Abstract 3677: Early effects of CSF-470 melanoma vaccine plus adjuvants in immune cell stimulation
Authors: Pampena Maria B. , Juliá Estefanía P. , Rocca Yamila S. , Blanco Paula A. , Barrio María M. , Mordoh José , Levy Estrella M. 
Published: July 2017

Abstract
Background: In a Phase II/III clinical trial (CASVAC-0401, NCT01729663), cutaneous melanoma (CM) patients (pts) stages II/III were randomized to receive CSF-470 vaccine or IFN-α (2:1). Vaccine CSF-470 is composed of four lethally-irradiated allogeneic CM cell lines; BCG (Bacillus Calmette-Guérin) and rhGM-CSF were used as adjuvants. At the end of the Phase II part of the study (30 pts), a higher distant-metastases-free-survival was observed in vaccinated pts. All vaccinated pts developed inflammation at the vaccination site. Therefore, in an attempt to understand these first steps of immune reaction, leukocytes migration, cytokines synthesis and monocyte phagocytosis promoted by the vaccine components were studied in vitro.
Methods: Cell migration of healthy donors’ leukocytes or mononuclear cells (MN) towards vaccine components was assayed in 0,5 μm transwells. Cytokines were measured by ELISA in supernatants of CSF-470 vaccine and MN (1:1, 0,5x106 each) cocultures. For phagocytosis assays, CM cell lines were labeled with PKH67 lipophilic dye, irradiated, and cocultured with purified monocytes. After 24 hs, cultures were stained with CD14 and double staining (CD14+PKH67+) was analyzed by flow cytometry.
Results: In migration assays, CSF-470 vaccine induced higher attraction of leukocytes as compared to culture medium (p=0.003, unpaired-T-test). GM-CSF exerted little attraction, although combined with BCG it increased the migration triggered by CSF-470 (vaccine vs vaccine plus adjuvants, p=0.034, unpaired-T-test). When using MN, CSF-470 alone or plus adjuvants, highly attracted monocytes, which represented 10%-65% of the migrating CD45+ population (before migration 6-15%) (p&lt;0.0001, paired-T-test). In the lymphocyte population, natural killer cells were mainly attracted towards the vaccine either with or without adjuvants, representing 12%-33% of total migrated lymphocyte population (before migration 4-25% ) (p=0.002, paired-T-test). Regarding cytokine production, 6 hs cocultures of CSF-470 vaccine plus adjuvants and MN, promoted TNFα production (385±217 pg/ml). IFNγ release was detected in CSF-470:MN cocultures after 48 hs (109,2 pg/ml). BCG appears to be the most important adjuvant for production of these proinflammatory cytokines. IL-10, but not IL-12p70, was also detected. Interestingly, approximately 60% of monocytes phagocytosed CSF-470 cells after 24 hs of coculture, either in the presence or absence of adjuvants.
Conclusions: Our in vitro results suggest that recruitment of leukocytes, mostly from the innate immune system, is an early event after CSF-470 vaccine + BCG + GM-CSF interaction with immune cells. Also, early release of TNFα and IFNγ proinflammatory cytokines and efficient vaccine phagocytosis by monocytes would favor subsequent antigen processing and presentation. Further experiments will be performed to address the initial steps triggered after CSF-470 vaccination.
Citation Format: Maria B. Pampena, Estefanía P. Juliá, Yamila S. Rocca, Paula A. Blanco, María M. Barrio, José Mordoh, Estrella M. Levy. Early effects of CSF-470 melanoma vaccine plus adjuvants in immune cell stimulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3677. doi:10.1158/1538-7445.AM2017-3677