Intratumoral injection of IFN-alpha dendritic cells after dacarbazine activates anti-tumor immunity: results from a phase I trial in advanced melanoma.

BACKGROUND: Advanced melanoma patients have an extremely poor long term prognosis and are in strong need of new therapies. The recently developed targeted therapies have resulted in a marked antitumor effect, but most responses are partial and some degree of toxicity remain the major concerns. Dendritic cells play a key role in the activation of the immune system and have been typically used as ex vivo antigen-loaded cell drugs for cancer immunotherapy. Another approach consists in intratumoral injection of unloaded DCs that can exploit the uptake of a wider array of tumor-specific and individual unique antigens. However, intratumoral immunization requires DCs endowed at the same time with properties typically belonging to both immature and mature DCs (i.e. antigen uptake and T cell priming). DCs generated in presence of interferon-alpha (IFN-DCs), due to their features of partially mature DCs, capable of efficiently up-taking, processing and cross-presenting antigens to T cells, could successfully carry out this task. Combining intratumoral immunization with tumor-destructing therapies can induce antigen release in situ, facilitating the injected DCs in triggering an antitumor immune response. METHODS: We tested in a phase I clinical study in advanced melanoma a chemo-immunotherapy approach based on unloaded IFN-DCs injected intratumorally one day after administration of dacarbazine. Primary endpoint of the study was treatment safety and tolerability. Secondary endpoints were immune and clinical responses of patients. RESULTS: Six patients were enrolled, and only three completed the treatment. The chemo-immunotherapy was well tolerated with no major side effects. Three patients showed temporary disease stabilization and two of them showed induction of T cells specific for tyrosinase, NY-ESO-1 and gp100. Of interest, one patient showing a remarkable long-term disease stabilization kept showing presence of tyrosinase specific T cells in PBMC and high infiltration of memory T cells in the tumor lesion at 21 months. CONCLUSION: We tested a chemo-immunotherapeutic approach based on IFN-DCs injected intratumorally one day after DTIC in advanced melanoma. The treatment was well tolerated, and clinical and immunological responses, including development of vitiligo, were observed, therefore warranting additional clinical studies aimed at evaluating efficacy of this approach. TRIAL REGISTRATION: Trial Registration Number not publicly available due to EudraCT regulations: https://www.clinicaltrialsregister.eu/doc/EU_CTR_FAQ.pdf.

Chimeric Antigen Receptor Immunotherapy for Solid Tumors: Choosing the Right Ingredients for the Perfect Recipe.

Chimeric antigen receptor T cell therapies are revolutionizing the clinical practice of hematological tumors, whereas minimal progresses have been achieved in the solid tumor arena. Multiple reasons have been ascribed to this slower pace: The higher heterogeneity, the hurdles of defining reliable tumor antigens to target, and the broad repertoire of immune escape strategies developed by solid tumors are considered among the major ones. Currently, several CAR therapies are being investigated in preclinical and early clinical trials against solid tumors differing in the type of construct, the cells that are engineered, and the additional signals included with the CAR constructs to overcome solid tumor barriers. Additionally, novel approaches in development aim at overcoming some of the limitations that emerged with the approved therapies, such as large-scale manufacturing, duration of manufacturing, and logistical issues. In this review, we analyze the advantages and challenges of the different approaches under development, balancing the scientific evidences supporting specific choices with the manufacturing and regulatory issues that are essential for their further clinical development.

Clinical and Antitumor Immune Responses in Relapsed/Refractory Follicular Lymphoma Patients after Intranodal Injections of IFNα-Dendritic Cells and Rituximab: a Phase I Clinical Trial.

PURPOSE: This study was aimed at evaluating the feasibility, safety, immunologic and clinical responses in patients with follicular lymphoma treated with monocyte-derived dendritic cells generated in the presence of IFNα and GM-CSF (IFN-DC) in combination with low doses of rituximab. PATIENTS AND METHODS: Firstly, we analyzed in vitro and in vivo the immunologic properties of IFN-DC against follicular lymphoma. Thus, we performed a phase I trial in 8 patients with refractory and relapsed follicular lymphoma based on sequential intranodal injections of low-dose of rituximab and unloaded IFN-DC and report the safety, clinical, and immunologic results of the enrolled patients. RESULTS: Preclinical studies indicated that IFN-DC can synergize with rituximab leading to increased cytotoxicity and T-cell tumor infiltration. The clinical evaluation showed that the combined treatment was totally safe. The overall response rate was 50%, PET-negative complete response rate 37%, and remission is still ongoing in 2/4 of responding patients (median follow-up 26 months, range 11-47). Notably, following the combined therapy all patients showed induction/enhancement of T-cell responses by CD107 degranulation or IFNγ ELISPOT assay against patient-specific tumor IGHV sequences. CONCLUSIONS: These results represent the proof-of-principle on the effectiveness of unloaded IFN-DC in inducing durable clinical responses and promoting induction of tumor-specific peripheral T cells, thus suggesting the occurrence of an effective endogenous antitumor vaccination. The overall findings indicate that some unique properties of IFN-DC can be successfully exploited to induce/enhance antitumor responses, thus representing a valuable antitumor strategy for novel and more effective combination therapies in patients with cancer.

Immunization of stage IV melanoma patients with Melan-A/MART-1 and gp100 peptides plus IFN-alpha results in the activation of specific CD8(+) T cells and monocyte/dendritic cell precursors.

The use of IFN-alpha in clinical oncology has generally been based on the rationale of exploiting its antiproliferative and antiangiogenic activities. However, IFN-alpha also exhibits enhancing effects on T-cell and dendritic cell functions, which may suggest a novel use as a vaccine adjuvant. We have carried out a pilot phase I-II trial to determine the effects of IFN-alpha, administered as an adjuvant of Melan-A/MART-1:26-35(27L) and gp100:209-217(210M) peptides, on immune responses in stage IV melanoma patients. In five of the seven evaluable patients, a consistent enhancement of CD8(+) T cells recognizing modified and native MART-1 and gp100 peptides and MART-1(+)gp100(+) melanoma cells was observed. Moreover, vaccination induced an increase in CD8(+) T-cell binding to HLA tetramers containing the relevant peptides and an increased frequency of CD45RA(+)CCR7(-) (terminally differentiated effectors) and CD45RA(-)CCR7(-) (effector memory) cells. In all patients, treatment augmented significantly the percentage of CD14(+) monocytes and particularly of the CD14(+)CD16(+) cell fraction. An increased expression of CD40 and CD86 costimulatory molecules in monocytes was also observed. Notably, postvaccination monocytes from two of the three patients showing stable disease or long disease-free survival showed an enhanced antigen-presenting cell function and capability to secrete IP10/CXCL10 when tested in mixed leukocyte reaction assays, associated to a boost of antigen and melanoma-specific CD8(+) T cells. Although further clinical studies are needed to show the adjuvant activity of IFN-alpha, the present data represent an important starting point for considering a new clinical use of IFN-alpha and new immunologic end points, potentially predictive of clinical response.

A good manufacturing practice method to ex vivo expand natural killer cells for clinical use.

BACKGROUND: Great interest has been raised recently by the design of new adoptive immunotherapeutic strategies based on the in vivo infusion of ex vivo-expanded and activated natural killer (NK) cells. The development of good manufacturing practice (GMP) methods for the efficient production of fully functional NK cells is mandatory for clinical application. MATERIALS AND METHODS: Peripheral blood mononuclear cells were obtained by leukapheresis and processed in the GMP facility. For NK-cell enrichment, a two-step immunomagnetic procedure consisting of CD3(+) T-cell depletion followed by CD56(+) cell positive selection was used. Isolated NK cells were suspended in serum-free medium containing autologous plasma, interleukin (IL)-2 and IL-15 in the presence of irradiated autologous feeder cells and cultured for 14 days at 37 °C. IL-2 and IL-15 were also added during the last 24 hours of culture. Expanded cells underwent full quality control testing for cytogenetic characteristics, viability, sterility, phenotype and endotoxin status; functional tests, such as degranulation assays and cytotoxicity, were performed on expanded NK cells before cryopreservation and after thawing. RESULTS: NK-cell populations expanded on average 15.7±4.7 fold by day 14, with a viability of 96% ±0.5. At the end of the incubation period, 97% ±1.1 of the expanded population was CD56(+) NK cells; these effector cells showed significant up-regulation of the activating receptors NKG2D and DNAM-1. Functional tests demonstrated that expanded NK cells are fully functional with no difference whether tested before cryopreservation or after thawing. DISCUSSION: These data provide the basis for developing new NK-cell-based immunotherapeutic strategies for the treatment of patients with cancer.

Evaluation of the effects of human leukocyte IFN-alpha on the immune response to the HBV vaccine in healthy unvaccinated individuals.

HBV vaccine needs 3 injections over 6 months to induce immunity. Thus, the use of adjuvants capable of inducing earlier immune protection would be highly desirable. Most adjuvants may act by inducing cytokines, and among them, type I interferons (IFNs), deserve a special attention in view of the potent immunomostimulatory activity observed in mouse models and on dendritic cell functions. The aim of the present trial was to evaluate the effects of IFN-alpha administered as an adjuvant of HBV vaccine in healthy unvaccinated individuals. No significant enhancing effect on the antibody response was observed, in spite of an early and transient upregulation of costimulatory molecule expression on peripheral blood mononuclear cells, which may be suggestive of an IFN-mediated activation of antigen presenting cells. We conclude that, under the conditions used in this trial, natural IFN-alpha does not act as an adjuvant of the HBV vaccine in healthy unvaccinated individuals.