Phase I/II Multicenter Trial of a Novel Therapeutic Cancer Vaccine, HepaVac-101, for Hepatocellular Carcinoma.

PURPOSE: Immunotherapy for hepatocellular carcinoma (HCC) shows considerable promise in improving clinical outcomes. HepaVac-101 represents a single-arm, first-in-human phase I/II multicenter cancer vaccine trial for HCC (NCT03203005). It combines multipeptide antigens (IMA970A) with the TLR7/8/RIG I agonist CV8102. IMA970A includes 5 HLA-A*24 and 7 HLA-A*02 as well as 4 HLA-DR restricted peptides selected after mass spectrometric identification in human HCC tissues or cell lines. CV8102 is an RNA-based immunostimulator inducing a balanced Th1/Th2 immune response. PATIENTS AND METHODS: A total of 82 patients with very early- to intermediate-stage HCCs were enrolled and screened for suitable HLA haplotypes and 22 put on study treatment. This consisted in a single infusion of low-dose cyclophosphamide followed by nine intradermal coadministrations of IMA970A and CV8102. Only patients with no disease relapse after standard-of-care treatments were vaccinated. The primary endpoints of the HepaVac-101 clinical trial were safety, tolerability, and antigen-specific T-cell responses. Secondary or exploratory endpoints included additional immunologic parameters and survival endpoints. RESULTS: The vaccination showed a good safety profile. Transient mild-to-moderate injection-site reactions were the most frequent IMA970A/CV8102-related side effects. Immune responses against ≥1 vaccinated HLA class I tumor-associated peptide (TAA) and ≥1 vaccinated HLA class II TAA were respectively induced in 37% and 53% of the vaccinees. CONCLUSIONS: Immunotherapy may provide a great improvement in treatment options for HCC. HepaVac-101 is a first-in-human clinical vaccine trial with multiple novel HLA class I- and class II-restricted TAAs against HCC. The results are initial evidence for the safety and immunogenicity of the vaccine. Further clinical evaluations are warranted.

Brilliant cresyl blue staining does not present cytotoxic effects on human luteinized follicular cells, according to gene/protein expression, as well as to cytotoxicity tests.

In vitro oocyte maturation presents many advantages and its success is related to the selection of fully grown oocytes. In animal models, staining of cumulus-oocyte complexes (COCs) with Brilliant Cresyl Blue (BCB) is widely used for this selection. However, a lack of information about the safety of BCB makes its applicability in humans questionable. Because granulosa and cumulus cells have a close relationship with the oocyte and taking into account that follicular cells are also exposed to BCB when this pre-selection method is applied, we aimed to evaluate the effects of BCB on human follicular cells exposed to BCB. Cytotoxicity tests (Sulforhodamine B and Neutral Red Uptake) and gene and protein expression of elements related to the cell cycle (BAX, BCL2, TP53 and CDKN1A), as well as to cell death and metabolism (GAPDH), glucose consumption, and estradiol and progesterone secretion, were examined in granulosa and cumulus cells with and without exposure to BCB. Regardless estradiol secretion and glucose consumption, all other evaluations presented similar results between control and treated (BCB) groups, independently of cell type. Therefore, our results demonstrate convincingly that BCB seems to be safe for use in humans and it should encourage future studies to evaluate the development of embryos derived from human oocytes selected by BCB staining.

Safety of brilliant cresyl blue staining protocols on human granulosa and cumulus cells.

The selection of human immature oocytes destined for in vitro maturation (IVM) is performed according to their cumulus-oocyte complex (COC) morphology. In animal models, oocyte pre-selection with brilliant cresyl blue (BCB) staining improves fertilization and blastocyst rates and even increases the number of calves born. As the granulosa cells and cumulus cells (GCs and CCs) have a close relationship with the oocyte and are available in in vitro fertilization (IVF) programs, applying BCB staining to these cells may help to elucidate whether BCB shows toxicity to human oocytes and to determine the safest protocol for this dye. GCs and CCs were isolated from 24 patients who underwent controlled ovarian stimulation. After 48 h, cells were exposed to: Dulbecco's Modified Eagle Medium (DMEM) with or without phenol red, DPBS and mDPBS for 60 min; 13, 20 and 26 µM BCB for 60 min; and 60, 90 or 120 min to 13 µM BCB. Cellular viability was tested using 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) and trypan blue assays. The 20 and 26 µM BCB exposures resulted in lower cell viability, similar to when cells were exposed to BCB for 90 or 120 min. GCs and CCs viabilities were equal among control group and 13 µM BCB group after 60 min. BCB staining was not toxic to GCs and CCs when the regime of 13 µM BCB for 60 min was used. Due to the close molecular/biochemical relationship between these cells and the gamete, we propose that it is unlikely that the use of BCB could interfere with the viability/health of human oocytes.

Selection of Rattus norvegicus oocytes for in vitro maturation by brilliant cresyl blue staining.

The objective of this work was to evaluate the rate of meiosis resumption and nuclear maturation of rat (Rattus norvegicus) oocytes selected for in vitro maturation (IVM) after staining of cumulus-oocyte complexes (COCs) with blue cresyl brilliant (BCB) using different protocols: exposure for 30, 60 or 90 min at 26 µM BCB (Experiment 1), and exposure for 60 min at 13, 20 or 26 µM BCB (Experiment 2). In Experiment 1, the selection of oocytes exposed to BCB for 60 min was found to be the most suitable, as meiosis resumption rates in the BCB(+) group (n = 35/61; 57.37%) were the closest to the observed in the control (not exposed) group (n = 70/90; 77.77%) and statistically higher than the values observed for the BCB(-) group (n = 3/41; 7.32%). Additionally, the more effective evaluation of diagnostic tests (sensitivity and negative predictive value 100%) was observed in COCs exposed for 60 min. In Experiment 2, the 13 µM BCB(+) group presented rates of meiosis resumption (n = 57/72; 72.22%) similar to the control group (n = 87/105; 82.86%) and higher than other concentration groups. However, this results of the analysis between BCB(-) oocytes was also higher in the 13 µM BCB group (n = 28/91; 30.78%) when compared with BCB(-) COCs exposed to 20 µM (n = 3/62; 4.84%) or 26 µM (n = 3/61; 4.92%) BCB. The nuclear maturation rate in the 13 µM BCB group was similar between BCB(+) or BCB(-) oocytes. The 20 µM BCB group had a lower rate of nuclear maturation of BCB(-) oocytes than other groups. Thus, our best results in the selection of Rattus norvegicus oocytes by staining with BCB were obtained using the concentration of 13 µM and 20 µM, and an incubation period of 60 min.