scholarly journals EFFECT OF ANTIGEN-PRIMED DENDRITIC CELL-BASED IMMUNOTHERAPY ON ANTITUMOR CELLULAR IMMUNE RESPONSE IN PATIENTS WITH COLORECTAL CANCER

2021 ◽  
Vol 23 (4) ◽  
pp. 963-968
Author(s):  
V. V. Kurilin ◽  
E. V. Kulikova ◽  
A. V. Sokolov ◽  
Yu. A. Kozhevnikov ◽  
D. D. Blinova ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Immune Response ◽  
Dendritic Cells ◽  
Cancer Patients ◽  
Peripheral Blood ◽  
T Regulatory Cells ◽  
Mononuclear Cells ◽  
Tumor Antigens ◽  
Autologous Tumor ◽  
Oncological Diseases

The problem of treatment of oncological diseases is one of the most urgent for modern medicine. Existing treatment approaches are based on a surgical, radiation, chemotherapeutic approach, and the use of immunotherapy methods aimed at markers and / or specific antigens of tumors.Approaches based on the mechanisms of cellular and molecular regulation of a specific antitumor immune response have shown their high efficiency (for example, antibodies against HER2 in breast cancer), but these approaches have a number of side and undesirable effects that limit their application. Considering the central role of the mechanisms of recognition of tumor antigens and their presentation to cytotoxic cells in effective tumor elimination, it is important to search for and develop approaches to restore these mechanisms in cancer pathology. Because maturation, differentiation of dendritic cells and their main function are impaired in oncological diseases, scientific research is underway to obtain mature dendritic cells and restore the natural way of antigen presentation to effector cells.The work carried out limited clinical studies (13 patients with colorectal cancer), a previously developed protocol for obtaining antigen-primed dendritic cells of patients with colorectal cancer and their joint culture with autologous mononuclear cells in vitro. From the peripheral blood of cancer patients, dendritic cells primed with autologous tumor antigens (tumor cell lysate), which were co-cultured with their own mononuclear cells in the presence of immunoregulatory cytokines (IL-12 and IL-18). The resulting cell suspensions were purified from the culture medium and cytokines and used for a course of immunotherapy (weekly, 20-30 million cells intravenously, dropwise), consisting of 3-5 injections. At different periods of immunotherapy (before the start of the course of immunotherapy, 3 months and 6 months after the end of immunotherapy), immunological parameters were assessed in the peripheral blood of patients (immunogram (CD3+, CD3+CD4+, CD3+CD8+, CD19, CD16+CD56+-cells), the relative content of T-regulatory cells (CD4+CD25+FoxP3+-cells), myeloid suppressor cells (CD14+HLA-DR- cells)) and assessed the cytotoxic activity of peripheral blood mononuclear cells of patients against cells of the tumor line of human colorectal cancer (Colo-320).The data obtained showed that in cancer patients, against the background of ongoing immunotherapy, the indicator of the direct cytotoxic test significantly increases, which makes it possible to judge the effective stimulation of the antitumor cellular immune response. This is also indicated by an increase in the relative number of CD16+CD56+-cells (NK-cells) 3 months after immunotherapy. The study of immunosuppressive cells in the blood of cancer patients showed the absence of significant changes in CD14+HLA-DR- -cells and T-regulatory cells.Thus, limited clinical studies of immunotherapy of patients with colorectal cancer based on autologous dendritic cells primed with lysate of autologous tumor cells demonstrated an increase in the antitumor cytotoxic immune response. 

scholarly journals Immune Characterization Of Metastatic Colorectal Cancer Patients Post Reovirus Administration

2020 ◽  
Author(s):  
Ruwan Parakrama ◽  
Elisha Fogel ◽  
Carol Chandy ◽  
Titto Augustine ◽  
Matt Coffey ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Flow Cytometry ◽  
Cancer Patients ◽  
Tumor Cells ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Sequencing Analysis ◽  
Peripheral Blood Mononuclear ◽  
Blood Mononuclear Cells

Abstract Background KRAS mutations are prevalent in 40-45% of patients with colorectal cancer (CRC) and targeting this gene has remained elusive. Viruses are well known immune sensitizing agents. The therapeutic efficacy of oncolytic reovirus in combination with chemotherapy is underway in a phase 1 study of metastatic CRC. This study evaluates the nature of immune response by determining the cytokine expression pattern in peripheral circulation along with the distribution of antigen presenting cells (APCs) and activated T lymphocytes. Further the study evaluates the alterations in exosomal and cellular microRNA levels along with the effect of reovirus on leukocyte transcriptome.Methods Reovirus was administered as a 60-minute intravenous infusion for 5 consecutive days every 28 days, at a tissue culture infective dose (TCID50) of 3x1010. Peripheral blood mononuclear cells (PBMC) were isolated from whole blood prior to reovirus administration and post-reovirus on days 2, 8, and 15. The expression profile of 25 cytokines in plasma was assessed (post PBMC isolation) on an EMD Millipore multiplex Luminex platform. Exosome and cellular levels of miR-29a-3p was determined in pre and post reovirus treated samples. Peripheral blood mononuclear cells were stained with fluorophore labelled antibodies against CD4, CD8, CD56, CD70, and CD123, fixed and evaluated by flow cytometry. The expression of granzyme B was determined on core biopsy of one patient. Finally, Clariom D Assay, was used to determine the expression of 847 immune-related genes when compared to pre reovirus treatment by RNA sequencing analysis. A change was considered if the expression level either doubled or halved and the significance was determined at a p value of 0.001. Results Cytokine assay indicated upregulation at day 8 for IL-12p40 (2.95; p=0.05); day 15 for GM-CSF (3.56; p=0.009), IFN-Ƴ (1.86; p=0.0004) and IL-12p70 (2.42; p=0.02). An overall reduction in IL-8, VEGF and RANTES/CCL5 was observed over the 15-day period. Statistically significant reductions were observed at Day 15 for IL-8 (0.457-fold, 53.3% reduction; p=0.03) and RANTES/CC5 (0.524-fold, 47.6% reduction; p=0.003). An overall increase in IL-6 was observed, with statistical significance at day 8 (1.98-fold; 98% increase, p=0.00007). APCs were stimulated within 48 hours and activated (CD8+ CD70+) T cells within 168 hours as determine by flow cytometry. Sustained reductions in exosomal and cellular levels of miR-29a-3p (a microRNA upregulated in CRC and associated with decreased expression of the tumor suppressor WWOX gene) was documented. Reovirus administration further resulted in increases in KRAS (33x) , IFNAR1 (20x), STAT3(5x), and TAP1 (4x) genes after 2 days; FGCR2A (23x) and CD244 (3x) after 8 days; KLRD1 (14x), TAP1 (2x) and CD244(2x) after 15 days. Reductions (>0.5x) were observed in VEGFA (2x) after 2 days; CXCR2 (2x), ITGAM (3x) after 15 days.Conclusions Reovirus has profound immunomodulatory properties that span the genomic, protein and immune cell distribution levels. This is the first study with reovirus in cancer patients that demonstrates these multi-layered effects, demonstrating how reovirus can function as an immune stimulant (augmenting the efficacy of immuno-chemo-therapeutic drugs), and an oncolytic agent. Reovirus thus functions bimodally as an oncolytic agent causing lysis of tumor cells, and facilitator of immune-mediated recognition and destruction of tumor cells.

scholarly journals IMMU-22. PHASE IB IMMUNOTHERAPY CLINICAL TRIAL WITH THE USE OF AUTOLOGOUS DENDRITIC CELLS PULSED WITH AN ALLOGENIC TUMORAL CELL LINES LYSATE IN PATIENTS WITH NEWLY DIAGNOSED DIFFUSE INTRINSIC PONTINE GLIOMA (DIPG)

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii364-iii364
Author(s):  
Andres Morales La Madrid ◽  
Jaume Mora ◽  
Ofelia Cruz ◽  
Vicente Santa-Maria Lopez ◽  
Sara Perez-Jaume ◽  
...  
Keyword(s):  
Immune Response ◽  
Dendritic Cells ◽  
Mononuclear Cells ◽  
Tumor Antigens ◽  
Progression Free Survival ◽  
Diffuse Intrinsic Pontine Glioma ◽  
Newly Diagnosed ◽  
Tumor Lysate ◽  
Pontine Glioma ◽  
Peripheral Blood Mononuclear

Abstract BACKGROUND Diffuse intrinsic pontine glioma (DIPG) is a lethal condition, and therefore novel approaches are needed. Monocyte-derived dendritic cells (mDCs) pulsed with tumor antigens, as professional antigen-presenting cells, are a promising strategy for immunotherapy of invasive brain tumors. METHODS Our Ib pilot study explored the use of immunotherapy with mDCs for the treatment of newly diagnosed DIPG. Patient′s mDCs were extracted after irradiation and were primed with an allogenic tumor lysate from five patients with K27M-mutated DIPGs. The principal goal of this study was to establish the feasibility and safety of the intradermic administration of these mDC vaccines in patients with DIPG. In the absence of progression, patients received maintenance boosts of tumor lysate. Additionally, we evaluated the non-specific and antitumoral immune response generated in peripheral blood mononuclear cells (PBMC) and in cerebrospinal fluid (CSF) cells. RESULTS Nine patients were included in the study (2016–2018). Vaccines fabrication was feasible and administered in all cases without grade 3 or 4 toxicities. KLH (9/9 patients) and antitumor (8/9 patients) specific responses were identified in PBMC. Immunological responses were also confirmed in T-lymphocytes from the CSF of two patients. Twenty-four month overall survival and progression free survival was 33.3% (95 % CI 13.2% to 84.0 %) and zero, respectively. DISCUSSION These results demonstrate that mDC vaccination is feasible, safe, and generates a DIPG-specific immune response detected in PBMC and CSF. There was a trend in improved OS when compared to historic controls. This strategy shows a promising immunotherapy backbone for future combination schemas.

scholarly journals Immune Characterization of Metastatic Colorectal Cancer Patients Post Reovirus Administration

2020 ◽  
Author(s):  
Ruwan Parakrama ◽  
Elisha Fogel ◽  
Carol Chandy ◽  
Titto Augustine ◽  
Matt Coffey ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Flow Cytometry ◽  
Cancer Patients ◽  
Tumor Cells ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Sequencing Analysis ◽  
Peripheral Blood Mononuclear ◽  
Blood Mononuclear Cells

Abstract Background: KRAS mutations are prevalent in 40-45% of patients with colorectal cancer (CRC) and targeting this gene has remained elusive. Viruses are well known immune sensitizing agents. The therapeutic efficacy of oncolytic reovirus in combination with chemotherapy is underway in a phase 1 study of metastatic CRC. This study evaluates the nature of immune response by determining the cytokine expression pattern in peripheral circulation along with the distribution of antigen presenting cells (APCs) and activated T lymphocytes. Further the study evaluates the alterations in exosomal and cellular microRNA levels along with the effect of reovirus on leukocyte transcriptome.Methods: Reovirus was administered as a 60-minute intravenous infusion for 5 consecutive days every 28 days, at a tissue culture infective dose (TCID50) of 3x1010. Peripheral blood mononuclear cells (PBMC) were isolated from whole blood prior to reovirus administration and post-reovirus on days 2, 8, and 15. The expression profile of 25 cytokines in plasma was assessed (post PBMC isolation) on an EMD Millipore multiplex Luminex platform. Exosome and cellular levels of miR-29a-3p was determined in pre and post reovirus treated samples. Peripheral blood mononuclear cells were stained with fluorophore labelled antibodies against CD4, CD8, CD56, CD70, and CD123, fixed and evaluated by flow cytometry. The expression of granzyme B was determined on core biopsy of one patient. Finally, Clariom D Assay, was used to determine the expression of 847 immune-related genes when compared to pre reovirus treatment by RNA sequencing analysis. A change was considered if the expression level either doubled or halved and the significance was determined at a p value of 0.001.Results:Cytokine assay indicated upregulation at day 8 for IL-12p40 (2.95; p=0.05); day 15 for GM-CSF (3.56; p=0.009), IFN-Ƴ (1.86; p=0.0004) and IL-12p70 (2.42; p=0.02). An overall reduction in IL-8, VEGF and RANTES/CCL5 was observed over the 15-day period. Statistically significant reductions were observed at Day 15 for IL-8 (0.457-fold, 53.3% reduction; p=0.03) and RANTES/CC5 (0.524-fold, 47.6% reduction; p=0.003). An overall increase in IL-6 was observed, with statistical significance at day 8 (1.98- fold; 98% increase, p=0.00007). APCs were stimulated within 48 hours and activated (CD8+ CD70+) T cells within 168 hours as determine by flow cytometry. Sustained reductions in exosomal and cellular levels of miR-29a-3p (a microRNA upregulated in CRC and associated with decreased expression of the tumor suppressor WWOX gene) was documented. Reovirus administration further resulted in increases in KRAS (33x) , IFNAR1 (20x), STAT3(5x), and TAP1 (4x) genes after 2 days; FGCR2A (23x) and CD244 (3x) after 8 days; KLRD1 (14x), TAP1 (2x) and CD244(2x) after 15 days. Reductions (>0.5x) were observed in VEGFA (2x) after 2 days; CXCR2 (2x), ITGAM (3x) after 15 days.Conclusions: Reovirus has profound immunomodulatory properties that span the genomic, protein and immune cell distribution levels. This is the first study with reovirus in cancer patients that demonstrates these multi- layered effects, demonstrating how reovirus can function as an immune stimulant (augmenting the efficacy of immuno-chemo-therapeutic drugs), and an oncolytic agent. Reovirus thus functions bimodally as an oncolytic agent causing lysis of tumor cells, and facilitator of immune-mediated recognition and destruction of tumor cells.

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