Cancer immunotherapy emerged as a novel therapeutic option that employs enhanced or amended native immune system to create a robust response against malignant cells. The systemic therapies with immune-stimulating cytokines have resulted in substantial dose-limiting toxicities. Targeted cytokine immunotherapy is being explored to overcome the heterogeneity of malignant cells and tumor cell defense with a remarkable reduction of systemic side effects. Cell-based strategies, such as dendritic cells (DCs), fibroblasts or mesenchymal stem cells (MSCs) seek to minimize the numerous toxic side effects of systemic administration of cytokines for extended periods of time. The usual toxicities comprised of a vascular leak, hypotension, and respiratory insufficiency. Natural and strong tropism of MSCs toward malignant cells made them an ideal systemic delivery vehicle to direct the proposed therapeutic genes to the vicinity of a tumor where their expression could evoke an immune reaction against the tumor. Compared with other methods, the delivery of cytokines via engineered MSCs is safer and renders a more practical, and promising strategy. Large numbers of genes code for cytokines have been utilized to reengineer MSCs as therapeutic cells. This review highlights the recent findings on the cytokine gene therapy for human malignancies by focusing on MSCs application in cancer immunotherapy.

Devitalized tumor cells either autologous or allogeneic have been used as anti-cancer vaccines with the purpose of facilitating the induction of an immune response able to destroy growing tumor cells since the identification of tumor antigens was deemed not to be necessary, particularly in the autologous system. Such vaccines were tested first in animal models and then in the clinics as unmodified tumor cells or after insertion of genes coding for factors known to increase the immune response against tumors. These vaccines were usually given by subcutaneous injections along with different immunological adjuvants. Such immunization approaches were found to be effective in mice when carried out in a tumor preventive setting but significantly less in the therapeutic context, that is, in the presence of an established tumor. By analyzing several clinical trials of vaccination using either autologous or allogeneic unmodified and gene-modified tumor cells published in the last 10 to 15 years, we conclude for a lack of sufficient evidence for efficacy of this strategy in inducing both a strong immune response and a therapeutic response. A potential variant of this strategy is the direct intratumoral injection of immunostimulatory genes delivered by vectors in vivo. But even this approach failed to provide a statistically significant clinical benefit for the cancer patients.We also point out the inherent drawbacks of the tumor cell-based vaccine strategy that include (a) a limited frequency by which human tumor lines can be obtained from clinical samples, (b) the low number of available cells for vaccination, (c) the release of immune-suppressive factors by tumor cells, and (d) the cost and time necessary for standardization and collecting/expanding a number of cells according to the approved regulatory requirements. Thus, taking into consideration the new developments in cancer vaccines, we believe that tumor cell-based vaccines should be dismissed as anti-cancer vaccines unless a clear benefit could be demonstrated by the few ongoing trials of combination with new immunomodulating reagents (eg, anti-CTLA4, PD-1, chemotherapy).

Tumor-associated dendritic cells (DCs) often induce T cell anergy or deletion and regulatory T cells instead of antitumor immunity. Although many tumor-associated Ags have been found, there is still no effective vaccine for cancer. Thus, novel rational strategies to enhance the immunogenicity of cancer-specific Ags are needed. Chromosome 1 open reading frame 190 (c1orf190), a gene that encodes a 239-aa hypothetical protein and contains multiple kinase phosphorylation sites, has a wide relationship with multiple signaling pathway molecules and can be regulated by multiple factors, such as TLR ligands. In this study, we demonstrate that c1orf190 can activate NF-κB, drive the production of cytokines, and promote the Ag-presenting function and the priming ability of DCs. Furthermore, c1orf190 can promote resistance of DCs to tumor-associated inhibition not only in the Ag-presenting function but also in the priming ability to induce Ag-specific T lymphocytes. Thus, c1orf190, an NF-κB activator, may be a candidate gene for regulating the function of DCs to resist tumor-associated factor-mediated dysfunction. We also found that c1orf190-mediated cytokine release is achieved by activating the canonical but not the noncanonical NF-κB pathway.

Delivery of cytokine genes at the tumor site in pre-clinical models has been shown to recruit host inflammatory cells followed by inhibition of tumor growth. This local effect is often accompanied by systemic protection mediated by the immune system, mainly by CD8(+) T and NK cells. On this basis, cytokine gene-transduced tumor cells have widely been used as vaccines in clinical trials, which have shown good safety profiles and some local responses but substantial lack of systemic efficacy. Are these findings the end of the story? Possibly not, if major improvements will be attained in the coming years. These should be directed at the level of gene selection and delivery, in order to identify the optimal cytokine and achieve efficient and durable cytokine expression, and at the level of improving immune stimulation, i.e. by co-administration of co-stimulatory molecules including B7 and CD40, or boosting the expression of tumor antigens or MHC class I molecules. Interestingly, some of the cytokines which have shown encouraging anti-tumor activity, including IFNs, IL-4, IL-12 and TNF-alpha, are endowed with anti-angiogenic or vasculotoxic effects, which may significantly contribute to local tumor control. Therapeutic exploitation of this property may result in the design of novel approaches which, by maximizing immune-stimulating and anti-angiogenic effects, could possibly lead to starvation of established tumors in patients.

Vaccination with autologous tumor cells genetically modified to express costimulatory molecules has shown utility for cancer immunotherapy in preclinical and limited clinical settings. Given the complicated nature of gene therapy, a practical alternative approach has been designed that relies on modification of the cell membrane with biotin and its "decoration" with a chimeric protein composed of the functional portion of human CD80 and core streptavidin (CD80-SA). We tested whether primary tumor cells resected from cancer patients can be decorated with CD80-SA and whether such cells serve as antigen-presenting cells (APCs) to generate autologous T cell responses ex vivo. Tumors and peripheral blood lymphocytes (PBLs) were collected from 14 lung, 9 colon, and 2 breast "treatment-naive" cancer patients presenting various clinical stages of the disease. Tumors were mechanically processed, irradiated, decorated with CD80-SA or control streptavidin (SA) protein, and used as APCs in ex vivo autologous T cell-proliferative and cytotoxicity assays. All tumor samples were modified with CD80-SA, albeit with various degrees of decoration ranging from 21.8 to 100%. CD80- SA-decorated cells generated significant proliferative responses in autologous T cells from 9 of 16 evaluable patients (p < 0.05). Proliferative responses were CD80-SA specific and heterogeneous, with stimulation indices ranging from 0.25 to 45. In 15 of 15 evaluable patients, CD80-SA-specific cytotoxic T cell responses against autologous tumors were generated, 11 of which were significant, with specific killing ranging from 5 to 70%. Taken together, these data demonstrate that primary tumor cells can be effectively decorated with CD80-SA and that such cells serve as APCs to induce autologous antitumor T cell responses.

The authors have previously reported a tumor cell vaccine modified with superantigen staphylococcal enterotoxin A (SEA) and its antitumor effect. The tumor cell vaccines modified with multiple immune activators frequently elicited stronger immune responses against established tumors than single-modified vaccines.

The authors explored the effectiveness of a tumor cell vaccine transduced with immune activators, dual-modified using the protein transfer technique. First, a glycosylphosphatidylinositol (GPI)-anchored murine B7.1 (mB7.1-GPI) and a transmembrane-anchored SEA (TM-SEA) were genetically generated. Then, the murine lymphoma EL4 cells were dual modified with the incorporation of mB7.1-GPI and TM-SEA onto the cell surface. Flow cytometry and laser confocal microscopy showed that the incorporation of B7.1 and SEA molecules onto EL4 cells was quite stable.

The dual-modified tumor cell vaccine EL4/mB7.1-GPI + TM-SEA elicited significantly stronger antitumor immune responses both in vitro and in vivo when compared with the single-modified tumor cell vaccines EL4/mB7.1-GPI and EL4/TM-SEA.

The results of the current study validated the novel approach for preparing tumor cell vaccines modified with dual immune active molecules using the protein transfer technique, and supported the feasibility and effectiveness of the dual-modified tumor cell vaccine.

Persistent activation of T-lymphocytes requires two signals: one is initiated by T-cell receptor binding to antigenic peptide presented by MHC molecules. In addition, binding of the B7 family members CD80 or CD86 on professional antigen presenting cells to CD28 on T cells is considered to provide an important costimulatory signal. Activation without costimulation induces T-cell unresponsiveness or anergy. To selectively localize costimulatory activity to the surface of tumor cells and enhance activation of tumor-specific T cells, we have developed a novel molecular design for bispecific costimulatory proteins with antibody-like structure. Within a single polypeptide chain we have assembled the IgV-like, CD28-binding domain of human CD86 (CD86(111)) together with hinge, CH2 and CH3 domains of human IgG1, and the scFv(FRP5) antibody fragment which recognizes the ErbB2 (HER2) protooncogene present at high levels on the surface of many human tumor cells. Upon expression in the yeast Pichia pastoris, the resulting CD86(111)-IgG-scFv(FRP5) protein could be purified as a homodimeric, tetravalent molecule from culture supernatants using single-step affinity chromatography. Bispecific binding of the molecule to ErbB2 on the surface of tumor cells and to the B7 counter receptor CTLA-4 was demonstrated by FACS analysis. Potent costimulatory activity of chimeric CD86(111)-IgG-scFv(FRP5) was confirmed by its ability to stimulate the proliferation of primary human lymphocytes pre-activated by low concentrations of anti-CD3 antibody. Our results suggest that such multivalent soluble proteins which combine specific targeting to tumor cells with costimulatory activity may become useful tools to elicit and/or improve T-cell mediated, tumor-specific immune responses.

The coexistence of tumor progression with a tumor-specific immune response constitutes a major paradox of tumor immunity. During the last decade, the presence of cytotoxic T lymphocytes (CTLs) recognising melanoma-associated antigens has been unequivocally demonstrated in numerous different in vivo and in vitro models. However, most often these melanoma-specific T lymphocytes do not control tumor growth. Several mechanisms that involve changes in melanoma phenotype and/or in T-cell differentiation and function could explain the inability of the immune response to control melanoma. In the last few years it has been demonstrated that cellular cytotoxicity is the result of a balance between activating signals triggered by the TCR and costimulatory molecules and inhibitory signals triggered by inhibitory receptors expressed by the CTL. Because the final outcome of the immune response against melanoma depends on the balance between activating and inhibitory signals, the expression de novo on melanoma cells of ligands for inhibitory NKRs and the down-regulation of costimulatory molecules may favor the escape of tumor cells from immunosurveillance. In this paper we review how altered expression of molecules required for T-cell costimulation could result in impaired lysis of melanoma. The modulation of antimelanoma T-cell responses by a group of receptors originally described on NK cells (NK-associated receptors) but which are now known also to be expressed on a subset of cytolytic effector cells is reviewed. We hypothesize that the expression of ligands for NKRs on melanoma cells may contribute to T-cell-mediated immune responses against melanoma either enhancing or inhibiting activation and differentiation to effector cells. Blocking inhibitory receptors or increasing activating receptors could result in new strategies to improve T-cell-mediated rejection of melanoma.

Leukaemic blast cells lack co-stimulatory molecules such as B7, necessary for T-lymphocyte activation. We have used modified CD80+ (B7-1+) tumour cells, with autologous, IL-2 producing, stromal marrow cells in a series of subcutaneous vaccinations to provide a localised environment for the enhancement of cytotoxic T-lymphocytes (CTL) in a patient with acute myeloid leukaemia (AML). Localised inflammation was evident after the fifth and sixth injections with a reduction in the number of circulating blasts in the following 2 weeks. Peripheral blood in vitro CTL activity increased 36-47% after five injections.CD4 T-lymphocytes (5.7%) expanded from post-injection skin biopsies, expressed intracellular IFNgamma and perforin when exposed to autologous B7-1+ blasts and when co-cultured with either B7-1+ or unmanipulated autologous blast cells showed proliferative responses. In this patient, co-injection of B7-1+ tumour cells, together with a local source of sustained IL-2, resulted in an autologous anti-leukaemic in vitro immune response.