Michael Papamichail - Academia.edu (original) (raw)

Papers by Michael Papamichail

British Journal of Cancer, 2003

HER-2/neu oncoprotein contains several major histocompatibility complex class I-restricted epitop... more HER-2/neu oncoprotein contains several major histocompatibility complex class I-restricted epitopes, which are recognised by cytotoxic T lymphocyte (CTL) on autologous tumours and therefore can be used in immune-based cancer therapies. Of these, the most extensively studied is HER-2(9 369). In the present report, we used dendritic cells pulsed with HER-2(9 369) to stimulate, in the presence of IL-7 and IL-12, the production of IFN-g by patients' CTL detected by the enzyme-linked immunosorbent spot-assay. Frequencies of peptide-specific precursors were estimated in HLA-A2, HLA-A3 and HLA-A26 patients with HER-2/neu-positive (þ) breast, ovarian, lung, colorectal and prostate cancers and healthy individuals. We found increased percentages of such precursors in HLA-A2 (25%) and HLA-A26 (30%) patients, which were significantly higher (60%) in HLA-A3 patients. Our results demonstrate for the first time that pre-existing immunity to HER-2(9 369) occurs in patients with colorectal, lung and prostate cancer. They also suggest that HER-2(9 369) can be recognised by CTL, besides HLA-A2, also in the context of HLA-A3 and HLA-A26, thus increasing the applicability of HER-2(9 369)-based vaccinations in a considerably broader patients' population.

Cancer Immunology, Immunotherapy, 2003

To broaden the applicability of adoptive cellular immunotherapy against HER-2/neu overexpressing ... more To broaden the applicability of adoptive cellular immunotherapy against HER-2/neu overexpressing human cancers, we constructed a chimeric scFv/c gene composed of the variable regions of a HER-2/neu specific monoclonal antibody (mAb) joined to the signaling c-chain of the Fc(e)RI receptor. The scFv(anti-HER-2/neu)/c chimeric gene was successfully expressed as functional surface receptor in the MD.45 cytolytic T-cell (CTL) hybridoma (MD.45-HER/c). Expression of the chimeric protein triggered IL-2 and IFN-c secretion in vitro upon encountering cell surface HER-2/neu and mediated non-major-histocompatibility-complex (MHC)-restricted HER-2/neu-specific target cell lysis. We also examined the in vivo activity of the MD.45-HER/c transduced cells. Severe combined immunodeficiency disease (SCID) mice that were given HER-2/neu positive (+) human tumor cell lines had significantly increased survival compared to mice treated with saline only, or with MD.45 cells transduced with a control anti-trinitrophenyl (anti-TNP) chimeric receptor gene (MD.45-TNP/c). These results demonstrate the feasibility of redirecting MD.45 CTL to react in vitro and in vivo with a variety of HER-2/neu + tumor cells by our gene transduction protocol. Moreover, they open the possibility of using the same chimeric gene for transducing primary lymphocytes and thus allowing adoptive immunotherapy against HER-2/neu + cancers. Keywords HER-2/neu AE Chimeric receptors AE Single chain Fv AE SCID mice AE Cancer immunotherapy

Cancer Immunology, …, 2010

HLA-mismatched natural killer (NK) cells have shown efficacy in acute myeloid leukemia, and their... more HLA-mismatched natural killer (NK) cells have shown efficacy in acute myeloid leukemia, and their adoptive transfer in patients with other malignancies has been proven safe. This phase I clinical trial was designed to evaluate safety (primary endpoint) and possible clinical efficacy (secondary endpoint) of repetitive administrations of allogeneic, in vitro activated and expanded NK cells along with chemotherapy in patients with advanced nonsmall cell lung cancer (NSCLC). Patients with unresectable, locally advanced/metastatic NSCLC receiving 1st/2nd line chemotherapy were eligible to receive 2-4 doses of activated NK cells from two relative donors. Donor's CD56 ? cells were cultured for 20-23 days with interleukin-15 (IL-15) and hydrocortisone (HC) and administered intravenously between chemotherapy cycles. Premedication with corticosteroids and/or H1 inhibitors was allowed. Sixteen patients (performance status 0-1) with adenocarcinoma (n = 13) or squamous cell carcinoma (n = 3) at stage IIIb (n = 5) or IV (n = 11) receiving 1st (n = 13) or 2nd (n = 3) line treatment were enrolled. Fifteen patients received 2-4 doses of allogeneic activated NK cells (0.2-29 9 10 6 /kg/dose, median 4.15 9 10 6 /kg/dose). No side effects (local or systemic) were observed. At a median 22-month follow-up (range, 16.5-26 months) 2 patients with partial response and 6 patients with disease stabilization were recorded. Median progression free survival and overall survival were 5.5 and 15 months, respectively. A 56% 1-year survival and a 19% 2-year survival were recorded. In conclusion, repetitive infusions of allogeneic, in vitro activated and expanded with IL-15/HC NK cells, in combination with chemotherapy are safe and potentially clinically effective.

Cancer Immunol Immunother, 1999

Anti-CD3 monoclonal antibody (mAb) activates in vitro peripheral blood mononuclear cells (PBMC) t... more Anti-CD3 monoclonal antibody (mAb) activates in vitro peripheral blood mononuclear cells (PBMC) to lyse a variety of tumor cell lines in a non-major histocompatibility-complex(MHC)-restricted manner [subsequently referred to as anti-CD3-activated killer (AAK) cytotoxicity]. Prothymosin a (ProTa) is a biological response modi®er that exerts its eects primarily on mononuclear cells, especially when these cells' eector functions are impaired. In this study, we report that ProTa enhances the AAK cytotoxicity in PBMC from healthy donors. This eect was more profound with cancer patients' PBMC, which were de®cient in their ability to respond with enhanced AAK cytotoxicity upon in vitro stimulation with anti-CD3. Thus, cancer patients' PBMC, activated with a combination of anti-CD3 and ProTa, exhibited increased AAK activity and eciently lysed both autologous tumor and Daudi targets. The ProTa eect on PBMC was demonstrated to involve stimulation of adhesion molecules (CD2, CD18, CD54, CD49f) and CD25 expression, up-regulation of perforin mRNA transcription, increased numbers of perforin-positive (+) cells and elevated production of interleukin-2 (IL-2), interleukin-1b (IL-1b) and tumor necrosis factor a (TNFa). Moreover, eector CD8 + and CD56 + cells pretreated with anti-CD3 and ProTa contained high cytoplasmic perforin levels and increased expression of IL-1b-and TNFa-speci®c receptors. The induction of autologous-tumor-reactive CD8 + and CD56 + lymphocytes in anti-CD3-activated PBMC by ProTa provides an alternative protocol aimed at the improvement of clinical results in cellular adoptive immunotherapy of cancer.

Haematologica, Apr 1, 2002

We demonstrate the efficacy of two cytokine-rich supernatants, one from peripheral blood cell cul... more We demonstrate the efficacy of two cytokine-rich supernatants, one from peripheral blood cell cultures stimulated with anti-CD3 and the other from monocyte-conditioned media, in enhancing the rapid (within 6 days) expansion and clonogenicity of CD34+, progenitor cells. We, thereby,increased the generation of mostly myeloid precursors able to support stem cell transplants.

Asco Meeting Abstracts, Sep 20, 2012

Cancer Immunol Immunother, 1995

We have recently reported that administration of ProT alpha to DBA/2 mice before the inoculation ... more We have recently reported that administration of ProT alpha to DBA/2 mice before the inoculation of syngeneic L1210 leukemic cells prolonged the survival of these animals by (a) inducing tumoricidal peritoneal macrophages, (b) enhancing natural killer (NK) and inducing lymphokine-activated killer (LAK) activities in splenocytes and (c) inducing the production of interleukin-2 and tumor necrosis factor alpha [Papanastasiou et al. (1992) Cancer Immunol Immunother 35:145; Baxevanis et al. (1994) Cancer Immunol Immunothera 38:281]. In this report we demonstrated that ProT alpha, when administered simultaneously with L1210 tumor cells, is capable of generating in DBA/2 animals tumor-specific CD8+ cytotoxic T lymphocytes (CTL). The ProT alpha-induced CD8+ CTL lysed their syngeneic L1210 targets in a major histocompatibility complex (MHC)-restricted fashion since monoclonal antibodies (mAb) against the H-2Kd allelic product could inhibit the cytotoxic response. Mice receiving only ProT alpha developed non-MHC-restricted cytotoxic activity (NK, and LAK activities) whereas those receiving ProT alpha and L1210 tumor cells developed both MHC-restricted (CTL) and non-MHC-restricted cytotoxic activities and survived longer. The ProT alpha-induced CD8+ CTL activity was regulated by ProT alpha-induced L1210-specific syngeneic CD4+ cells. This was shown in two different ways: first, CD8(+)-cell-mediated cytotoxic responses against L1210 targets were associated with L1210-specific and MHC-restricted proliferative responses of syngeneic CD4+ cells and, second, CD4+ cells from mice that had received both ProT alpha and L1210 tumor cells could enhance in vitro the otherwise weak, MHC-restricted and L1210-specific cytotoxicity of syngeneic CD8+ cells from mice that had received only L1210 cells. Our data suggest that ProT alpha is capable of inducing nonspecific, as well as tumor-specific CTL responses in vivo. This is of importance since ProT alpha may prove to be useful in clinical protocols aimed at cancer immunotherapy.

Asco Meeting Abstracts, Sep 10, 2014

Breast Cancer Research and Treatment, Aug 1, 2003

We describe, here, a rapid flow cytometry technique for the detection and quantification of estro... more We describe, here, a rapid flow cytometry technique for the detection and quantification of estrogen (ER) and progesterone (PgR) receptors in several human cell lines and in clinical samples obtained from breast cancer tumors. ER and PgR quantitation can be very useful in patients with breast cancer as their role in diagnosis and prognosis is well established. However ligand binding assays and immunohistochemical assays are difficult to measure heterogeneity in individual cells. On the other hand, flow cytometry is a convenient tool for quantification in individual cells. Flow cytometric results with breast cancer cell lines and clinical samples were compared to those obtained by quantitative biochemical ER and PgR performed by the standard dextran-coated charcoal biochemical assay. The latter assay is affected by the level of endogenous steroids. This is also the case in the routine measurement of ER/PgR in patient's tumor cells whereby estradiol molecules in patient's serum produced negative or low values in the biochemical assay. The mAbs used in our flow cytometric method bind to their specific ER or PgR independently of whether they are preoccupied by their ligands and they produce reliable results. With the use of beads calibrated in MESF (Molecules of Equivalent Soluble Fluorochrome) units, the ER and PgR can be measured on a per cell basis. The flow cytometric method showed a strong correlation with biochemical receptor assessments of either ERα (ERαDCC, r = 0.918, p = 0.073) or PgR (PgRDCC, r = 0.75, p = 0.001). This study demonstrates that ERα and PgR can be detected by flow cytometry on a per cell basis in intact cells, and can be quantitated reliably in terms of MESF without the limitations of competition with serum's estradiol molecules.

Evidence is presented which indicates the presence on murine lymphocytes of a membrane receptor f... more Evidence is presented which indicates the presence on murine lymphocytes of a membrane receptor for determinants on the hinge region of human IgG. These determinants are exposed following pepsin scission of IgG molecule, i.e. on the F(ab')2 fragment. The evidence for a hinge receptor derives, in vivo, from splenic localization of F(ab')2 in germinal centres and, in vitro, from immunofluorescent binding studies. The sequential immunofluorescent pattern for the uptake of human F(ab)2 fragments into murine spleen germinal centres was identical with that previously observed for heat-aggregated human IgG, but F(ab')2 fragments appeared to be retained in the germinal centres for a shorter time than aggregated IgG. Experiments with nude mice and T cell-deprived mice showed that the localization of F(ab')2 fragments does not require T cells. Competition experiments suggest that the receptor for F(ab')2 may bear little relation to the receptor for aggregated IgG. The relevance of such a lymphocyte membrane receptor to the immunopathology of rheumatoid arthritis is discussed in the light of previous findings that a proportion of the serum IgG of patients with rheumatoid arthritis has a structural anomaly compared with control IgG, characterized exposure of new determinants at the hinge region.

Journal of chemotherapy (Florence, Italy)

The effect of gamma-interferon (IFN-gamma) on the induction of interleukin-2 (IL-2) activated kil... more The effect of gamma-interferon (IFN-gamma) on the induction of interleukin-2 (IL-2) activated killer cell activity was studied: (I) in peripheral blood lymphocytes (LAK cells) from cancer patients and healthy donors, (II) in lymphocytes infiltrating solid tumors (TIL) from melanoma and breast cancer patients, and (III) in pleural effusion associated lymphocytes (EAL) from patients with lung adenocarcinoma. The coculture of LAK, TIL and pleural effusion mononuclear cells (MNC) with several doses of IFN-gamma (10, 50, 250, and 1250 U/ml) and a low dose of IL-2 (10 U/ml) for 5 days resulted in a synergistic effect on the cytotoxicity of these cells against several tumor cell lines. Furthermore there was a potentiation in the proliferation of MNC after a 5-day culture. The induction of lymphocyte cytotoxicity by a combination of IFN-gamma with low doses of IL-2 may be helpful in designing more effective cancer immunotherapeutic protocols with LAK, TIL or EAL.

The Journal of Immunology

ABSTRACT

Anticancer research

We recently demonstrated that supernatants from cultures of peripheral blood mononuclear cells (P... more We recently demonstrated that supernatants from cultures of peripheral blood mononuclear cells (PBMC) activated with anti-CD3-specific antibody (ACD3S) can induce, upon brief exposure, tumor-reactive lymphocytes in cancer patients. Here, we report that ACD3S can also induce rapid and stable maturation of dendritic cells (DC) which can be used as antigen presenting cells in in vitro protocols and for cancer immunotherapy in vivo. A short (4-day) priming of CD14+ monocytes with granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) followed by only a 24 hour-incubation in ACD3S, is sufficient to generate fully mature and stable DC. These DC (i) stimulated strong T cell proliferative responses in the mixed lymphocyte reaction, (ii) when pulsed with unfractionated peptides from autologous tumor membrane extracts activated CD4+ T cells which proliferated in response to the autologous tumor and CD8+ cytotoxic T cells (CTL) which specifically lyse autologous tum...

British journal of cancer, Jan 22, 2003

Chimeric receptors comprising of the T-cell receptor-zeta cytoplasmic signalling chain fused to a... more Chimeric receptors comprising of the T-cell receptor-zeta cytoplasmic signalling chain fused to an extracellular ligand-binding domain of a single-chain antibody (scFv) have served as effective tools for redirecting cytotoxic T lymphocytes (CTL) against tumour cells. In this report, we constructed a chimeric scFv/zeta gene composed of the variable regions of an HER-2/neu-specific monoclonal antibody (MAb) joined to the TCR-zeta chain. The scFv(anti-HER-2/neu)/zeta chimeric gene was successfully expressed as a functional surface receptor in the MD.45 CTL hybridoma (MD.45-HER/zeta). More importantly, the scFv(anti-HER-2/neu)/zeta receptor was functionally active, since it triggered cytokine secretion by the MD.45-HER/zeta cells upon recognition of HER-2/neu-positive (+) tumour cell lines, or primary tumour cells from patients with HER-2/neu(+) cancers. The MD.45-HER/zeta-transduced cells also lysed HER-2/neu(+) target cells in vitro with high specificity. We tested the antitumour effi...

Cancer immunology, immunotherapy : CII, 2002

We have developed culture conditions for the efficient expansion of cytotoxic effector cells from... more We have developed culture conditions for the efficient expansion of cytotoxic effector cells from peripheral blood mononuclear cells (PBMC) by the timed addition of cytokine-rich supernatants collected from allogeneic PBMC cultures stimulated with anti-CD3 monoclonal antibody (mAb) (allogeneic CD3 supernatants; ACD3S). These cytotoxic effectors belonged primarily to CD56(+) natural killer (NK) cells, and the cell subset with the greatest cytotoxic activity was an otherwise rare population of CD3(+)CD56(+) cells (NKT cells) that expand dramatically under these conditions. CD3(+)CD56(+) cytotoxic effectors were generated from the PBMC of 16 patients with several types of cancer. The CD3(+)CD56(+) cell subset expanded significantly and efficiently lysed NK- as well as lymphokine-activated killer (LAK)-sensitive targets. More importantly, ACD3S-activated CD3(+)CD56(+) cells were capable of efficiently lysing autologous tumor cells including metastatic colorectal, ovarian, breast, lung a...

British journal of cancer, Jan 16, 2001

HER2/neu-derived peptides inducing MHC class II-restricted CD4+ T helper lymphocyte (Th) response... more HER2/neu-derived peptides inducing MHC class II-restricted CD4+ T helper lymphocyte (Th) responses, although critical for tumour rejection, are not thoroughly characterized. Here, we report the generation and characterization of CD4+ T cell clones specifically recognizing a HER-2/neu-derived peptide (776-788) [designated HER2(776-788)]. Such clones yielded specific proliferative and cytokine [gamma-interferon(IFN)-gamma] responses when challenged with autologous dendritic cells (DCs) loaded with HER2(776-788). By performing blocking studies with monoclonal antibodies (MAbs) and by using DCs from allogeneic donors sharing certain HLA-DR alleles, we found that HER2(776-788) is a promiscuous peptide presented, at least, by DRB5*0101, DRB1*0701 and DRB1*0405 alleles. One TCRV beta 6.7+ clone recognized the HLA-DRB5*0101+ FM3 melanoma cell line transfected with a full length HER-2/neu cDNA. Moreover, this clone recognized the HER-2/neu+ SKBR3 breast cancer cell line induced to express HL...

Stem Cells and Development, 2014

Mesenchymal stem cells (MSCs) are a population of cells harboring in many tissues with the abilit... more Mesenchymal stem cells (MSCs) are a population of cells harboring in many tissues with the ability to differentiate toward many different lineages. Unraveling the molecular profile of MSCs is of great importance due to the fact that these cells are very often used in preclinical and clinical studies. We have previously reported the expression of insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1) an oncofetal mRNA-binding protein-in different stem cell types such as bone marrow (BM)-MSC and umbilical cord blood (UCB)-hematopoietic stem cells. Here, we demonstrate that MSCs of adipose tissue, BM, and UC origin have a differential pattern of IGF2BP1 and ten-eleven-translocate 1/2 (TET1/2) expression that could correlate with their proliferation potential. Upon IGF2BP1 interference, a significant reduction of cell proliferation is observed, accompanied by reduced expression of c-MYC and GLI1 and increased p21. We also present, for the first time, evidence that IGF2BP1 is epigenetically regulated by TET1 and TET2 demethylases. Specifically, we show that TET1 directly binds to the promoter of IGF2BP1 gene and affects the hydroxymethylation status of its promoter. These results indicate that IGF2BP1 and TET1/2 contribute to the stemness of MSCs, at least regarding their proliferative potential.