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Cell transplants in therapeutic studies do not preserve their long-term function inside the donor body. In mesenchymal stem cell (MSC) transplants, transplanted cells disperse through the body and are prone to degradation by immune cells after the transplant process. Various strategies, such as usage of the immunosuppressive drugs to eliminate allograft rejection, are designed to increase the efficiency of cell therapy. Another strategy is the construction of biomimetic encapsulates using polymeric materials, which isolate stem cells and protect them from environmental effects. In this study, fibroblasts (L929) and MSCs were investigated for their improved viability and functionality once encapsulated inside the alginate microbeads under in vitro conditions for up to 12 days of incubation. Thus, uniform and injectable (

Introduction The efficacy of SARS-CoV2 standard single donor convalescent plasma varied according to the application time and most importantly the amount of antibody that is administered. Single donor plasma has some drawbacks; such as the insufficient levels of neutralizing antibody activities, the requirements of blood group compatibility, and the risk of infection transmission. In this study, the efficacy and safety of pathogen inactivated, isohemagglutinin-depleted (concentrated) and pooled convalescent plasma was investigated. Methods In this study, ACB-IP 1.0 convalescent plasma product was prepared as follows; first, convalescent plasma was collected from different donors, then pathogen-inactivation was carried-out, and isohemagglutinins were cryodepleted, respectively. Finally, concentrated convalescent plasma product was pooled and stored until use. A total of sixteen patients were treated with two different convalescent plasma products. Nine patients were treated with standard single donor convalescent plasma and seven were treated with pathogen-free, concentrated, pooled convalescent plasma (ACB-IP 1.0) between 01 April 2020 and 31 December 2020. The outcomes of these two plasma products were compared regarding SARS-CoV2 antibody titers, neutralizing antibody activities, length of hospitalization and mortality rates. Results Five out of six single donor plasma SARS-CoV2 antibody titers remained below 12 s/co, but the antibody titers of all ACB-IP 1.0 plasma were above 12 s/co. SARS-CoV2 total antibody titers of ACB-IP 1.0 plasma were statistically higher than the antibody titers of single donor plasma. Mean total plasma neutralizing antibody activity of ACB-IP 1.0 plasma (1.5421) was found statistically higher than single donor plasma (0.9642) in 1:256 dilution (ρ=0.0087) The mortality rate of the patients treated with ACB-IP 1.0 plasma showed statistically lower (p: 0,033) than the patients treated with single donor plasma. The administration of either single donor plasma or ACB-IP 1.0 plasma to the patients within eight days significantly shortened the length of hospitalization compared to administration of either plasma to the patients later than eight days (ρ= 0,0021) Discussion Pathogen-free, concentrated, pooled convalescent plasma may resolve the bias in SARS-CoV2 antibody titers and neutralizing antibody activities, without requiring blood group compatibility that allows patient accessibility in a shorter time and has safe plasma characteristic. This study indicates that ACB-IP 1.0 may be a superior product compared to standard single donor plasma. (Patent Application No: PY2020-00232)

ObjectiveChimeric antigen receptor T (CAR-T) cell therapies already made an impact on the treatment of B cell malignancies. Although CAR-T cell therapies are promising, there are concerns with commercial products regarding their affordability and sustainability. In this preliminary study, results of the first productional and clinical data of academic CAR-T cell (ISIKOK-19) from Turkey are presented.Materials and MethodsA pilot clinical trial (NCT04206943) designed to assess the safety and feasibility of ISIKOK-19 T-cell therapy in patients with relapsed and refractory CD19+ tumors was conducted and participating patients received ISIKOK-19 infusions between October 2019 and July 2021. Production data of the first 8 patients and the clinical outcome of 7 patients who received ISIKOK-19 cell infusion is presented in this study.ResultsNine patients were enrolled for the trial (ALL n=5 and NHL n=4) but only 7 patients could receive the treatment. Two out of three ALL patients and three out of four NHL patients had complete/partial response (ORR 72%). Four patients (57%) had CAR-T-related toxicities (CRS, CRES, and pancytopenia). Two patients were unresponsive and had progressive disease following CAR-T therapy. Two patients with partial response had progressive disease during follow-up.ConclusionProduction efficacy and fulfilling the criteria of quality control were satisfactory for academic production. Response rates and toxicity profiles are acceptable for this heavily pretreated/refractory patient group. ISIKOK-19 cells appear to be a safe, economical, and efficient treatment option for CD19 positive tumors. The findings of this study need to be supported by the currently ongoing clinical trial of ISIKOK-19.

IntroductionThe efficacy of SARS-CoV2 standard single donor convalescent plasma varied according to the application time and most importantly the amount of antibody that is administered. Single donor plasma has some drawbacks; such as the insufficient levels of neutralizing antibody activities, the requirements of blood group compatibility, and the risk of infection transmission. In this study, the efficacy and safety of pathogen inactivated, isohemagglutinin-depleted (concentrated) and pooled convalescent plasma was investigated.MethodsIn this study, ACB-IP 1.0 convalescent plasma product was prepared as follows; first, convalescent plasma was collected from different donors, then pathogen-inactivation was carried-out, and isohemagglutinins were cryodepleted, respectively. Finally, concentrated convalescent plasma product was pooled and stored until use.A total of sixteen patients were treated with two different convalescent plasma products. Nine patients were treated with standard single donor convalescent plasma and seven were treated with pathogen-free, concentrated, pooled convalescent plasma (ACB-IP 1.0) between 01 April 2020 and 31 December 2020.The outcomes of these two plasma products were compared regarding SARS-CoV2 antibody titers, neutralizing antibody activities, length of hospitalization and mortality rates.ResultsFive out of six single donor plasma SARS-CoV2 antibody titers remained below 12 s/co, but the antibody titers of all ACB-IP 1.0 plasma were above 12 s/co. SARS-CoV2 total antibody titers of ACB-IP 1.0 plasma were statistically higher than the antibody titers of single donor plasma. Mean total plasma neutralizing antibody activity of ACB-IP 1.0 plasma (1.5421) was found statistically higher than single donor plasma (0.9642) in 1:256 dilution (ρ=0.0087)The mortality rate of the patients treated with ACB-IP 1.0 plasma showed statistically lower (p: 0,033) than the patients treated with single donor plasma. The administration of either single donor plasma or ACB-IP 1.0 plasma to the patients within eight days significantly shortened the length of hospitalization compared to administration of either plasma to the patients later than eight days (ρ= 0,0021)DiscussionPathogen-free, concentrated, pooled convalescent plasma may resolve the bias in SARS-CoV2 antibody titers and neutralizing antibody activities, without requiring blood group compatibility that allows patient accessibility in a shorter time and has safe plasma characteristic. This study indicates that ACB-IP 1.0 may be a superior product compared to standard single donor plasma.(Patent Application No: PY2020-00232)

Current evidence strongly suggests that aberrant activation of the nuclear factor kappa B (NF-kB) signaling cascade is connected to carcinogenesis. The matrix metalloproteinases (MMP) which are also the key agents for tumor metastasis may be potent candidates for tumor diagnosis in clinics. In this in vitro study, we hypothesized that metformin with an effective dose can inhibit tumor cell proliferation and metastasis by modulating the expressions of MMP-2 and -9 and interfering with NF-kB signaling in primary breast cancer cells (PBCCs). 300 000 cells per ml were obtained from biopsies of breast tumors from five human donors. The cell viability and proliferation were tested. Immunocytochemistry was performed for MMP-2, MMP-9, and NF-kB, and enzyme-linked immunosorbent assay for NF-kB activity, quantitative real-time PCR for RELA/p65, IkBα, MMP-2, and MMP-9. Three different doses of metformin (5, 10, and 25 mM) (Met) reduced the viability and proliferation of PBCCs in a dose-dependent manner, maximum inhibition was observed at 25 mM Met. The expression of RELA/p65 was not affected by 25 mM Met. Nuclear immunoreactivity and activity of NF-kB reduced while cytoplasmic NF-kB (p65) elevated by 25 mM Met compared to non-treatment (P < 0.05). The expression and immunoreactivity of MMP-9 but not MMP-2 were decreased by 25 mM Met treatment, compared with the non-treatment (P < 0.05). Metformin may have an essential antitumor role in the invasion and metastasis pathways of PBCCs by downregulating the MMP-9 expression blocking both the activity and nuclear translocation of NF-kB.

The SARS-CoV-2 virus caused the most severe pandemic around the world, and vaccine development for urgent use became a crucial issue. Inactivated virus formulated vaccines such as Hepatitis A, oral polio vaccine, and smallpox proved to be reliable approaches for immunization for prolonged periods. During the pandemic, we produced an inactivated SARS-CoV-2 vaccine candidate, having the advantages of being manufactured rapidly and tested easily in comparison with recombinant vaccines. In this study, an inactivated virus vaccine that includes a gamma irradiation process for the inactivation as an alternative to classical chemical inactivation methods so that there is no extra purification required has been optimized. The vaccine candidate (OZG-38.61.3) was then applied in mice by employing the intradermal route, which decreased the requirement of a higher concentration of inactivated virus for proper immunization, unlike most of the classical inactivated vaccine treatments. Hence, the novelty of our vaccine candidate (OZG-38.61.3) is that it is a non-adjuvant added, gamma-irradiated, and intradermally applied inactive viral vaccine. Efficiency and safety dose (either 1013 or 1014 viral copy per dose) of OZG-38.61.3 was initially determined in Balb/c mice. This was followed by testing the immunogenicity and protective efficacy of OZG-38.61.3. Human ACE2-encoding transgenic mice were immunized and then infected with a dose of infective SARS-CoV-2 virus for the challenge test. Findings of this study show that vaccinated mice have lower SARS-CoV-2 viral copy number in oropharyngeal specimens along with humoral and cellular immune responses against the SARS-CoV-2, including the neutralizing antibodies similar to those shown in Balb/c mice without substantial toxicity. Subsequently, plans are being made for the commencement of Phase 1 clinical trial of the OZG-38.61.3 vaccine for the COVID-19 pandemic.

The COVID-19 outbreak caused by SARS-CoV-2 has created an unprecedented health crisis since there is no coronavirus vaccine in the market due to the novelty of this virus. Therefore, SARS-CoV-2 vaccines have become very important to reduce morbidity and mortality. At this point, inactivated vaccines are important because the straightforward process of existing infrastructure used for several licensed human vaccines can be used for SARS-CoV-2. Inactive vaccines provide an antigenic presentation similar to that when they encounter invasive virus particles of the immune system. In this study, in vitro and in vivo safety and efficacy analyzes of lyophilized vaccine candidates inactivated by gamma-irradiation were performed. Our candidate OZG-3861 version 1 (V1) is an inactivated SARS-CoV-2 virus vaccine, and SK-01 version 1 (V1) is the GM-CSF adjuvant added vaccine candidate. We applied the candidates intradermal to BALB/c mice to assess the toxicity and immunogenicity of the OZG-3861 V1 and SK-01 V1. Here, we report our preliminary results in vaccinated mice. When considered in terms of T and B cell responses, it was observed that especially the vaccine models containing GM-CSF as an adjuvant caused significant antibody production with neutralization capacity in absence of the antibody-dependent enhancement feature. Another finding showed that the presence of adjuvant is more important in T cell response rather than B cell. The vaccinated mice showed T cell response upon restimulation with whole inactivated SARS-CoV-2 or peptide pool. This study encouraged us to start the challenge test using infective SARS-CoV-2 viruses and our second version of gamma-irradiated inactivated vaccine candidates in humanized ACE2+ mice.

Dornase alfa, the recombinant form of the human DNase I enzyme, breaks down neutrophil extracellular traps (NET) that include a vast amount of DNA fragments, histones, microbicidal proteins and oxidant enzymes released from necrotic neutrophils in the highly viscous mucus of cystic fibrosis patients. Dornase alfa has been used for decades in patients with cystic fibrosis to reduce the viscoelasticity of respiratory tract secretions, to decrease the severity of respiratory tract infections, and to improve lung function. Previous studies have linked abnormal NET formations to lung diseases, especially to acute respiratory distress syndrome (ARDS). It is well known that novel coronavirus disease 2019 (COVID-19) pneumonia progresses to ARDS and even multiple organ failure. High blood neutrophil levels are an early indicator of COVID-19 and predict severe respiratory diseases. Also it is reported that mucus structure in COVID-19 is very similar to that in cystic fibrosis due to the accumulation of excessive NET in the lungs. In this study, we showed the recovery of three individuals with COVID-19 after including dornase alfa in their treatment. We followed clinical improvement in the radiological analysis (two of three cases), oxygen saturation (SpO(2)), respiratory rate, disappearance of dyspnoea, coughing and a decrease in NET formation and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral load after the treatment. Also here, we share our preliminary results suggesting that dornase alfa has an anti-viral effect against SARS-CoV-2 infection in a green monkey kidney cell line, Vero, and a bovine kidney cell line, MDBK, without determined cytotoxicity on healthy peripheral blood mononuclear cells. (C) 2020 The Authors. Published by Elsevier Ltd.

Relapsed and refractory CD19-positive B-cell acute lymphoblastic leukemia (ALL) and non-Hodgkin lymphoma (NHL) are the focus of studies on hematological cancers. Treatment of these malignancies has undergone recent transformation with the development of new gene therapy and molecular biology techniques, which are safer and well-tolerated therapeutic approaches. The CD19 antigen is the most studied therapeutic target in these hematological cancers. This study reports the results of clinical-grade production, quality control, and in vivo efficacy processes of ISIKOK-19 cells as the first academic clinical trial of CAR-T cells targeting CD19-expressing B cells in relapsed/refractory ALL and NHL patients in Turkey.We used a lentiviral vector encoding the CD19 antigen-specific antibody head (FMC63) conjugated with the CD8-CD28-CD3ζ sequence as a chimeric antigen receptor (CAR) along with a truncated form of EGFR (EGFRt) on human T-lymphocytes (CAR-T). We preclinically assessed the efficacy and safety of the manufactured CAR-T cells, namely ISIKOK-19, from both healthy donors’ and ALL/NHL patients’ peripheral blood mononuclear cells.We showed significant enhancement of CAR lentivirus transduction efficacy in T-cells using BX-795, an inhibitor of the signaling molecule TBK1/IKKƐ, in order to cut the cost of CAR-T cell production. In addition, ISIKOK-19 cells demonstrated a significantly high level of cytotoxicity specifically against a CD19+ B-lymphocyte cancer model, RAJI cells, in NOD/SCID mice.This is the first report of preclinical assessment of efficacy and safety analysis of CAR-T cells (ISIKOK-19) targeting CD19-expressing B cells in relapsed/refractory ALL and NHL patients in Turkey.Relaps/refrakter CD19-pozitif B-hücreli akut lenfoblastik lösemi (ALL) ve Hodgkin dışı lenfoma (NHL), hematolojik kanserlerle ilgili çalışmaların odak noktasıdır. Bu malignitelerin tedavisi, daha güvenilir ve iyi tolere edilen terapötik yaklaşımlar olan, yeni gen terapisi ve moleküler biyoloji tekniklerinin geliştirilmesi ile son zamanlarda dönüşüm geçirmiştir. CD19 antijeni, bu hematolojik kanserlerde en çok çalışılan terapötik hedeftir. Bu çalışma, Türkiye’de relaps/refrakter ALL ve NHL hastalarında, ilk akademik klinik deneme için kullanılması amacıyla, CD19 eksprese eden B hücrelerini hedefleyen CAR-T (ISIKOK-19) hücrelerinin klinik sınıf üretimi, kalite kontrolü ve in vivo etkinlik süreçlerinin sonuçlarını bildirmektedir.Bu çalışmada, insan T-lenfositleri (CAR-T) üzerinde kesilmiş EGFR (EGFRt) formu ile birlikte kimerik bir antijen reseptörü (CAR) olarak CD8-CD28-CD3ζ sekansı ile konjuge CD19 antijene özgü antikor başını (FMC63) eksprese eden bir lentiviral vektör kullandık. Hem sağlıklı donörlerin hem de ALL/NHL hastalarının periferal kan mononükleer hücrelerinden üretilen CAR-T (ISIKOK-19) hücrelerinin etkinliğini ve güvenliğini klinik öncesi olarak değerlendirdik.CAR-T hücre üretiminin maliyetini düşürmek için sinyal molekülü TBK1/IKKƐ’nın bir inhibitörü olan BX-795 kullanarak T-hücrelerinde CAR lentivirüs transdüksiyon etkinliğinin önemli ölçüde arttığını gösterdik. Ek olarak, ISIKOK-19 CAR-T hücrelerinin, NOD/SCID farelerinde özellikle bir CD19 + Blenfosit kanser modeli olan RAJI hücrelerine karşı önemli ölçüde ve yüksek seviyede sitotoksisite gösterdiğini değerlendirdik.Bu çalışma, Türkiye’de relaps/refrakter ALL ve NHL hastalarında CD19 eksprese eden B-hücrelerini hedefleyen CAR-T hücrelerinin ilk akademik klinik çalışması olarak ISIKOK-19 hücrelerinin klinik sınıf üretimi, kalite kontrolü ve in vivo etkinlik süreçlerinin sonuçlarını bildirmektedir.