gastric cancer lines. Furthermore, 3 of 19 tested HER2/neu‐derived peptide epitopes [HER2(9106), HER2(9369), HER2(9689)], which all bound HLA‐A2.1 with high (IC50 < 50 nM) affinity, were able to sensitize HLA‐A2+ C1R/A2 cells to be recognized by the gastric cancer‐specific CTLs, demonstrating the immunodominance of these epitopes. In conclusion, our findings implicate HER2/neu‐derived epitopes as potential candidates for novel immunotherapy and vaccine strategies against gastric cancer. Int. J. Cancer 78:202–208, 1998.

Designer T cells by T cell receptor replacement

T cell receptor (TCR) gene transfer is a convenient method to produce antigen‐specific T cells for adoptive therapy. However, the expression of two TCR in T cells could impair their function or cause unwanted effects by mixed TCR heterodimers. With five different TCR and four different T cells, either mouse or human, we show that some TCR are strong – in terms of cell surface expression – and replace weak TCR on the cell surface, resulting in exchange of antigen specificity. Two strong TCR are co‐expressed. A mouse TCR replaces human TCR on human T cells. Even though it is still poorly understood why some TCRα/β combinations are preferentially expressed on T cells, our data suggest that, in the future, designer T cells with exclusive tumor reactivity can be generated by T cell engineering.

A safeguard eliminates T cell receptor gene-modified autoreactive T cells after adoptive transfer

By transfer of T cell receptor (TCR) genes, antigen specificity of T cells can be redirected to target any antigen. Adoptive transfer of TCR-redirected T cells into patients has shown promising results. However, this immunotherapy bears the risk of autoreactive side effects if the TCR recognizes antigens on self-tissue. Here, we introduce a safeguard based on a TCR-intrinsic depletion mechanism to eliminate autoreactive TCR-redirected T cells in vivo. By the introduction of a 10-aa tag of the human c-myc protein into murine (OT-I, P14) and human (gp100) TCR sequences, we were able to deplete T cells that were transduced with these myc-tagged TCRs with a tag-specific antibody in vitro. T cells transduced with the modified TCR maintained equal properties compared with cells transduced with the wild-type receptor concerning antigen binding and effector function. More importantly, therapeutic in vivo depletion of adoptively transferred T cells rescued mice showing severe signs of autoimmune insulitis from lethal diabetes. This safeguard allows termination of adoptive therapy in case of severe side effects.

Engineering antigen-specific primary human NK cells against HER-2 positive carcinomas

NK cells are promising effectors for tumor adoptive immunotherapy, particularly when considering the targeting of MHC class I low or negative tumors. Yet, NK cells cannot respond to many tumors, which is particularly the case for nonhematopoietic tumors such as carcinomas or melanoma even when these cells lose MHC class I surface expression. Therefore, we targeted primary human NK cells by gene transfer of an activating chimeric receptor specific for HER-2, which is frequently overexpressed on carcinomas. We found that these targeted NK cells were specifically activated upon recognition of all evaluated HER-2 positive tumor cells, including autologous targets, as indicated by high levels of cytokine secretion as well as degranulation. The magnitude of this specific response correlated with the level of HER-2 expression on the tumor cells. Finally, these receptor transduced NK cells, but not their mock transduced counterpart, efficiently eradicated tumor cells in RAG2 knockout mice as visualized by in vivo imaging. Taken together, these results indicate that the expression of this activating receptor overrides inhibitory signals in primary human NK cells and directs them specifically toward HER-2 expressing tumor cells both in vitro and in vivo.

Vaccination with a plasmid DNA encoding HER-2/neu together with low doses of GM-CSF and IL-2 in patients with metastatic breast carcinoma: a pilot clinical trial

BackgroundAdjuvant trastuzumab (Herceptin) treatment of breast cancer patients significantly improves their clinical outcome. Vaccination is an attractive alternative approach to provide HER-2/neu (Her2)-specific antibodies and may in addition concomitantly stimulate Her2-reactive T-cells. Here we report the first administration of a Her2-plasmid DNA (pDNA) vaccine in humans.Patients and MethodsThe vaccine, encoding a full-length signaling-deficient version of the oncogene Her2, was administered together with low doses of GM-CSF and IL-2 to patients with metastatic Her2-expressing breast carcinoma who were also treated with trastuzumab. Six of eight enrolled patients completed all three vaccine cycles. In the remaining two patients treatment was discontinued after one vaccine cycle due to rapid tumor progression or disease-related complications. The primary objective was the evaluation of safety and tolerability of the vaccine regimen. As a secondary objective, treatment-induced Her2-specific immunity was monitored by measuring antibody production as well as T-cell proliferation and cytokine production in response to Her2-derived antigens.ResultsNo clinical manifestations of acute toxicity, autoimmunity or cardiotoxicity were observed after administration of Her2-pDNA in combination with GM-CSF, IL-2 and trastuzumab. No specific T-cell proliferation following in vitro stimulation of freshly isolated PBMC with recombinant human Her2 protein was induced by the vaccination. Immediately after all three cycles of vaccination no or even decreased CD4+ T-cell responses towards Her2-derived peptide epitopes were observed, but a significant increase of MHC class II restricted T-cell responses to Her2 was detected at long term follow-up. Since concurrent trastuzumab therapy was permitted, λ-subclass specific ELISAs were performed to specifically measure endogenous antibody production without interference by trastuzumab. Her2-pDNA vaccination induced and boosted Her2-specific antibodies that could be detected for several years after the last vaccine administration in a subgroup of patients.ConclusionThis pilot clinical trial demonstrates that Her2-pDNA vaccination in conjunction with GM-CSF and IL-2 administration is safe, well tolerated and can induce long-lasting cellular and humoral immune responses against Her2 in patients with advanced breast cancer.Trial registrationThe trial registration number at the Swedish Medical Products Agency for this trial is Dnr151:785/2001.

A Synthetic Peptide Homologous to Functional Domain of Human IL-10 Down-Regulates Expression of MHC Class I and Transporter Associated with Antigen Processing 1/2 in Human Melanoma Cells

Tumor cells treated with IL-10 were shown to have decreased, but peptide-inducible expression of MHC class I, decreased sensitivity to MHC class I-restricted CTL, and increased NK sensitivity. These findings could be explained, at least partially, by a down-regulation of TAP1/TAP2 expression. In this study, IT9302, a nanomeric peptide (AYMTMKIRN), homologous to the C-terminal of the human IL-10 sequence, was demonstrated to mimic these previously described IL-10 effects on MHC class I-related molecules and functions. We observed a dose-dependent down-regulation of MHC class I at the cell surface of melanoma cells after 24-h treatment with IT9302. The IL-10 homologue peptide also caused a dose-dependent inhibition of the IFN-γ-mediated surface induction of MHC class I in a melanoma cell line. We demonstrated, using Western blot and flow cytometry, that IT9302 inhibits the expression of TAP1 and TAP2 proteins, but not MHC class I H chain or low molecular protein molecules. Finally, peptide-treated melanoma cells were shown to be more sensitive to lysis by NK cells in a dose-dependent way. Taken together, these results demonstrate that a small synthetic peptide derived from IL-10 can mimic the Ag presentation-related effects mediated by this cytokine in human melanomas and increase tumor sensitivity to NK cells, which can be relevant in the designing of future strategies for cancer immune therapy.

Protective Efficacy of a DNA Influenza Virus Vaccine Is Markedly Increased by the Coadministration of a Schiff Base-Forming Drug

ABSTRACT Effective vaccination against heterologous influenza virus infection remains elusive. Immunization with plasmid DNA (pDNA) expressing conserved genes from influenza virus is a promising approach to achieve cross-variant protection. However, despite having been described for more than a decade, pDNA vaccination still requires further optimization to be applied clinically as a standard vaccination approach. We have recently described a simple and efficient approach to enhance pDNA immunization, based on the use of tucaresol, a Schiff base-forming drug. In this report we have tested the ability of this drug to increase the protection conferred by pDNA vaccination against influenza virus infection. Our results demonstrate that a significant protection was achieved in two strains of mice by using the combination of pDNA and tucaresol. This protection was associated with an elevated humoral and cellular response and a switch in the type of the T helper cell (Th) immune response from type 2 to type 1. This vaccine combination represents a promising strategy for designing a clinical study for the protection from influenza and similar infections.

Immunization with heat shock protein 70 from methylcholanthrene-induced sarcomas induces tumor protection correlating with in vitro T cell responses

Abstract. The cytosolic members of the heat shock protein 70 (hsp70) family have in recent years been shown to elicit protective immunity mediated via binding to antigenic peptides in tumor and viral animal models. In this study we have used the methylcholanthrene (MC)-induced sarcomas MC57S and MC57X, previously shown to express individually distinct MHC-I associated peptides recognized by tumor necrosis factor-alpha (TNF-α) producing CD8+ T cells. With hsp70 purified from tumor or liver tissue, we were able to confirm that tumor-derived hsp70 elicited in vivo protection against a challenge with the same tumor as that used for hsp70 isolation. We also observed that immunizing with hsp70 isolated from tumor tissue resulted in a significantly better protection than immunizing with hsp70 isolated from the liver tissue of healthy mice. In two out of three experiments however, immunization with liver-derived hsp70 as well as tumor-derived hsp70 resulted in a significantly delayed tumor outgrowth as compared to saline-injected controls. In vitro, T cell lines from mice immunized with tumor-derived hsp70 could recognize tumor cells from the same MC57 tumor as that used for the hsp70 purification, resulting in TNF-α production. In sera from hsp70-immunized mice we observed undetectable or only very low levels of anti-hsp70 antibodies, suggesting that it is possible to repeatedly immunize the mice with no significant interference from neutralizing antibodies. We therefore conclude that hsp70 immunization can lead to protection against the tumor it was purified from, with a low risk of eliciting neutralizing antibodies that could affect further immunizations.

DNA Immunization of HLA Transgenic Mice with a Plasmid Expressing Mycobacterial Heat Shock Protein 65 Results in HLA Class I- and II-Restricted T Cell Responses That Can Be Augmented by Cytokines

Infection with Mycobacterium tuberculosis (MTB) remains a major cause of morbidity and mortality world-wide. An effective vaccination strategy is the immunization with plasmid DNA (pDNA), expressing an antigen (Ag) from a pathogen in vivo, which results in specific immune response against the encoded protein as well as the pathogen itself or cells infected with it. To test the ability to induce HLA-restricted T cell immune response against a mycobacterial antigen in humans by pDNA vaccination, we have used transgenic mice that express HLA class I (A*0201/Kb) or HLA class II (DRB1*0301) molecules. pDNA immunization with mycobacterial heat shock protein 65 (Mhsp65)-expressing plasmid (P3M.65) resulted in HLA-II-restricted, Ag-specific T cell-mediated immune responses characterized by proliferation and cytokine production. These T cell responses could be further augmented by the coinjection of P3M.65 and plasmid expressing murine GM-CSF. Furthermore, coimmunizing HLA-I transgenic mice with P3M.65 and a plasmid expressing murine IFN-gamma induced a specific cytotoxic T lymphocyte response restricted by HLA-A2. These results represent the first evidence of a concomitant in vivo induction of HLA class I- as well as class II-restricted T cell responses by pDNA immunization, which is induced or augmented by the codelivery of cytokine-expressing plasmids, supporting its potential use in clinical trials.