Cell Dose Research Articles

Influence of Nutrient Medium Composition for Culturing Bacteria and the Dose of <i>B. subtilis</i> 10-4 Biologics on Growth Indicators and Productivity of Wheat Plants

Inoculation of crop seeds with selective strains of growth-stimulating bacteria is an environmentally friendly, low-cost way to increase their yield, but may depend on some biotechnological factors affecting their physiological activity. These include the conditions for the cultivation of bacteria and the dose of the drug. The work analyzed the effectiveness of inoculation of Bashkir spring wheat with 28 different preparations of the Bacillus subtilis strain 10-4: 1 - an aqueous suspension of bacterial cells washed off potato-glucose agar (PGA), 2 - a liquid culture containing cells with exometabolites obtained by cultivating bacteria in potato- glucose broth (PGB), 3 - liquid culture of bacterial cells grown in legume-glucose broth (LGB). At the same time, the growth-stimulating effect of applying a high dose (108 cells/ml) and a low dose (104-105 cells/ml) of bacteria in the inoculum was evaluated. When comparing the preparations obtained from potato-glucose medium, it was revealed that the positive growth effect (an increase in the number of wheat plant roots) was caused only by a high dose of the drug cells in the PGA and both doses of the drug in the PGB, but a high dose of the drug in the PGB inhibited seed germination up to 74% (in the control 100%). When comparing the preparations obtained during the cultivation of bacteria in PGB and LGB, it was revealed that the best growth effect (total root length and shoot height) was greater than control in plants inoculated with a small dose of the drug in PGB and a large dose of the drug in LGB. The stimulating effect of a small dose of the drug in LGB was less and extended only to the root system. The results of laboratory experiments coincided with the effectiveness of the studied drugs in the field. The use of a drug obtained in BGO and administered at a dose of 108 cells / ml, and a drug obtained in PGB and administered at a dose of 105 cells/ml, increased grain yield by 1.6-1.7 times with a decrease in the intensity of leaf-stem diseases by 32 and 11% compared with the untreated control. The drug obtained in LGB and introduced in a small dose did not provide either an increase in yield or a protective effect compared to the control. Possible causes of the action of B. subtilis 10-4 strain preparations related to the production of phytohormones by it, depending on the composition of the medium, and with different adaptive capacity of bacteria when cultured on nitrogen-rich and less nitrogen-rich media are discussed.

Survivin (BIRC5) Peptide Vaccine in the 4T1 Murine Mammary Tumor Model: A Potential Neoadjuvant T Cell Immunotherapy for Triple Negative Breast Cancer: A Preliminary Study

A triple negative breast cancer model using the murine 4T1 tumor cell line was used to explore the efficacy of an adjuvanted survivin peptide microparticle vaccine using tumor growth as the outcome metric. We first performed tumor cell dose titration studies to determine a tumor cell dose that resulted in sufficient tumor takes but allowed multiple serial measurements of tumor volumes, yet with minimal morbidity/mortality within the study period. Later, in a second cohort of mice, the survivin peptide microparticle vaccine was administered via intraperitoneal injection at the study start with a second dose given 14 days later. An orthotopic injection of 4T1 cells into the mammary tissue was performed on the same day as the administration of the second vaccine dose. The mice were followed for up to 41 days with subcutaneous measurements of tumor volume made every 3-4 days. Vaccination with survivin peptides was associated with a peptide antigen-specific gamma interferon enzyme-linked immunosorbent spot response in the murine splenocyte population but was absent from the control microparticle group. At the end of the study, we found that vaccination with adjuvanted survivin peptide microparticles resulted in statistically significant slower primary tumor growth rates in BALB/c mice challenged with 4T1 cells relative to the control peptideless vaccination group. These studies suggest that T cell immunotherapy specifically targeting survivin might be an applicable neoadjuvant immunotherapy therapy for triple negative breast cancer. More preclinical studies and clinical trials are needed to explore this concept further.

Stem cells for neonatal brain injury – Lessons from the bench

Neonatal brain injury resulting from various intractable disorders including intraventricular hemorrhage and hypoxic ischemic encephalopathy still remains a major cause of mortality and morbidities with few effective treatments. Recent preclinical research results showing the pleiotropic neuroprotective effects of stem cell therapy, specifically mesenchymal stem cells (MSCs), suggest that MSCs transplantation might be a promising new therapeutic modality for neuroprotection against the currently intractable and devastating neonatal brain injury with complex multifactorial etiology. This review summarizes recent advances in preclinical stem cell research for treating neonatal brain injury with a focus on the important issues including the mechanism of neuroprotection, and determining the ideal cell source, route, timing and dose of MSCs transplantation.

Evaluation of Transplanted Stem Cell Dynamic Variables in the Bone Marrow of Children with Malignancies

Objective: According to risk classifications, the transplant of healthy hematopoietic stem cells in the bone marrow is a priority treatment option for hematopoietic diseases. The effectiveness of hematopoietic stem cell transplantation after bone marrow transplantation is largely dependent on the capacity of the bone marrow microenvironment to accept transplanted cells. Detailed analysis of hematopoietic stem cell kinetics after transplantation is very important, as initial transplantation of hematopoietic stem cells affects prognosis. In this study, hematopoietic stem and progenitor cell percentage values were evaluated together with other transplantation-related parameters to determine the capacity of stem cell dynamic variables in the bone marrow. Methods: Dynamic variables in hematopoietic stem cell fate in bone marrow transplantation include many parameters; hematopoietic stem cell transplantation rates in the apheresis product of 13 patients and cell counts per kilogram and engraftment times, CD34+ cell rates, CD34+ cell counts per kilogram, chimerism rates, Graft-versus-Host Disease grades, white blood cell counts, and post-transplant relapse status. In the apheresis products collected from the patients before transplantation, hematopoietic stem and progenitor cells were identified by the immunophenotypic barcode "CD34+, CD38-, CD45RA-, CD90+, CD49f+", and data analysis was performed with the BD FACSDivaTM software by studying on the BD FACSCanto II flow cytometer device. Results: According to the results of the pearson correlation analysis (non-parametric correlations), a strong positive and significant relationship was found between the number of hematopoietic stem cell transplantation per kg and the percentage of hematopoietic stem cell transplantation of the patients (r=.733, p<.05). According to the results of the analysis, there was a moderately significant and positive relationship (r=.631, p<.05) between the percentage of hematopoietic stem cell transplantation and platelet engraftment time, while there was a strong inverse relationship between erythrocyte engraftment time (r=-.730, p<.01). A strong positive and significant relationship was found between the number of Hematopoietic stem cell transplantation cells per kilogram infused into the patients and the platelet engraftment times (r=.780, p<.05). Conclusion: This study confirms that a mean cell dose of hematopoietic stem and progenitor cells ≥ 2.57 x 106/kg infused provides rapid short- and long-term platelet engraftment in pediatric patients undergoing autologous and allogeneic transplantation. This study is important in terms of revealing the possible relationship between pulling forward the platelet engraftment time and the number and percentage of Hematopoietic stem and progenitor cells in our transplanted patients. It is also important that a statistically significant inverse relationship was determined between HSPC values and mean erythrocyte engraftment times.

Abstract P2-08-01: Licochalcone A from licorice reprograms metabolic and antioxidant pathways in the breast leading to a tumor preventive environment

Abstract Background: Increased adiposity is a risk factor for postmenopausal breast cancer. It is often accompanied by chronic low-grade inflammation and elevated levels of reactive oxygen species, which drive breast tumorigenesis. Risk reducing drugs such as selective estrogen receptor modulators and aromatase inhibitors, which have demonstrated efficacy, have had a significantly low acceptance among women at high risk for breast cancer. This hesitancy is mainly due to the adverse side effects of these medications such as vasomotor symptoms, osteoporosis, thromboembolism, and uterine cancer. Therefore, alternative strategies with lower toxicity and greater acceptability are needed. We have previously shown that licochalcone A (LicA) from licorice (Glycyrrhiza inflata), which has osteogenic effects, suppresses aromatase expression and activity, enhances the activity of detoxifying enzymes, and reduces estrogen genotoxic metabolism in cell lines and animal models. However, its effects on the breast tissue of high-risk women are understudied. We hypothesize that LicA creates a tumor preventive environment in the breast by locally modulating adipogenesis and antioxidant/anti-inflammatory responses leading to decreased proliferation. Methods: We prepared microstructures from fresh tissue of contralateral unaffected mastectomy specimens of 6 postmenopausal women with incident unilateral breast cancer. We exposed these to DMSO (control) and LicA (5 µM) for 24 h. Employing total RNA sequencing, we examined differential gene expression between treated and control samples. Up-regulated and down-regulated genes were analyzed using Enrichr gene ontology (GO) pathway analysis. Enriched pathways with combined enrichment scores > 4 and FDR < 0.05 were considered statistically significant. Metabolism flux analysis was performed (FDR < 0.05). Live cell imaging to monitor proliferation of pre-malignant DCIS.COM, DCIS.COM/ER+ PR+; and malignant MDA-MB-231 (ER- PR-), MCF-7 (ER+ PR+), MCF-7aro, and BRCA1 defective HCC-1937, and HCC3153 cells was conducted using IncuCyte. Single versus repeated dosing of various concentrations of LicA were also evaluated. Results: We observed significant (P < 0.05) upregulation up to 8-fold of antioxidant genes, consistent with significant upregulation of NRF2, the major regulator of antioxidant pathways. This was accompanied by significant (P < 0.05) downregulation of NF-kB dependent inflammatory pathways. In addition, we observed the significant (P < 0.05) downregulation, ranging from 4 to 32-fold of cholesterol biosynthesis and transport, steroid hormone biosynthesis, as well as lipid metabolism genes, consistent with the profound downregulation of SREBF1 and SREBF2, which encode the master regulator of adipogenesis, SREBP. Metabolic flux results demonstrated a robust increase (FDR < 0.05) in the pentose phosphate shunt and NAD(P)H generation without enhancing ribose 5 phosphate formation, confirming an antioxidant and anti-proliferative environment. Likewise, LicA suppressed proliferation of pre-malignant and malignant cells dose- and time-dependently. Repeated dosing of lower concentrations of LicA (< 10 µM) demonstrated sustained antiproliferative effects even in the aggressive cancer cell lines. Conclusion: Our data suggest that LicA can generate a tumor-preventive breast microenvironment by reprogramming metabolic pathways involved in steroid and lipid homeostasis and antioxidant responses. These observations along with its low toxicity, suggest that LicA is a good candidate for further investigation as a breast cancer prevention agent. Citation Format: Atieh Hajirahimkhan, Elizabeth T. Bartom, Sriram Chandrasekaran, Susan Clare, Seema Khan. Licochalcone A from licorice reprograms metabolic and antioxidant pathways in the breast leading to a tumor preventive environment. [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-08-01.

Association of CD34+ Cell Dose with Progression-free Survival after Allogeneic Peripheral Blood Hematopoietic Cell Transplantation in Children with Hematologic Malignancies

A higher CD34+ cell dose is associated with improved engraftment but may also be associated with an increased risk of complications after allogeneic hematopoietic stem cell transplantation, including graft-versus-host disease (GVHD). We retrospectively analyze the impact of CD34+ cell dose on OS, PFS, neutrophil engraftment, platelet engraftment, treatment-related mortality, and GVHD grading. For analyses, CD34+ cell dose was stratified into low (< 8.5×106/kg) and high (> 8.5×106/kg). A subgroup analysis of higher CD34+ cell dose leads to prolonged OS and PFS, but statistical significance was achieved only for PFS (OR 0.36; 95%CI 0.14-0.95; P=0.04). This study reinforced that CD34+ cell dose at the time of allo-HSCT retained a positive impact on PFS.

Nitrogen and copper-doped saffron-based carbon dots: Synthesis, characterization, and cytotoxic effects on human colorectal cancer cells

AimDoped carbon dots (CDs) have attracted tremendous attention in cancer therapy. We aimed to synthesize copper, nitrogen-doped carbon dots (Cu, N-CDs) from saffron and investigated their effects on HCT-116 and HT-29 colorectal cancer (CRC) cells. Main methodsCDs were synthesized by hydrothermal method and characterized by transmission electron microscopy (TEM), energy-dispersive X-ray (EDX), Fourier transform infrared (FT-IR) spectroscopy, ultraviolet-visible (UV–Vis) absorption spectroscopy, and fluorescence spectroscopy. HCT-116 and HT-29 cells were incubated with saffron, N-CDs, and Cu, N-CDs for 24 and 48 h for cell viability. Cellular uptake and intracellular reactive oxygen species (ROS) were evaluated by immunofluorescence microscopy. Oil Red O staining was used to monitor lipid accumulation. Apoptosis was evaluated using acridine orange/propidium iodide (AO/PI) staining and quantitative real-time polymerase chain reaction (Q-PCR) assay. The expression of miRNA-182 and miRNA-21 was measured by Q-PCR, while the generation of nitric oxide (NO) and lysyl oxidase (LOX) activity was calculated by colorimetric methods. Key findingsCDs were successfully prepared and characterized. Cell viability decreased in the treated cells dose- and time-dependently. HCT-116 and HT-29 cells uptook Cu, N-CDs with a high level of ROS generation. The Oil Red O staining showed lipid accumulation. Concomitant with an up-regulation of apoptotic genes (p < 0.05), AO/PI staining showed increased apoptosis in the treated cells. In comparison to control cells, NO generation, and miRNA-182 and miRNA-21 expression significantly changed in the Cu, N-CDs treated cells (p < 0.05). SignificanceThe results indicated that Cu, N-CDs could inhibit CRC cells through the induction of ROS generation and apoptosis.

Early results from a phase 1, multicenter trial of PSCA-specific GoCAR T cells (BPX-601) in patients with metastatic castration-resistant prostate cancer (mCRPC).

140 Background: Prostate stem cell antigen (PSCA) is expressed in &gt;80% of metastatic prostate cancers. BPX-601 is an autologous PSCA-directed chimeric antigen receptor (CAR)-T cell immunotherapy engineered to express a rimiducid-inducible MyD88/CD40 costimulation switch to enhance T cell potency and persistence. Safety and activity of BPX-601 with rimiducid in mCRPC and pancreatic cancer is being assessed in an ongoing, phase 1/2 clinical trial (NCT02744287). Results from the first two prostate cancer cohorts are reported. Methods: Eligible mCRPC patients (pts) had progressed on ≥ 2 prior therapies including an androgen receptor antagonist and taxane. Using a 3+3 design, pts received lymphodepleting chemotherapy followed by a single-dose of 5 x 106 BPX-601 cells/kg and single or weekly doses of 0.4 mg/kg rimiducid infused over 2 hours beginning 7 days following cell infusion. Primary objective of phase 1 is to determine safety, tolerability and MTD or RP2D. Secondary objectives include characterization of clinical efficacy, PK of rimiducid and long-term safety. Biomarkers indicative of GoCAR-T cell expansion in blood, immune cell activity, and infiltration to tumor are being monitored. Results: As of Sept 2022, 8 pts received BPX-601 5x106 cells/kg; 3 and 5 pts received a single or weekly (range: 1-30) doses of rimiducid. Most common ≥ grade 3 adverse events were myelosuppression, attributed to lymphodepletion. All patients developed cytokine release syndrome (6 G1, 2 G3). Immune-effector cell associated neurotoxicity syndrome occurred and resolved in 2 pts (1 G1, 1 G4). One pt experienced a DLT of neutropenic sepsis (G5) with possible hemophagocytic lymphohistiocytosis (eg, IL-18, ferritin, M-CSF, fractalkine, MIP-1β and IL-1RA levels were elevated). Of 7 evaluable pts, PSA50 response was observed in 3 pts at Day 28. Preliminary RECIST-based results demonstrated partial response in 1, stable disease in 3, 1 progressive disease; at data cut off 2 had not reached imaging timepoint. One patient continues on study with SD after &gt;9 months, with persistent evidence of rimiducid responsiveness. Peripheral blood BPX-601 cells expanded to an average of 3466 vector copies / μg DNA +/- 3109 during the first week with continued re-expansion to an average of 30234 copies/ug DNA +/- 67031) following rimiducid treatment. Serum IFN-γ, GM-CSF and IL-6 rapidly increased over 24 hours following rimiducid treatment (mean IFN-γ increase 26.9-fold ± 14.2) and subsequently diminished over 2 days. Conclusions: BPX-601, a PSCA-directed GoCAR-T cell product, has preliminary evidence of biologic activity with toxicity characteristic of previously reported CAR-T studies. Markers of rimiducid-induced GoCAR-T cell activation and proliferation were observed. Exploration of escalating weekly rimiducid doses &gt; 0.4 mg/kg and BPX-601 cell doses is planned. Clinical trial information: NCT02744287 .

The Choice of Either Conventional Chemotherapy or Inotuzumab Ozogamicin as Bridging Regimen Does Not Appear To Impact Clinical Response to CD19-Directed CAR-T Therapy in Pediatric B-ALL

Bridging therapy (BT) given during the period between T-cell collection and initiation of lymphodepleting chemotherapy is indicated for most children with B-cell acute lymphoblastic leukemia (B-ALL) undergoing treatment with tisagenlecleucel (tisa-cel), a CD19-directed chimeric antigen receptor T-cell (CAR-T) therapy. Both conventional chemotherapy agents and B-cell directed antibody-based therapies such as antibody-drug conjugates and bispecific T-cell engagers have been used as systemic forms of BT. The purpose of this retrospective study was to evaluate if there are detectable differences in clinical outcomes based on the type of BT given (conventional chemotherapy or inotuzumab). A retrospective analysis was performed on all patients treated with tisa-cel at Cincinnati Children's Hospital Medical Center for B-ALL with bone marrow disease (with or without extramedullary disease). Patients who did not receive systemic BT were excluded. Only 1 patient received blinatumomab as BT and was therefore not included in this analysis to focus the analysis on the use of inotuzumab. Pre-infusion characteristics and post-infusion outcomes were collected. Fisher's exact test was used for categorical variables, and t-test or Mann-Whitney test was used for continuous parametric and non-parametric variables respectively. Mantel-Cox was used for survival analyses. Thirty-two patients received BT before CD19 CAR-T for medullary leukemia; 24 received conventional chemotherapy, and 8 received inotuzumab ozogamicin (InO). Cohorts were evenly matched regarding CAR-T indication, recipient age, and median CAR-T cell dose. There were no significant differences between the groups for attaining a minimal residual disease (MRD)–negative complete response after CAR-T, the percentage of patients who maintained prolonged B-cell aplasia, or the median duration of B-cell aplasia. Thirty-seven percent of patients in the conventional chemotherapy group and 43% in the antibody-based therapy group relapsed, with a median time to relapse in both groups of 5 months. No differences in event-free survival, the cumulative incidence of relapse, or overall survival were seen between the two groups. Initial response to tisa-cel, relapse rate, and survival were similar between patients who received BT with conventional chemotherapy or InO therapy. Because low disease burden at the time of infusion is a positive prognostic factor, choice of bridging regimen should focus on therapy that is anticipated to effectively lower disease burden and minimize treatment-related toxicity. Given the limitations associated with the single center retrospective analysis, a larger, multicenter study is needed to further explore these findings.

Higher doses of tisagenlecleucel are associated with improved outcomes: a report from the pediatric real-world CAR consortium.

Remarkable complete response rates have been shown with tisagenlecleucel, a chimeric antigen receptor (CAR) T-cell therapy targeting CD19, in patients up to age 26 years with refractory/relapsed B-cell acute lymphoblastic leukemia; it is US Food and Drug Administration approved forthis indication. Currently, patients receive a single dose of tisagenlecleucel across a wide doserange of 0.2 to 5.0× 106 and 0.1 to 2.5× 108 CAR T cells per kg for patients ≤50 and >50 kg, respectively. The effect of cell dose on survival and remission is not yet well established. Our primary goal was to determine if CAR T-cell dose affects overall survival (OS), event-free survival (EFS), or relapse-free-survival (RFS) in tisagenlecleucel recipients. Retrospective data were collected from Pediatric Real World CAR Consortium member institutions and included 185 patients infused with commercial tisagenlecleucel. The median dose of viable transduced CAR T cells was 1.7× 106 CAR T cells per kg. To assess the impact of cell dose, we divided responders into dose quartiles: 0.134 to 1.300× 106 (n= 48 [27%]), 1.301 to 1.700× 106 (n= 46 [26%]), 1.701 to 2.400×106 (n= 43 [24%]), and 2.401 to 5.100× 106 (n= 43 [24%]). OS, EFS, and RFS were improved in patients who received higher doses of tisagenlecleucel (P= .031, .0079, and .0045, respectively). Higher doses of tisagenlecleucel were not associated with increased toxicity. Because the current tisagenlecleucel package insert dose range remains broad, this work has implications in regard totargeting higher cell doses, within the approved dose range, to optimize patients' potential for long-standing remission.

Unique Reduced-Intensity Conditioning Haploidentical Peripheral Blood Stem Cell Transplantation Protocol for Patients with Hematologic Malignancy

Reduced-intensity conditioning (RIC) haploidentical (haplo-) hematopoietic stem cell transplantation (HSCT) requires more hematopoietic progenitor and stem cells (HPSCs) to promote engraftment and immune reconstitution and needs a stronger graft-versus-leukemia effect. Peripheral blood stem cells (PBSCs) offer advantages over bone marrow; however, the use of higher-dose non-T cell-depleted (non-TCD) in vitro PBSCs may increase the occurrence of severe graft-versus-host disease (GVHD). This prospective, single-arm clinical study was performed to investigate using high-dose non-TCD in vitro PBSCs as the graft source, using fludarabine/Ara-C/busulfan (FAB) as the conditioning regimen, using rabbit antithymocyte globulin to remove T cells in vivo, and enhancing GVHD prophylaxis with an IL-2 receptor antagonist in RIC-haplo-HSCT in patients with hematologic malignancies age 50 to 70 years or <50 years with comorbidities (Hematopoietic Cell Transplantation Comorbidity Index score ≥2) classified as intermediate to high risk. The primary endpoint was day 100 acute GVHD (aGVHD). A total of 47 patients were enrolled; the median age was 52 years (range, 30 to 68 years), the median duration of follow-up was 34 months (range, 2 to 99 months), and the medium-infused doses of mononuclear cells, CD34+ cells, and CD3+ cells were 15.93×108/kg, 8.68×106/kg, and 5.57×108/kg, respectively. The cumulative incidence of grade II-IV aGVHD at day 100 was 30.3% (95% confidence interval [CI], 15.9% to 44.8%), and that of grade III-IV aGVHD was 10.2% (95% CI, .6% to 19.8%). The 2-year cumulative incidence of chronic GVHD (cGVHD) was 34.9% (95% CI, 19.0% to 50.8%). The 2-year cumulative incidences of localized and extensive cGVHD were 26.1% (95% CI, 11.80% to 40.40%) and 8.7% (95% CI, 3.26% to 20.65%), respectively. The 2-year cumulative incidence of relapse was 17.3% (95% CI, 5.1% to 29.5%), the 2-year overall survival rate was 71.2% (95% CI, 57.9% to 84.5%), and the 2-year disease-free survival rate was 66.2% (95% CI, 52.1% to 80.3%). The incidence of aGVHD was not high, and the overall efficacy was good. This study demonstrates that this unique RIC-haplo-PBSC transplantation protocol was effective in treating hematologic malignancies. Nonetheless, larger prospective multicenter clinical trials and experimental studies should be performed to further confirm our findings.

Novel extragenic genomic safe harbors for precise therapeutic T-cell engineering

AbstractCell therapies that rely on engineered immune cells can be enhanced by achieving uniform and controlled transgene expression in order to maximize T-cell function and achieve predictable patient responses. Although they are effective, current genetic engineering strategies that use γ-retroviral, lentiviral, and transposon-based vectors to integrate transgenes, unavoidably produce variegated transgene expression in addition to posing a risk of insertional mutagenesis. In the setting of chimeric antigen receptor (CAR) therapy, inconsistent and random CAR expression may result in tonic signaling, T-cell exhaustion, and variable T-cell persistence. Here, we report and validate an algorithm for the identification of extragenic genomic safe harbors (GSH) that can be efficiently targeted for DNA integration and can support sustained and predictable CAR expression in human peripheral blood T cells. The algorithm is based on 7 criteria established to minimize genotoxicity by directing transgene integration away from functionally important genomic elements, maximize efficient CRISPR/Cas9–mediated targeting, and avert transgene silencing over time. T cells engineered to express a CD19 CAR at GSH6, which meets all 7 criteria, are curative at low cell dose in a mouse model of acute lymphoblastic leukemia, matching the potency of CAR T cells engineered at the TRAC locus and effectively resisting tumor rechallenge 100 days after their infusion. The identification of functional extragenic GSHs thus expands the human genome available for therapeutic precision engineering.

GMP development and preclinical validation of CAR-T cells targeting a lytic EBV antigen for therapy of EBV-associated malignancies.

Epstein-Barr virus (EBV) is a widely spread pathogen associated with lymphoproliferative diseases, B/ T/ NK cell lymphomas, nasopharyngeal carcinoma (NPC) and gastric carcinoma (GC). EBV lytic reactivations contribute to the genomic instability, inflammation and tumorigenesis of NPC, promoting cancer progression. Patients with NPC refractory to standard therapies show dismal survival. EBV gp350 is an envelope protein detectable in NPC specimens intracellularly and on the cell membrane of malignant cells, and is a potential viral antigen for T cell-directed immunotherapies. The potency of T cells engineered with a chimeric antigen receptor (CAR) targeting gp350 against EBV+ lymphoproliferative disease was previously shown. Here, we advanced towards preclinical and non-clinical developments of this virus-specific CAR-T cell immunotherapy against NPC. Different gp350CAR designs were inserted into a lentiviral vector (LV) backbone. A construct expressing the scFv 7A1-anti-gp350 incorporating the CD8 transmembrane and CD28.CD3ζ signaling domain (ZT002) was selected. High titer ZT002 (~1x108 TU/ml) was manufactured in HEK 293T/17 suspension cells in serum free media as large-scale production under good manufacturing practices (GMP). A LV multiplicity of infection (MOI) of 1 resulted in high frequencies of functional gp350CAR+ T cells (>70%) at a low (<2) vector copy numbers in the genome. ZT002 was therefore used to establish gp350CAR-T batch run production methods. GMP upscaling and validation of T cell transduction and expansion in several runs resulted in average 3x109 gp350CAR-T cells per batch. >80% CD3+ gp350CAR-T cells bound to purified gp350 protein. In vitro cytotoxicity and cytokine secretion assays (IFN-γ and TNF-α) confirmed the specificity of gp350CAR-T cells against gp350+ NPC, GC and lymphoma cell targets. Immunocompromised B-NDG mice (NOD.CB17-PrkdcscidIl2rgtm1/Bcgen) were challenged s.c. with a EBV+ NPC C666.1 cell line expressing gp350 and then treated with escalating doses of gp350CAR-T cells or with non-transduced T cells. gp350CAR-T cells promoted antitumor responses, bio-distributed in several tissues, infiltrated in tumors and rejected gp350+ tumor cells. These results support the use of gp350CAR-T cells generated with ZT002 as an Innovative New Drug to treat patients with solid and liquid EBV-associated malignancies.

57 - Conditioning Intensity, Modality, and Niche Clearance, Not Cell Dose, Determine Myeloid CNS Engraftment after Transplant

57 - Conditioning Intensity, Modality, and Niche Clearance, Not Cell Dose, Determine Myeloid CNS Engraftment after Transplant

TRANSPLANTATION OF SUBRETINAL STEM CELL-DERIVED RETINAL PIGMENT EPITHELIUM FOR STARGARDT DISEASE: A Phase I Clinical Trial.

To assess the safety of injecting human embryonic stem cell retinal pigment epithelial cell dose to treat Stargardt disease. In this prospective, Phase I clinical trial, human embryonic stem cell retinal pigment epithelial cells in suspension were injected into the subretinal space in eyes with the worse best-corrected visual acuity (BCVA). After vitrectomy/posterior hyaloid removal, a partial retinal detachment was created and the human embryonic stem cell retinal pigment epithelial cells were administered. Phacoemulsification with intraocular lens implantation was performed in eyes with lens opacity. All procedures were optical coherence tomography-guided. The 12-month follow-up included retinal imaging, optical coherence tomography, visual field/electrophysiologic testing, and systemic evaluation. The main outcome was the absence of ocular/systemic inflammation or rejection, tumor formation, or toxicity during follow-up. The mean baseline BCVAs in the phacoemulsification and no phacoemulsification groups were similar (1.950 ± 0.446 and 1.575 ± 0.303, respectively). One year postoperatively, treated eyes showed a nonsignificant increase in BCVA. No adverse effects occurred during follow-up. Intraoperative optical coherence tomography was important for guiding all procedures. This surgical procedure was feasible and safe without cellular migration, rejection, inflammation, or development of ocular or systemic tumors during follow-up.

Prognostic Factors for the Long-Term Survival after Hematopoietic Stem Cell Transplantation in Patients with Hodgkin Lymphoma.

This study investigated the possible prognostic factors for the long-term survival (Cure Rate) of Hodgkin Lymphoma patients who underwent HSCT. This retrospective cohort study analyzed 116 Patients diagnosed with Hodgkin Lymphoma who received autologous hematopoietic stem cell transplantation (Auto-HSCT) between the years 2007 and 2014 and followed up until 2017. The information regarding patients' survival had been collected using phone calls, and their pre-transplant information was available in the archived documents. Prognostic effects were investigated using long-term survival models. Patients with obesity had five times higher odds of long-term survival (cure) than the others (P=0.06). Also, the recurrence experience after HSCT negatively impacted the curing potential by 78% (P=0.05). Also, with 32 years as the change point, patients younger than 32 had 76% fewer odds of surviving long-term (P=0.03), and Poor transfused stem cell dose of CD34+ (<0.16 × 106 cells/ml) reduced the odds of long-term survival by 92% (P=0.01). According to the statistical models used in this study, obesity can increase the curing potential of Hodgkin lymphoma after transplantation. Meanwhile, aging, poor transfused CD34+ cells, and recurrence after HSCT were associated with lower survival following HSCT.

Intraoperative transfusion is a risk factor for cerebral injury after cardiac surgery in children: a prospective observational study

INTRODUCTION: Donor blood components are able to initiate a systemic inflammatory response syndrome (SIRS) and potentiate neuroinflammation with subsequent cerebral damage. OBJECTIVE: To study the effect of transfusion on the development of cerebral damage during the surgical correction of congenital heart defects in children. MATERIALS AND METHODS: 78 patients aged from 1 to 78 months, weighing from 3.3 to 21.5 kg, were studied. All patients underwent correction of a septal defect under cardiopulmonary bypass. All patients were divided to group 1 — without the use of transfusion and group 2 — with the use of red blood cell transfusion. Cerebral damage markers (S-100-β protein, neuron-specific enolase (NSE) and glial fibrillar acidic protein (GFAP)) and SIRS (interleukins 1 (ILb-1), 6 (IL-6), 10 (IL-10) and tumor necrosis factor alpha (TNF-α) were studied. Markers ware studied at three control points: 1 — before the start of surgery, 2 — immediately after end of cardiopulmonary bypass, 3 — 16 hours after the end of the operation. RESULTS: The peak concentration of most markers in the blood in both groups of patients was noted at the 2nd control point. The concentration of all markers of cerebral damage was significantly higher in the transfusion group at the 2nd control point: S-100-β protein (ng/ml) — 509.90 [379.30–871.70] and 717.10 [517.90–1195.33] (р = 0.024); NSE (ng/ml) — 17.55 [11.19–26.41] and 34.05 [17.06–44.90] (р = 0,023); GFAP (ng/ml) — 0.1190 [0.1135–0.1245] and 0.1231 [0.1138–0.1493]. Correlations were found between markers of cerebral damage and SIRS, the strongest of which was the relationship between NSE and TNF-α at the 3rd control point — Rho = 0.43 (p = 0.0001). A correlation of S-100-β protein with transfusion volume was observed at the 2nd (Rho = 0.48, p = 0.00065) and 3rd control points (Rho = 0.36, p = 0.01330). CONCLUSIONS: The influence of the fact of transfusion and the dose of red blood cell on the development of cerebral damage during cardiac surgery in children has been proven.

Unrelated Donor Cord Blood Transplantation in Children: Lessons Learned Over 3 Decades

Four decades ago, Broxmeyer et al. demonstrated that umbilical cord blood (CB) contained hematopoietic stem cells (HSC) and hypothesized that CB could be used as a source of donor HSC for rescue of myeloablated bone marrow. In 1988, Gluckman et al. reported the first successful matched sibling cord blood transplant (CBT) in a child with Fanconi Anemia. In 1991, Rubinstein et al. established an unrelated donor CB bank, and in 1993, the first unrelated CBT used a unit from this bank. Since that time, >40 000 CBTs have been performed worldwide. Early outcomes of CBT were mixed and demonstrated the importance of cell dose from the CB donor. We hypothesized that improvements in CB banking and transplantation favorably impacted outcomes of CBT today and performed a retrospective study combining data from Eurocord and Duke University in 4834 children transplanted with a single unrelated CB unit (CBU) from 1993 to 2019. Changes in standard transplant outcomes (overall survival [OS], disease free survival [DFS], acute and chronic graft-versus-host disease [GvHD], treatment related mortality [TRM], and relapse) over 3 time periods (1: <2005; 2: 2005 to <2010; and 3: >2010 to 2019) were studied. Increased cell dose and degree of HLA matching were observed over time. OS, times to engraftment, and DFS improved over time. The incidence of TRM and GvHD decreased while the incidence of relapse remained unchanged. Relative contributions of cell dose and HLA matching to transplant outcomes were also assessed and showed that HLA matching was more important than cell dose in this pediatric cohort.

Tanshinone IIA Inhibits Triple-Negative Breast Cancer Cells MDA-MB-231 via G Protein-Coupled Estrogen Receptor- (GPER-) Dependent Signaling Pathway.

Due to the lack of classic estrogen receptors, there has been a shortage of targeted therapy for triple-negative breast cancer (TNBC), resulting in a poor prognosis. However, the newly discovered G protein-coupled estrogen receptor (GPER) has been found to be expressed in TNBC cells. Salvia miltiorrhiza (Danshen) is an essential Chinese medicine for gynecological disorders, and its component tanshinone IIA (Tan IIA) exerts an anticancer effect. Therefore, this study attempted to investigate whether GPER is involved in the inhibitory effect of Tan IIA on TNBC. We applied various databases and GO pathway analysis to predict the possible mechanism of Tan IIA. We identified 39 overlapping targets, including c-Jun, c-Fos, and caspase-3, and enriched cell cycle-related pathways. Next, we demonstrated the strong binding ability of Tan IIA to GPER by molecular docking assay. In the subsequent validation tests, Cell Counting Kit-8 (CCK8) assay showed that Tan IIA inhibited proliferation of MDA-MB-231 cells time and dose dependently without affecting normal cells. Using Transwell plate, flow cytometry, and Western blot assays, we showed that Tan IIA inhibited migration and induced apoptosis of MDA-MB-231 dose dependently. Importantly, protein expressions of GPER, epidermal growth factor receptor (EGFR), extracellular regulated protein kinases (ERK), c-Fos, and c-Jun were all decreased by Tan IIA dose dependently. Administration of GPER inhibitor partly abolished these effects. Furthermore, nuclear translocation of c-Fos and c-Jun as well as cell cycle-related proteins was downregulated by Tan IIA dose dependently. In summary, Tan IIA could inhibit the proliferation and migration of MDA-MB-231 cells and induce apoptosis, and the possible mechanism may be the regulation of GPER-mediated pathways, suggesting that GPER could be a therapeutic target for TNBC.

Research Highlights.

Research Highlights.