Improved efficacy of a dendritic cell-based vaccine against a murine model of colon cancer: The helper protein effect

Purpose Targeted immunotherapy using dendritic cells (DCs) has been employed in numerous investigations aiming at combating neoplasms. We previously showed that copulsing of an antigen with a helper protein could considerably enhance antigen presenting capacity of ex vivo-generated DCs. In this study, we attempted to administer an effective treatment in a murine model of colon cancer with DCs pulsed with the mixture of a tumor-specific gp70-derived peptide (AH1) and a helper protein, ovalbumin (OVA). Materials and Methods First, the presence of gp70 in CT26 tumor cells and tumor tissues was verified using immunofluorescence and Western blot analyses. Next, DCs were purified from normal mice, loaded ex vivowith AH1 and OVA (DC-Pep-OVA), and injected into tumor-bearing mice. Tumor volume, in vitro antigen (Ag)-specific proliferation of splenic cells, and survival rate were measured to determine the efficacy of DC-Pep-OVA. As the control groups, tumor-bearing mice were vaccinated with DC-Pep, unpulsed DC, and DCs loaded with a mixture of OVA and an irrelevant peptide (P15), or were not vaccinated at all. Results DC-Pep-OVA showed superior efficacy over other groups, as indicated by smaller tumor volume, higher Ag-specific proliferation rate of splenic cells, and prolonged survival. Conclusion Overall, in the present study we showed for the first time that DCs copulsed with AH1 (tumor Ag) and OVA (helper molecule) could be considered as potentially robust weapons for use in future antitumor immunotherapies. © 2015 by the Korean Cancer Association

Newborn screening for presymptomatic diagnosis of complement and phagocyte deficiencies

The clinical outcomes of primary immunodeficiencies (PIDs) are greatly improved by accurate diagnosis early in life. However, it is not common to consider PIDs before the manifestation of severe clinical symptoms. Including PIDs in the nation-wide newborn screening programs will potentially improve survival and provide better disease management and preventive care in PID patients. This calls for the detection of disease biomarkers in blood and the use of dried blood spot samples, which is a part of routine newborn screening programs worldwide. Here, we developed a newborn screening method based on multiplex protein profiling for parallel diagnosis of 22 innate immunodeficiencies affecting the complement system and respiratory burst function in phagocytosis. The proposed method uses a small fraction of eluted blood from dried blood spots and is applicable for population-scale performance. The diagnosis method is validated through a retrospective screening of immunodeficient patient samples. This diagnostic approach can pave the way for an earlier, more comprehensive and accurate diagnosis of complement and phagocytic disorders, which ultimately lead to a healthy and active life for the PID patientsThis work was supported by the Swedish Research Council (VR) and grants provided by the Stockholm County Council (ALF)

Feasibility study of injectable intervertebral disc-mimetic cell encapsulated microgels for nucleus pulposus regeneration

INTRODUCTION Low back pain is a common and widespread health problem affecting a significant percentage of adults (up to 80%) worldwide1. Mesenchymal stem cells (MSCs) have shown new hope to regenerate the early stages of intervertebral disc degeneration. However, there are debates as to whether transplanted cells in cell suspensions can survive in the harsh environment of degenerated IVDs, which may limit the effectiveness of cell therapy. An appropriate cell delivery system is necessary to ensure the efficacy of MSCs in vivo2. Herein, hybrid injectable microgels based on hyaluronic acid (HA) and collagen type II (COLII) were developed for cell delivery to the IVD, mimicking the polysaccharide-protein composition of the native NP extracellular matrix. The study also uses an ex vivo papain-induced bovine tail disc degeneration model to investigate this cell therapy. EXPERIMENTAL METHODS Tyramine-grafted HA and COLII were prepared using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and n-hydroxysulfosuccinimide (NHS) chemistry3. HA-Tyr and COLII-Tyr were dissolved in phosphate buffered saline (PBS) and mixed at different ratios followed by adding horseradish peroxidase (HPR). The polymer solution was extruded through a syringe pump and microdroplets were generated using a flicking-based vibrating nozzle system. The droplets fell into a H2O2 solution and formed microgels. The hydrogels were characterized in terms of their mechanical properties and gelation time. The shape and size distribution of the microgels were analyzed using phase contrast and scanning electron microscopy (SEM). RESULTS AND DISCUSSION With this novel approach, uniform microgels could be fabricated with variable size in the range of 300–500 µm and narrow distribution in all compositions (Figure 1). We are currently optimizing the injection conditions of microgels into NP tissue as a function of microgel composition and needle gauge. CONCLUSION This study presents the initial optimization process to produce enzymatically crosslinked HA-COLII microgels. The goal is to utilize these microgels as building blocks mimicking the extracellular matrix for NP tissue regeneration. REFERENCES Wöltje M. et al., Biomimetics. 12;8(2):152, 2023. Xu H. et al., Biofabrication. 31;13(1):015010, 2020. Jooybar E. et al., Acta Biomater. 1;83:233-44, 2019. ACKNOWLEDGMENTS This research was funded by the Council for Development of Stem Cell Sciences and Technologies (#57940), Iran National Science Foundation: INSF (# 99002288) and an SNF-bridge project # 40B2-0_211510/1

Generation of CD19-targeted chimeric antigen receptor T cells

Background: Current advancements in the field of chimeric antigen receptor (CAR) therapy, particularly U.S. FDA approval of Kymriah and Yescarta, heralds a new era of cancer treatment. This rapid progress in technology has urged more countries and institutions to keep pace with the fast-growing and developing technology of producing CAR T cell-based therapies in the race to develop new cancer-targeting drugs. Hence, for stepping in line with global advances and to pave the way for subsequent preclinical and clinical studies, we have established a development protocol for a cancer-targeting CAR T cell; we have chosen CD19 CAR T cell as a well-defined model to set-up T cell expansion, activation, and viral transduction as the prerequisites for diverse CAR T cell therapies. Methods: T cells from peripheral blood mononuclear cells (PBMCs) were activated and expanded. CD19 CAR lentiviral particles were produced in the Lenti-X� 293T Cell Line using PolyFect Transfection Reagent. Results: Activation protocol resulted in (65 ± 4; P = 0.046) increase in the rate of activated T cells 24 hours after the initiation of the procedure. The expansion methodology resulted in a high purity of the T cell population (96 ± 3) in the pool of PBMCs within 14 days of the procedure. Finally, 35 ± 6 of T cells were transduced with CD19 lentivirus with MOI of 3. Conclusion: Collectively, the results of this study prove that we have successfully overcome the first hurdle on the road to reach CAR T cell technology which is the prerequisite for developing preclinical and clinical phases of CAR therapy in settings with basic resources. © 2018 The Author(s)

Various ways to improve whole cancer cell vaccines

Immunotherapy based on whole cancer cell vaccines is regarded as a promising avenue for cancer treatment. However, limited efficacy in the first human clinical trials calls for more optimized whole cancer cell vaccines and better patient selection. It is suggested that whole cancer cell vaccines consist preferably of immunogenically killed autologous cancer stem cells associated with dendritic cells. Adjuvants should stimulate both immune effector cells and memory cells, which could be achieved through their correct dosage and timing of administration. There are indications that whole cancer cell vaccination is less effective in patients who are immunocompromised, who have specific genetic defects in their immune or cancer cells, as well as in patients in an advanced cancer stage. However, such patients form the bulk of enrolled patients in clinical trials, prohibiting an objective evaluation of the true potential of whole cancer cell immunotherapy. Each key point will be discussed