Zeinab Abdel-Wahab

A phase I clinical trial of immunotherapy with interferon‐γ gene‐modified autologous melanoma cells

Tumor cells transduced with cytokine genes provide immunogenic vaccines for cancer immunotherapy.

Induction of Human Dendritic Cell Maturation Using Transfection with RNA Encoding a Dominant Positive Toll-Like Receptor 4

Maturation of dendritic cells (DC) is critical for the induction of Ag-specific immunity. Ag-loaded DC matured with LPS, which mediates its effects by binding to Toll-like receptor 4 (TLR4), induce Ag-specific CTL in vitro and in vivo in animal models. However, clinical use of LPS is limited due to potential toxicity. Therefore, we sought to mimic the maturation-inducing effects of LPS on DC by stimulating TLR4-mediated signaling in the absence of exogenous LPS. We developed a constitutively active TLR4 (caTLR4) and demonstrated that transfection of human DC with RNA encoding caTLR4 led to IL-12 and TNF-α secretion. Transfection with caTLR4 RNA also induced a mature DC phenotype. Functionally, transfection of DC with caTLR4 RNA enhanced allostimulation of CD4+ T cells. DC transfected with RNA encoding the MART (Melan-A/MART-1) melanoma Ag were then used to stimulate T cells in vitro. Cotransfection of these DC with caTLR4 RNA enhanced the generation of MART-specific CTL. This CTL activity was superior to that seen when DC maturation was induced using either LPS or a standard mixture of cytokines (TNF-α, IL-6, IL-1β, and PGE2). We conclude that transfection of DC with RNA encoding a functional signaling protein, such as caTLR4, may provide a new tool for studying TLR signaling in DC and may be a promising approach for the induction of DC maturation for tumor immunotherapy.

Pulsing of Dendritic cells with cell lysates from either B16 melanoma or MCA-106 fibrosarcoma yields equally effective vaccines against B16 tumors in mice

Background and Objectives: Dendritic cells (DC) pulsed in vitro with a variety of antigens have proved effective in producing specific antitumor effects in vivo. Experimental evidence from other laboratories has confirmed that shared antigens can be encountered in histologically distinct tumors. In our experiments, we set out to evaluate the immunotherapeutic potential of vaccines consisting of DC pulsed with MCA‐106 fibrosarcoma or B16 melanoma cell lysates and to determine whether a cross‐reactivity exists between the two tumors.

Modulation of chemotherapy resistance in regional therapy: a novel therapeutic approach to advanced extremity melanoma using intra-arterial temozolomide in combination with systemic O6-benzylguanine.

This study investigated whether the therapeutic index of regional melanoma therapy using parenteral temozolomide could be improved by chemomodulation with O6-benzylguanine (O6BG), an inhibitor of the DNA repair enzyme O6-alkylguanine-DNA alkyltransferase (AGT). Using a nude rat s.c. human melanoma xenograft model of the extremity, tumors were analyzed for AGT level 2 to 3 hours after the i.p. injection of 3.5 to 70.0 mg/kg O6BG to inhibit AGT activity. Survival studies were conducted using animals that were treated with a 15-minute isolated limb infusion with 10% DMSO in PBS (control), temozolomide alone, or temozolomide in conjunction with single or multiple doses of i.p. O6BG. Tumor volume and toxicity level were monitored every other day. Administration of 3.5 mg/kg O6BG depleted tumor AGT activity by 93.5% (P < 0.01). Groups treated with regional temozolomide alone (350 mg/kg), systemic temozolomide with O6BG, or vehicle combined with O6BG showed no significant tumor responses compared with controls. Whereas use of regional temozolomide alone at a higher dose (750 mg/kg) showed some degree of tumor response, regional temozolomide given in conjunction with multiple dosages of O6BG showed a marked (P < 0.01) reduction in tumor growth with minimal toxicity. Our findings suggest that AGT modulation by the administration of O6BG in combination with temozolomide regional chemotherapy leads to a significant improvement in melanoma antitumor responses. Clinical trials using chemotherapy modulation may improve response rates in future regional infusion and perfusion drug trials. [Mol Cancer Ther 2006;5(3):732–8]

Optimizing a Novel Regional Chemotherapeutic Agent against Melanoma: Hyperthermia-Induced Enhancement of Temozolomide Cytotoxicity

Purpose: Previous preclinical studies have shown that regional temozolomide therapy via isolated limb infusion is more effective than melphalan, the current drug of choice for regional chemotherapy for advanced extremity melanoma. The aim of this study was to determine whether hyperthermia could further augment the efficacy of temozolomide, an alkylating agent, against melanoma and improve its therapeutic index in a rat model of isolated limb infusion. Experimental Design: Athymic rats bearing s.c. human melanoma xenografts (DM6) in their hind limbs were randomized to a 15-minute isolated limb infusion procedure with or without temozolomide at room temperature, normothermic (37.5°C), or hyperthermic (43°C) conditions. Results: The concomitant administration of hyperthermia during an infusion with temozolomide led to the greatest increase in tumor growth delay, decreased proliferative index, and increased cell death. Isolated limb infusion treatment with a low dose (350 mg/kg) of temozolomide was ineffective at producing tumor growth delay (P = 0.07). Similarly, temozolomide infusion under normothermia yielded minimal tumor growth delay (P = 0.08). In contrast, the combination of hyperthermia plus temozolomide treatment produced marked tumor growth delay of 10.4 days (P = 0.02) with minimal toxicity. The addition of heat to temozolomide treatment yielded the smallest proliferative index (P = 0.001), while markedly increasing the level of apoptosis 48 hours after isolated limb infusion. Conclusion: This study, the first to examine the interaction between hyperthermia and temozolomide, shows a strong, synergistic antitumor effect when hyperthermia is combined with temozolomide for regional treatment of melanoma confined to an extremity. The mechanism of this synergy seems to be through an augmentation, by hyperthermia, of the antiproliferative and proapoptotic effects of temozolomide.

Immunological memory induced by genetically transduced tumor cells

AbstractBackground: Recent studies have demonstrated the usefulness of gene-modified tumor cells for immunotherapy. Using the tumorigenic murine fibrosarcoma, MCA 106, we investigated the effects of localized interferon-γ (IFNg) secretion on tumorigenicity and on long-term memory. Methods: The murine IFNg (MuIFNg) gene was introduced into tumor cells. High and low IFNg-secreting clones were isolated. C57BL/6 mice were injected subcutaneously (s.c.) with either parental (P), high or low IFNg-secreting (H- or L-IFNg) cells, and tumor growth was assessed weekly. Spleens were harvested on different days postinjection (p.i.) to assess in vitro cytolytic activity. In parallel, tissues from injection sites were stained with macrophage-, CD4-, and CD8-detecting antibodies. Mice were injected s.c. with H-IFNg MCA106 tumor. After 150 days the animals were rechallenged s.c. with MCA106P in one leg and with irrelevant syngeneic tumor in the other. Results: Both P- and L-IFNg cells had similar growth, whereas the H-IFNg cells never grew. Only splenocytes from the H-IFNg animals showed in vitro CTL activity persisting until day 30 p.i. Histological data revealed a macrophage and CD4+ infiltrate much earlier in the H-IFNg group compared with the P group. Only the irrelevant, syngeneic tumor grew in animals previously injected with H-IFNg cells, whereas both P and irrelevant syngeneic tumors grew in controls. Conclusions: Transduction of MCA106 cells with the MuIFNg gene diminished in vivo tumorigenicity in proportion to the amount of IFNg secreted. Immunization with H-IFNg cells elicited a host response characterized by macrophages and CD4+ cells. Long-term tumor-specific memory was seen after immunization with H-IFNg cells.

Modulation of specific active immunization against murine melanoma using recombinant cytokines

Specific active immunization with tumour cells and IL-1beta or IL-2 was examined in a murine model. Mice were treated with irradiated B16 melanoma, IL-1beta or IL-2 only, or with B16 plus cytokines prior to i.v. challenge with viable B16. Lung metastases were recorded after 28 days. Treatment with cytokine alone was not protective. Treatment with B16 alone afforded moderate protection. Treatment with B16 in combination with either cytokine resulted in a significant level of B16 specific protection which was dependent on the dose of cytokine used. Multiple immunizations with B16 provided limited protection which was significantly improved with IL-2. Immunization with B16 in combination with both cytokines at doses that alone failed to enhance immunity resulted in significant protection, suggesting that the two cytokines act at least additively. These studies demonstrate the significant benefit of specific active immunization with tumour cells in combination with low doses of IL-1beta or IL2.

Cell surface reactive human monoclonal antibody directed to human melanoma-associated gangliosides.

An IgM human monoclonal antibody (human MAb) was generated by fusing lymph node cells Isolated from a surgical specimen of malignant melanoma with the heteromyeloma cell line SHMD-33. The antibody, designated 7c11.e8, reacted with surface antigens on human melanoma cells as shown by live cell immunofluorescence and absorption assays. The MAb 7c11.e8 reacted with DSI, SPG, GM4, GM3 and GD3 In enzyme-linked Immunosorbent assays (ELISA), and did not react with GD2, GM1, GM2, GD1a, GD1b, GT1b and a number of neutral glycosphingollplds. The main binding epitope for the MAb was, therefore, the terminal N-acetylneuramlnlc acid 2–3 Gal linked by a β1–1 bond to the ceramlde, or a β-4 bond to glucose or glucosamine. As shown by immunohlstochemlcal assays, 7c11.e8 antigen was expressed on all melanoma tumour tissues, and on a few samples of colon carcinoma, normal colon, skin, spinal cord, kidney and liver. However, other normal organs such as breast, lung, small Intestine, stomach and lymph nodes did not react with the MAb. In the presence of human serum the antibody Initiated a strong lysis of melanoma tumour cells In complement-dependent cellular cytotoxicity (CDCC) assays. This study demonstrates that It Is possible to Isolate human monoclonal antibodies directed to cell surface antigens using viable cell assays In the screening protocol. The preferential binding of 7c11.e8 to melanoma tissues and the reactivity with two of the major melanoma gangliosides (GM3 and GD3) suggest that 7c11.e8 may provide a useful reagent for diagnosis and therapy of malignant melanoma.

Immunotherapy of Human Melanoma With Gene-Modified Tumor Cell Vaccines.

The incidence of melanoma in the United States is increasing at a faster rate than that of any other cancer. The prognosis for metastatic disease is poor, and more effective treatments for disseminated disease are needed. Since melanoma is one of the more immunogenic tumors, strategies have focussed on immune recognition. In vitro studies suggest that potent tumor-specific cytotoxic T cells can be induced against human melanoma. Melanoma specific T-cell activation depends on appropriate presentation to the immune system of recently defined melanoma-associated antigens presented in the context of self-HLA gene products. Full T-cell activation requires the co- stimulation by B7-CD28 interactions at the T-cell surface and the elaboration of immune cytokines to promote T-cell growth. Data from animal models of tumor-specific immunization with tumor cells engineered to express immune cytokines or the B7 co-stimulatory molecule suggest that gene therapy for human melanoma may be an effective means to treat disseminated disease.

Generation of human IgG, IgA, and IgM anti-melanoma monoclonal antibodies utilizing lymphocytes of an actively immunized melanoma patient

Active specific immunotherapy with irradiated allogeneic melanoma cells has been shown to enhance the humoral immune response in melanoma patients. An increased titer of melanoma-binding antibodies was demonstrated in sera of immunized patients. Lymph node cells and splenocytes isolated from an actively immunized melanoma patient were fused with the human-murine heteromyeloma cell lines SHMD-33, SPM4-0, and SBC-H20. A group of human anti-melanoma monoclonal antibodies (MABs) were generated from the SHMD-33 fusion. Isolated MABs (one IgG2, one IgA, and two IgM) have been stable in cultures for more than 12 months and have produced human immunoglobulins at 0.2-0.9 Ug/ml/day. As shown by solid phase radioimmunoassays, the MABs react with autologous tumor cells and allogeneic melanoma tumors, including the cell line that was used for immunotherapy. In immunocytochemical assays, all four MABs react with a number of melanoma tumor cell lines. The IgG2 and IgA MABs stained preferentially melanoma tumor cells. In contrast, the IgM MABs cross-reacted with a broad panel of tumor cells from colon, prostate, pancreas, lung, and other human tumors. The MABs appear to be directed to intracellular rather than membrane-associated antigens as shown by immunofluorescence assays on live and permeabilized cells. The IgG2 antibody recognizes a 70 kDa antigen in melanoma cell lysates by Western immunoblotting. The target antigens for the other MABs have not yet been defined. Stability in culture and strong binding to melanoma tumor cells provide the basis for evaluating the potential of these human MABs. The IgG2 MAB, in particular, may prove useful for diagnostic and therapeutic applications in humans.(ABSTRACT TRUNCATED AT 250 WORDS)