Therapeutic response in patients with advanced malignancies treated with combined dendritic cell-activated T cell based immunotherapy and intensity-modulated radiotherapy.

Successful cancer immunotherapy is confounded by the magnitude of the tumor burden and the presence of immunoregulatory elements that suppress an immune response. To approach these issues, 26 patients with advanced treatment refractory cancer were enrolled in a safety/feasibility study wherein a conventional treatment modality, intensity modulated radiotherapy (IMRT), was combined with dendritic cell-based immunotherapy. We hypothesized that radiation would lower the tumor burdens, decrease the number/function of regulatory cells in the tumor environment, and release products of tumor cells that could be acquired by intratumoral injected immature dendritic cells (iDC). Metastatic lesions identified by CT (computed tomography) were injected with autologous iDC combined with a cytokine-based adjuvant and KLH (keyhole limpet hemocyanin), followed 24 h later by IV-infused T-cells expanded with anti-CD3 and IL-2 (AT). After three to five days, each of the injected lesions was treated with fractionated doses of IMRT followed by another injection of intratumoral iDC and IV-infused AT. No toxicity was observed with cell infusion while radiation-related toxicity was observed in seven patients. Five patients had progressive disease, eight demonstrated complete resolution at treated sites but developed recurrent disease at other sites, and 13 showed complete response at various follow-up times with an overall estimated Kaplan-Meier disease-free survival of 345 days. Most patients developed KLH antibodies supporting our hypothesis that the co-injected iDC are functional with the capacity to acquire antigens from their environment and generate an adaptive immune response. These results demonstrate the safety and effectiveness of this multimodality strategy combining immunotherapy and IMRT in patients with advanced malignancies.

Ex-vivo expanded human NK cells express activating receptors that mediate cytotoxicity of allogeneic and autologous cancer cell lines by direct recognition and antibody directed cellular cytotoxicity.

BACKGROUND: The possibility that autologous NK cells could serve as an effective treatment modality for solid tumors has long been considered. However, implementation is hampered by (i) the small number of NK cells in peripheral blood, (ii) the difficulties associated with large-scale production of GMP compliant cytolytic NK cells, (iii) the need to activate the NK cells in order to induce NK cell mediated killing and (iv) the constraints imposed by autologous inhibitory receptor-ligand interactions. To address these issues, we determined (i) if large numbers of NK cells could be expanded from PBMC and GMP compliant cell fractions derived by elutriation, (ii) their ability to kill allogeneic and autologous tumor targets by direct cytotoxicity and by antibody-mediated cellular cytotoxicity and (iii) defined NK cell specific receptor-ligand interactions that mediate tumor target cell killing. METHODS: Human NK cells were expanded during 14 days. Expansion efficiency, NK receptor repertoire before and after expansion, expression of NK specific ligands, cytolytic activity against allogeneic and autologous tumor targets, with and without the addition of chimeric EGFR monoclonal antibody, were investigated. RESULTS: Cell expansion shifted the NK cell receptor repertoire towards activation and resulted in cytotoxicity against various allogeneic tumor cell lines and autologous gastric cancer cells, while sparing normal PBMC. Blocking studies confirmed that autologous cytotoxicity is established through multiple activating receptor-ligand interactions. Importantly, expanded NK cells also mediated ADCC in an autologous and allogeneic setting by antibodies that are currently being used to treat patients with select solid tumors. CONCLUSION: These data demonstrate that large numbers of cytolytic NK cells can be generated from PBMC and lymphocyte-enriched fractions obtained by GMP compliant counter current elutriation from PBMC, establishing the preclinical evidence necessary to support clinical trials utilizing autologous expanded NK cells, both directly and in combination with monoclonal antibodies in future cell-based immunotherapy in select solid tumors.

Combining conventional therapies with intratumoral injection of autologous dendritic cells and activated T cells to treat patients with advanced cancers.

Dendritic cells (DCs) are potent antigen-presenting cells that have been used in cancer immunotherapy. To take advantage of the ability of DCs to acquire antigenic materials from their environment and generate primary as well as recall immune responses, 37 patients with advanced cancers were enrolled in a series of protocols based on direct intratumoral injection of immature DCs. To augment antigen uptake and antitumor immune response, DC injection was combined with radiotherapy or chemotherapy and/or injection of activated T cells. Treatments were well tolerated with no adverse reactions. Clinical responses were based on Response Evaluation Criteria in Solid Tumors, with the majority of patients showing stable disease. One of two patients who also received local radiation achieved a sustained complete response at injected and metastatic sites. The clinical responses observed in cancer patients with advanced disease suggest potential effectiveness of combination strategies and establish the basis for the current treatment protocol that is underway.

Combination of monocyte-derived dendritic cells and activated T cells which express CD40 ligand: a new approach to cancer immunotherapy.

Interactions between dendritic cells (DCs) and activated T cells are critically important for the establishment of an effective immune response. To develop the basis for a new DC-based cancer vaccine, we investigated cell-to-cell interactions between human monocyte-derived DCs and autologous T cells that are activated to express the CD40 ligand (CD40L). Peripheral blood monocytes were cultured in the presence of granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin 4 (IL-4) to induce differentiation of DCs. Activated T cells (ATs) consisted of autologous peripheral blood lymphocytes that had been activated with phytohemagglutinin (PHA) and then stimulated with calcium ionophore to up-regulate expression of CD40L. Coculture of these DCs and ATs induced significant production of interleukin 12 (IL-12) and also enhanced the production of interferon gamma (IFN-gamma). The production of IL-12 was blocked by an anti-CD40L antibody or by separation of the DC and AT fractions by a permeable membrane. Furthermore, coculture of DCs and ATs induced DCs to upregulate CD83 expression and stimulated migration of DCs toward the macrophage inflammatory protein 3-beta (MIP-3beta). ATs also migrated toward the MIP-3beta. These results suggest a combination of DCs and ATs as a potentially effective therapeutic strategy.