GMP Grade Research Articles

Novel CT-guided 188-rhenium brachytherapy device for local primary and secondary lung malignancies.

96 Background: Stereotactic brachytherapy for extensive local tumors offers a very effective treatment option locally without significant complications in medically impaired patients. In this context, we have recently developed a new potential anticancer agent from Poly-L-Lysins dendrimer as a delivery nano system loaded with diffusible Imidazolic probes complexed with 188-Rhenium for targeting in particular hypoxic tumors resistant to conventional cancer treatments. The aim of the study is assessment the safety profile and therapeutic efficacy of anticancer agent derived from [188Re]rhenium-ligand as radioactive ligand loaded 5th generation poly-L-lysine dendrimer in patients with unresectable Lung Malignancies. Methods: The experiment agent “ 188Re-ImDendrim” is consisting of 5th generation poly-L-lysine dendrimer (20 nM) mixed with nitro-imidazole-methyl-1,2,3-triazol-methyl-di-(2-pycolyl) amine at GMP grade and labelled with [188Re]-rhenium. The study was approved by Shanghai East Hospital ethics committee. 5 patients received “188Re-ImDendrim” directly into lung tumors under CT-guidance, at an activity level of 162 MBq/cc of tumor (range 2 to 7 cm; mean diameter, 4 cm) . For voluminous tumors ( > 65 cc) the dose is given in divided injection spaced 2 weeks apart (Tumor of 115cc: 2 administrations, Tumor of 180 cc: 3 administrations). At H0.5, H 4, H24, H36 , H72 post-administration, the patient get a SPECT control. The response to treatment is evaluated thanks to PET/CT Standardized Uptake Values (SUVs). Results: Stereotactic administrations of “188-Rhenium-ImDendrim” were successfully carried out in all patients under local anesthesia. The radioactive product diffuses homogeneously in the tumor volume and remains 72 hours post-administration with no significant diffusion out site of injection. The One of the 5 patients reported discrete transitive hemoptysis as adverse events. All targeted tumors were responding at 12 weeks, with two complete responses. Conclusions: Percutaneous single and iterative administrations of this novel 188-Rhenium-Imdendrim brachytherapy device into lung cancers are safe and well tolerated. The initial data on therapeutic response are promising. Clinical trial information: EC.D (BG) 016.03.1.

GMP-compliant sorting of dendritic cells for CCR7 expression improves therapeutic efficacy in cancer by enhancing lymph node migration and antigen-specific T cell activation

Background & Aim Dendritic cells (DCs) are a potentially powerful cell therapy due to their ability to induce T cell activation to defined antigens; however, this potential has not yet been realised in clinical trials. Monocyte-derived DCs (moDCs) are most commonly used in cancer therapy, although the rare blood DC subsets have also been explored. Crucially, cell function requires the chemokine receptor CCR7, which guides DCs from the site of antigen uptake to the lymph nodes. CCR7 expression, and therefore migration, is variable after isolation of DCs from blood or ex vivo culture, and may account for the limited efficacy seen in clinical trials. We hypothesised that selection of CCR7+ DCs by sorting would to improve cell function and therapeutic use of DCs. Methods, Results & Conclusion We have previously shown that murine CCR7+ bone marrow DCs (BMDCs) can be sorted using a novel chemokine-based system and show enhanced lymph node migration. This CCL19-fluorophore construct was shown to also detect expression of CCR7 on human DCs, and significantly enrich CCR7+ cells to high purity and viability (>90%) using magnetic bead and fluorescence-based sorting methods. Both murine and human CCR7+ DCs were more mature than CCR7- DCs by expression of CD80 and CD86 and production of T cell chemoattractants such as CCL17 and CCL22. In vitro, CCR7+ DCs were also most effective in stimulating antigen-specific T cells, especially memory T cells producing IFNγ, TNFα and IL-2. In vivo, sorted murine CCR7+ BMDCs, but not unsorted DCs, were sufficient to reduce subcutaneous and metastatic tumour growth in the B16F10.ova mouse model. Increased migration of injected DCs to lymph nodes led to an elevated overall number of tumour-specific T cells in CCR7+ DC-receiving mice, but this was not seen in those receiving unsorted DCs. In agreement with in vitro data, CCR7+ DCs increased production of mature tumour-specific T cells, particularly with the desirable memory (CD62L+CD44+) phenotype, suggesting their potency in a human therapeutic context. As production of this chemokine construct is possible at GMP grade, this protocol is amenable to GMP translation using the MACSQuant Tyto cell sorter. Purity and viability of CCR7+ DCs remained consistently high between the original and GMP-ised protocols, with an improved cell yield versus bead sorting. Overall, this novel sorting process is compliant with current GMP manufacture and creates a realistic DC product with enhanced function and potential therapeutic benefit.

British Society for Gene and Cell Therapy Autumn Conference Friday 23 November 2018 Regent's University Conference Centre, London, UK www.bsgct.org

British Society for Gene and Cell Therapy Autumn Conference Friday 23 November 2018 Regent's University Conference Centre, London, UK www.bsgct.org

An Innovative Standard Operation Procedure for Isolating GMP-Grade CD4+CD25+ T Cells from Non-Mobilized Leukapheresis.

This SOP describes a closed system for isolating GMP-grade CD4+CD25+ T cells from non-mobilized leukapheresis collections (nMLCs), independent of a clean room in a certified GMP premises, by using CliniMACS format GMP grade reagents (CD25-labeled magnetic beads with/without pre-depletion of CD8+ T cells and CD19+ B cells), a GMP grade-A laminar hood and CliniMACS cell processing system.

Microfibrous scaffolds from poly(l-lactide-co-ε-caprolactone) blended with xeno-free collagen/hyaluronic acid for improvement of vascularization in tissue engineering applications

Success of 3D tissue substitutes in clinical applications depends on the presence of vascular networks in their structure. Accordingly, research in tissue engineering is focused on the stimulation of angiogenesis or generation of a vascular network in the scaffolds prior to implantation. A novel, xeno-free, collagen/hyaluronic acid-based poly(l-lactide-co-ε-caprolactone) (PLC/COL/HA) (20/9.5/0.5 w/w/w) microfibrous scaffold was produced by electrospinning. Collagen types I and III, and hyaluronic acid were isolated from human umbilical cords and blended with the GMP grade PLC. When compared with PLC scaffolds the PLC/COL/HA had higher water uptake capacity (103% vs 66%) which may have contributed to the decrease in its Young's Modulus (from 1.31 to 0.89 MPa). The PLC/COL/HA better supported adipose tissue-derived mesenchymal stem cell (AT MSC) adhesion; within 24 h the cell number on the PLC/COL/HA scaffolds was 3 fold higher. Co-culture of human umbilical vein endothelial cells and AT MSCs induced capillary formation on both scaffold types, but the PLC/COL/HA led to formation of interconnected vessels whose total length was 1.6 fold of the total vessel length on PLC. Clinical use of this scaffold would eliminate the immune response triggered by xenogeneic collagen and transmission of animal-borne diseases while promoting a better vascular network formation.

CD19 Redirected CAR NK Cells Are Equally Effective but Less Toxic Than CAR T Cells

Based on the clinical success observed in acute lymphoblastic leukemia (ALL) with chimeric antigen receptor engineered T (CAR T), we hypothesized that combining the specificity of a CAR with the innate allo-reactivity of KIR-mismatched NK cells might provide a powerful tool for adoptive cell therapy. The use of a third-party bank of CAR-NK cells offers the advantage of an immediate availability to be exploited in the allogenic setting and could be associated with a lower toxicity profile than CAR-T cells.In order to overcome regulatory and manufacturing hurdles associated with generation of CAR-NK cells, we developed a feeder-free culture resulting in a 3.2-log expansion after 20 days of culture. Specifically, natural cytotoxicity receptors (NCR) expressed on NK cells are stimulated in the presence of pleiotropic cytokines and expanded in GMP grade bioreactors. Expanded NK cells from healthy donors preserve a high percentage of CD56+ CD57- cells (85±13%), associated with high proliferative capability, and maintain the surface expression and the responsiveness of NCR and CD16.We proved that NK cells generated from 10 different healthy donors have high ability to recognize and eliminate different tumor types, including acute myeloid leukemia (AML) and ALL.After genetic modification with a retroviral vector encoding a CAR specific for CD19 antigen, transduction of activated NK cells averaged 38%±15% and the CAR.CD19 expression was stable over extended in vitro culture (60 days). Detailed phenotypic characterization of CAR-NK cells showed that CAR expression was not limited to the more mature NKG2A-/KIR+ cells, but rather was distributed across different NK subsets.We also demonstrated that NK and CAR-NK cells display significant anti-leukemia activity towards CD19+ leukemia and lymphoma cell lines (LCL 721.221, DAUDI and BV173) and primary blasts obtained from patients with B-cell precursor ALL (Bcp-ALL). Co-culture experiments using a 1:5 E/T ratio, showed that, while the anti-tumor activity was already remarkable with non-modified effector NK cells (60±30%, 71±33% and 54±23% of residual LCL 721.221, DAUDI and BV173 cells, respectively; p<0.05 vs T cells), it reached the highest level when CAR-NK cells were used as effectors (7±9%, 16±30% and 22±16% of residual LCL 721.221, DAUDI and BV173 cells, respectively; p<0.05 vs non-transduced NK cells). Importantly, INF-g production was significantly lower upon CAR-NK activation compared to CAR-T cells (DAUDI 384±194 ng/ml vs 1860±678 ng/ml, p=0.002). Functional analysis on primary Bcp-ALL blasts, demonstrate that CAR-NK cells exert high degree of leukemia control (on average 2.1±2% vs 5.4±1.6% with non-modified NK cells as effectors; p=0.04).An in vivo model of leukemia xenograft immunodeficient mice was used to evaluate whether CAR-NK cells are associated with a lower toxicity profile compared to CAR-T cells. While the in vivo antileukemia activity was superimposable between CAR-T and CAR-NK cells (mouse bioluminenscence at 20 days, 4.9x105 vs 6.6x105 photons/second, respectively; p=n.s. Figure A), mice treated with two i.v. infusions (day 0 and day 15) of 10x106 CAR.CD19 NK cells had a 100% overall survival (OS of 5 out of 5 mice) at 50 days compared to 20% of mice (1 out of 5) receiving 10x106 CAR.CD19 T cells (Figure B; p=0.01). Cytokine plasma level monitoring, performed on day +7 and +30 after effector cell infusion in the absence of leukemia persistence (as evidenced by a lack of bioluminescence signal), showed that mice engrafted with CD19+ leukemia and treated with CAR.CD19-NK cells have lower levels of circulating hIFN-g cytokine compared to mice treated with CAR.CD19-T cells at both day 7 (42±82 vs 330±346 ng/ml; p=0.05) and day 30 (0.9±0.7 vs 4148±667 ng/ml; p=0.05).These in vitro and in vivo data demonstrate the feasibility of clinical scale feeder-free expansion of non-modified NK cells and stably transduced CAR-NK cells. Both non-modified and gene-modified cells were capable of significant tumor killing, suggesting a multi-modal adoptive cell approach to treatment of leukemia. Since NK cells have been shown to be safely used in third-party setting (St. Jude Children's Research Hospital, USA; NCT00640796), we suggest that ex-vivo expanded, feeder-free NK cells can be universally applied for ‘off-the-shelf’ immuno-gene-therapy, and that their innate allo-reactivity can be safely harnessed to potentiate allogeneic cell therapy. [Display omitted] DisclosuresLocatelli:Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; bluebird bio: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Miltenyi: Honoraria; Bellicum: Consultancy, Membership on an entity's Board of Directors or advisory committees.

A fully defined static suspension culture system for large-scale human embryonic stem cell production

Human embryonic stem cells (hESCs) play an important role in regenerative medicine due to their potential to differentiate into various functional cells. However, the conventional adherent culture system poses challenges to mass production of high-quality hESCs. Though scientists have made many attempts to establish a robust and economical hESC suspension culture system, there are existing limitations, including suboptimal passage methods and shear force caused by dynamic stirring. Here, we report on an efficient large-scale culture system, which enables long-term, GMP grade, single-cell inoculation, and serial expansion of hESCs with a yield of about 1.5 × 109 cells per 1.5-L culture, while maintaining good pluripotency. The suspension culture system was enlarged gradually from a 100-mm dish to a 1.8-L culture bag with methylcellulose involvement to avoid sphere fusion. Under the optimal experimental protocol, this 3D system resolves current problems that limit mass production and clinical application of hESCs, and thus can be used in commercial-level hESC production for cell therapy and pharmaceutics screening in the future.

Immunological Properties of Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells.

SummaryAge-related macular degeneration is caused by dysfunction and loss of retinal pigment epithelium (RPE) cells, and their transplantation may rescue visual functions and delay disease progression. Human embryonic stem cells (hESCs) may be an unlimited source of RPE cells for allotransplantation. We analyzed the immunomodulatory properties of hESC-derived RPE (hESC-RPE) cells, and showed that they inhibited T cell responses. Co-culture experiments showed that RPE cells inhibited interfon-γ secretion and proliferation of activated T cells. Furthermore, hESC-RPE cells enhanced T cell apoptosis and secretion of the anti-inflammatory cytokine interleukin-10 (IL-10). In addition, RPE cells altered the expression of T cell activation markers, CD69 and CD25. RPE cells transplanted into RCS rats without immunosuppression survived, provided retinal rescue, and enhanced IL-10 blood levels. Our data suggest that hESC-RPE cells have immunosuppressive properties. Further studies will determine if these properties are sufficient to alleviate the need for immunosuppression therapy after their clinical allotransplantation.

Automated Synthesis of 68Ga-DOTA-TOC with a Cationic Purification System: Evaluation of Methodological and Technical Aspects in Routine Preparations.

Gallium-68 is a PET isotope available in each nuclear medicine departments, even those not equipped with a cyclotron, since it is easily obtained by eluting compact and transportable generator system. The preparation of Ga-68 DOTA-labeled compounds is performed by remotely controlled automated systems developed in order to ensure production efficiency, reproducibility of the results, fast reaction time, to facilitate the synthesis and minimize the radiation exposure. Many automatic synthesis systems are available on the radiopharmaceutical market, however, they requires some technical adaptations for routine use. We reported the [68Ga]Ga-DOTA-TOC production by automated cassette-based theranostic synthesizer system used in combination with a disposable GMP grade cassette system for cationic purification. The synthesizer is integrated with the 68Ge/68Ga generator systems and it allows to perform elution, eluate purification and radiolabeling in about 38 minutes. We have performed in 2 year (January 2016 - January 2018) over 100 [68Ga]Ga-DOTA-TOC preparations. The average synthesis yield of radiopharmaceutical production was 54.4 ± 2.3 % and the radiochemical purity average was found 96.94 ± 0.74 %. Only three [68Ga]Ga-DOTA-TOC preparations have failed. The methodology and the adopted technical solutions allowed to obtain a high quality radiopharmaceutical product as required by the European Pharmacopoeia.

Multicenter Assessment of Animal-free Collagenase AF-1 for Human Islet Isolation

Animal-free (AF) SERVA Collagenase AF-1 and Neutral Protease (NP) AF GMP Grade have recently become available for human islet isolation. This report describes the initial experiences of 3 different islet transplant centers. Thirty-four human pancreases were digested using 1 vial of the 6 different lots of Collagenase AF-1 (2,000–2,583 PZ-U/vial) supplemented with 4 different lots of NP AF in a range of 50 to 160 DMC-U per pancreas. Isolation, culture, and quality assessment were performed using standard techniques as previously described. All data are presented as mean ± standard error of the mean (SEM). Variability of pancreas weight was associated with a wide range of collagenase and NP activities, ranging from 12.7 to 46.6 PZ-U/g (26.0 ± 1.5 PZ-U/g) and 0.4 to 3.0 DMC-U/g (1.5 ± 0.1 DMC-U/g), respectively. Postpurification islet yield was 296,494 ± 33,620 islet equivalents (IEQ) equivalent to 3,274 ± 450 IEQ/g with a purity of 55.9% ± 3.2%. Quality assessment performed after 2 to 4 d of culture demonstrated a viability of 88.1% ± 1.5% and a stimulation index of 3.7 ± 0.7. Eighteen of the 34 preparations were transplanted into type 1 diabetic patients equivalent to a transplantation rate of 52.9%. Six preparations, which were infused into patients as first transplant, could be analyzed and increased the fasting C-peptide level from 0.11 ± 0.08 pretransplant to 1.23 ± 0.24 and 2.27 ± 0.31 ng/mL 3 and 6 mo posttransplant (P < 0.05), respectively. Insulin requirements were simultaneously reduced at the same time from 39.2 ± 3.8 IU/d before transplantation to 10.8 ± 4.1 and 4.0 ± 2.3 IU/d, after 3 and 6 mo posttransplant (P < 0.05), respectively. This study demonstrates the efficiency of AF SERVA Collagenase AF-1 and NP AF for clinical islet isolation and transplantation. The new plant-based production process makes these products a safe new option for the islet field.

Adipose-derived stem cells: Comparison between two methods of isolation for clinical applications.

BackgroundAdipose-derived stem cells are recognized as being an effective mesenchymal stem cell population with enormous potential in different fields of regenerative medicine and stem cell therapy. Although there is unanimous agreement on the harvesting procedure for adipose tissue, there are various protocols for adipose-derived stem cell isolation. The aim of this study was compare two methods of adipose-derived stem cells (ASCs) isolation, one based on a mechanical + enzymatic (ME) procedure and the other one exclusively mechanical (MC), in order to determine which one was superior to the other in accordance with current European and US legislation.MethodsWe reported step by step the two different methods ASCs isolation by comparing them. The ME procedure included the use of a centrifuge, an incubator and collagenase digestion solution (Collagenase NB 6 GMP Grade 17458; Serva GmbH, Heidelberg, Germany). The MC procedure was performed by vibrating shaker and centrifuge, both placed in a laminar airflow bench.ResultsWith the ME procedure, a mean of 9.06 × 105 ASCs (range, 8.4 to 9.72 × 105; SD ± 6.6 × 105) was collected, corresponding to 25.9% of the total number of harvested cells. With the MC procedure, a mean of 5 × 105 ASCs (range: 4.0 to 6.0 × 105; SD, ±1 × 105) was collected, corresponding to 5% of the total number of harvested cells.ConclusionBased on data collected, from the same amount of lipoaspirate the ME procedure allowed to isolate a greater number of ASCs (25,9%) compared to the MC one (5%).

Abstract 4607: Specific inhibition of PGE2-EP4 signaling by E7046 promotes anti-tumor activity of checkpoint blockade agents through boosting cytotoxic T cell activity

Abstract Purpose: Immunotherapies targeting immune checkpoint receptors have shown great promise for a subset of cancer patients; however, robust and safe combination therapies are still needed. In the tumor microenvironment, prostaglandin E2 receptor type 4 (EP4) signaling has been implicated in both protumoral myeloid cell differentiation and cytotoxic T cell exhaustion. We evaluated the combination of the EP4 antagonist E7046 (clinical trial NCT02540291) with anti-PD1 or anti-CTLA4 in preclinical tumor models, and also interrogated the relationship between PGE2 pathway activation and cancer patient survival. Materials/Methods: Mouse syngeneic tumor models CT-26 and 4T1 were used for pharmacological investigation. GMP grade E7046 was administered to tumor-bearing animals by oral gavage. Co-culture of EG7-OVA and OT1 cells in an antigen-specific cytotoxic T cell (CTL) activation assay provided mechanistic insights. For translational validation, transcripts of five major genes involved in PGE2 synthesis, transport and degradation were compared between malignant and normal tissues across all TCGA tumor types, and correlation of their expression with overall survival was assessed. Results: In the CT26 tumor model, the combination of E7046 and anti-PD1 resulted in significantly more tumor-free animals compared with either agent alone. In the 4T1 tumor model, the combination of E7046 and anti-CTLA4 was also more effective in suppressing tumor growth and tumor rejection compared with anti-CTLA4 alone, and was accompanied by a markedly increased accumulation of GZMB+CD8T+ CTLs in the treated tumors. Consistent with those findings, addition of anti-PD1 antibody promoted OVA-specific CTL activation in vitro while addition of PGE2 strongly inhibited it, as measured by IFNγ secretion. Inclusion of E7046 dose-dependently reversed the PGE2-induced suppressive activity in the presence of anti-PD1 antibody. Among major human PGE2 pathway genes, TCGA analysis showed that PTGES1 was upregulated and HPGD downregulated across a broad range of tumor types. In contrast, COX1, COX2 and PGT showed less difference between malignant and normal tissues. Importantly, these differences of one or multiple PGE2 pathway genes were strongly associated with patient survival in certain cancer types. Conclusions: A subset of human cancer types displays upregulated PGE2 pathway that is associated with a poorer prognosis. PGE2-EP4 signaling potently suppresses antigen-specific CTL activation in the presence of PD1 signaling blockade. The combination of EP4 antagonist E7046 with either anti-PD1 or anti-CTLA4 demonstrated superior anti-tumor activity compared with anti-PD1 or anti-CTLA4 alone. This increased activity was accompanied by increased CTL activation. Citation Format: Diana I. Albu, David Verbel, Yuan Huang, Donna Kolber-Simonds, Zichun Wang, Xulong Wang, Zoltan Dezso, Christy Ingersoll, Kuan-Chun Huang, Janna Hutz, Mary Woodall-Jappe, Xingfeng Bao. Specific inhibition of PGE2-EP4 signaling by E7046 promotes anti-tumor activity of checkpoint blockade agents through boosting cytotoxic T cell activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4607. doi:10.1158/1538-7445.AM2017-4607

Tattooing Vaccines Combining Biomedical Engineering for RNA Immunization

Producing recombinant antigens in GMP grade for vaccine purposes can be very difficult and in general, a time-consuming process [1]. Overcoming these two problems was one of the most important goals of DNA vaccines [2]. Producing and purifying plasmids that encode antigens has many advantages, namely easy storage and high stability [3]. However, there are different roadblocks concerning DNA for use in vaccines.

Long-Term Efficacy of GMP Grade Xeno-Free hESC-Derived RPE Cells Following Transplantation.

PurposeRetinal pigment epithelium (RPE) dysfunction underlies the retinal degenerative process in age-related macular degeneration (AMD), and thus RPE cell replacement provides an optimal treatment target. We characterized longitudinally the efficacy of RPE cells derived under xeno-free conditions from clinical and xeno-free grade human embryonic stem cells (OpRegen) following transplantation into the subretinal space of Royal College of Surgeons (RCS) rats.MethodsPostnatal (P) day 20 to 25 RCS rats (n = 242) received a single subretinal injection of 25,000 (low)-, 100,000 (mid)-, or 200,000 (high)-dose xeno-free RPE cells. BSS+ (balanced salt solution) (vehicle) and unoperated eyes served as controls. Optomotor tracking (OKT) behavior was used to quantify functional efficacy. Histology and immunohistochemistry were used to evaluate photoreceptor rescue and transplanted cell survival at 60, 100, 150, and 200 days of age.ResultsOKT was rescued in a dose-dependent manner. Outer nuclear layer (ONL) was significantly thicker in cell-treated eyes than controls up to P150. Transplanted RPE cells were identified in both the subretinal space and integrated into the host RPE monolayer in animals of all age groups, and often contained internalized photoreceptor outer segments. No pathology was observed.ConclusionsOpRegen RPE cells survived, rescued visual function, preserved rod and cone photoreceptors long-term in the RCS rat. Thus, these data support the use of OpRegen RPE cells for the treatment of human RPE cell disorders including AMD.Translational RelevanceOur novel xeno-free RPE cells minimize concerns of animal derived contaminants while providing a promising prospective therapy to the diseased retina.

The Osteogenic Potential of Human Nondifferentiated and Pre-differentiated Mesenchymal Stem Cells Combined with an Osteoconductive Scaffold - Early Stage Healing.

Despite the huge research into stem cells and their regenerative properties for bone healing, there are still unanswered questions including the recipient's respond to the presence of the stem cells, the fate of stem cells inside the bone defect and the possible advantage in utilizing pre-differentiated cells. To address these problems, we used human multipotent mesenchymal stromal/stem cells (MSCs), GMP Grade, in a rat model of bone formation. In a "bioreactor concept" approach seven Wistar rats were implanted with 0.2 g of synthetic bone scaffold seeded with 2 × 106 MSCs, seven Wistar rats were implanted with 0.2 g of synthetic bone scaffold seeded with 1 × 106 predifferentiated osteoblasts and 1 × 106 pre-differentiated endothelial cells and 14 Wistar rats were implanted with 0.2 g of synthetic bone scaffold without seeded cells into an intramuscular pocket on the left side of their back. The right side of each rat was used as a control, and 0.2 g of synthetic bone scaffold was implanted into the intramuscular pocket alone. To see the early stage healing the samples were harvested 14 days after the implantation, MSCs were detected by positive DAPI and MTCO2 staining in 43% of all the samples implanted with MSCs, and no inflammation signs were present in any implanted animal. New vessels could be found in both groups implanted with MSCs, but not in the control group of animals. However, hematoxylin-eosin staining could not detect newly created bone within the implant in any of the groups. These results were in line with COLL1 staining, where we could detect positive staining only in three cases, all of which were implanted with un-differentiated MSCs. According to our findings, there were no benefits of using the pre-differentiated of MSC.

Next Generation Veto Cells for Non-Myeloablative Haploidentical HSCT: Combining Anti-Viral and Graft Facilitating Activity

Transplantation of T cell-depleted BM (TDBM) under mild conditioning, associated with minimal toxicity and reduced risk of GVHD, offers an attractive therapeutic option for patients with nonmalignant hematologic disorders or can be used to induce immune tolerance to subsequent organ transplantation. However, overcoming TDBM rejection after reduced conditioning remains a challenge. We previously demonstrated that this barrier can be overcome using donor-derived naïve CD8 T cells if they are expanded against third-party alloantigens under culture conditions inducing a central memory phenotype. Thus, such anti-3rd party CD8 T cells (Tcm) exhibit marked veto activity and reduced risk for GVHD due to the lower frequency of anti-host clones achieved during the culture period. In the present study, we tested the feasibility of generating veto Tcm by stimulation against well-defined peptides including viral antigens. For proof of concept, we used OT1 mice that express a transgenic (Tg) TCR designed to recognize ovalbumin (OVA) residues 257-264 in the context of H2Kb MHC-I. Prior to harvest of OT1 CD8+ T cells, mice were immunized twice with OVA-peptide. Mice were sacrificed 7 to 14 days after immunization, their spleens and lymph nodes removed and crushed, and magnetic bead sorting utilized to isolate the memory cells (CD8+CD44+). The resulting population was subjected to third-party stimulation by co-culture with irradiated splenocytes generated from spleens of OVA-expressing mice, under cytokine deprivation. hIL-15 (10ng/ml) was added to the culture 60 hrs after culture initiation to induce the cells to express a Tcm like phenotype, as previously described for anti-3rd party Tcm generated from WT naïve CD8 T cells (WT Tcm).When tested in vivo, the OT-1 Tcm (H2Kb) were able to enhance engraftment of allogeneic T cell depleted BMT (H2Kb) in sub-lethally irradiated (5.5 Gy TBI) Balb/c recipients (H2Kd), in analogy to the chimerism induced by WT Tcm. This initial successful experiment was then followed by an experiment, which more closely resembled the human setting, in which the CD8+CD44+ cells were isolated from WT C57BL/6 OVA-immunized mice and subsequently introduced to co-culture with irradiated splenocytes generated from spleens of OVA-expressing mice. Results showed that Tcm grown from such a starting population of CD8+CD44+ cells were also able to achieve marked donor chimerism when administered with megadose of T cell depleted (TCD) alloSCT in sublethally irradiated Balb/c recipients (Fig.1A). Notably, infusion of 5x106 Tcm into sublethally irradiated mice (5.5. GY TBI) without BMT, did not cause any GVHD as measured by weight loss, in contrast to CD8+CD44+ T cells used to generate the Tcm (Fig.1B). These results demonstrate that CD8+CD44+ derived from memory CD8 T cells by expansion against cognate peptides exhibit markedly reduced risk for GVHD compared to freshly isolated memory cells, while retaining their veto activity and inducing tolerance.Finally, based on these proof of concept studies, we were able to translate this approach and generate human anti-viral CD8 veto cells with central memory phenotype. Thus, CD8+CD45RO+ memory T cells were selected by depletion of CD4+CD56+CD45RA+ cells from PBMC of normal donors and then co-cultured with donor DCs pulsed with a viral peptide mixture of three prominent viruses (EBV, CMV and Adenovirus). In three large scale experiments using leukapheresis preparations of normal CMV and EBV positive donors, with GMP grade reagents, more than 1x109 Tcm could be attained by the end of 9 days of culture (average expansion of Tcm=13.5±4 fold) with greater than 90% purity of CD45RO+CD62L+CD8+ T cells (Fig 2).The harvested anti-viral Tcm exhibited more than 3 log depletion of alloreactivity, compared to fresh CD8 T cells, as measured by limit dilution analysis of cytotoxic T cell precursors against host type target cells (Fig. 3).In conclusion, our results suggest that potent veto CD8 Tcm can be generated from the memory pool of donors positive for viral reactivity by stimulation against viral antigens. Such veto Tcm could be most attractive for haploidenitcal transplantation of TCDBM, as they can enable engraftment following non-myeloablative conditioning and at the same time provide anti-viral protection.*E.B.L and N.O.G contributed equally [Display omitted] [Display omitted] [Display omitted] DisclosuresBachar Lustig:Yeda LTD: Patents & Royalties. Or Geva:Yeda LTD: Patents & Royalties. Lask:Yeda LTD: Patents & Royalties. Gidron:Yeda LTD: Patents & Royalties. Kagan:Yeda LTD: Patents & Royalties. Reisner:Cell Source LTD: Consultancy, Equity Ownership, Patents & Royalties, Research Funding.

An important role for adenine, cholera toxin, hydrocortisone and triiodothyronine in the proliferation, self-renewal and differentiation of limbal stem cells in vitro

The cornea is a self-renewing tissue located at the front of the eye. Its transparency is essential for allowing light to focus onto the retina for visual perception. The continuous renewal of corneal epithelium is supported by limbal stem cells (LSCs) which are located in the border region between conjunctiva and cornea known as the limbus. Ex vivo expansion of LSCs has been successfully applied in the last two decades to treat patients with limbal stem cell deficiency (LSCD). Various methods have been used for their expansion, yet the most widely used culture media contains a number of ingredients derived from animal sources which may compromise the safety profile of human LSC transplantation. In this study we sought to understand the role of these components namely adenine, cholera toxin, hydrocortisone and triiodothyronine with the aim of re-defining a safe and GMP compatible minimal media for the ex vivo expansion of LSCs on human amniotic membrane. Our data suggest that all four components play a critical role in maintaining LSC proliferation and promoting LSC self-renewal. However removal of adenine and triiodothyronine had a more profound impact and led to LSC differentiation and loss of viability respectively, suggesting their essential role for ex vivo expansion of LSCs. Replacement of each of the components with GMP-grade reagents resulted in equal growth to non-GMP grade media, however an enhanced differentiation of LSCs was observed, suggesting that additional combinations of GMP grade reagents need to be tested to achieve similar or better level of LSC maintenance in the same manner as the traditional LSC media.

Human Multipotent Mesenchymal Stromal Cells in the Treatment of Postoperative Temporal Bone Defect: An Animal Model.

Canal wall down mastoidectomy is one of the most effective treatments for cholesteatoma. However, it results in anatomical changes in the external and middle ear with a negative impact on the patient's quality of life. To provide complete closure of the mastoid cavity and normalize the anatomy of the middle and external ear, we used human multipotent mesenchymal stromal cells (hMSCs), GMP grade, in a guinea pig model. A method for preparing a biomaterial composed of hMSCs, hydroxyapatite, and tissue glue was developed. Animals from the treated group were implanted with biomaterial composed of hydroxyapatite and hMSCs, while animals in the control group received hydroxyapatite alone. When compared to controls, the group implanted with hMSCs showed a significantly higher ratio of new bone formation (p = 0.00174), as well as a significantly higher volume percentage of new immature bone (p = 0.00166). Our results proved a beneficial effect of hMSCs on temporal bone formation and provided a promising tool to improve the quality of life of patients after canal wall down mastoidectomy by hMSC implantation.

367 Phase I clinical trial for Recessive Dystrophic Epidermolysis Bullosa using genetically corrected autologous keratinocytes

Recessive Dystrophic Epidermolysis Bullosa (RDEB) is an inherited genetic skin disorder caused by mutations in the COL7A1 gene encoding type VII collagen (C7). The Current therapy is limited to supportive palliation. We report the results from the first Phase I clinical trial of ex vivo gene therapy for the treatment of severe RDEB (NCT01263379). 4 adult, male RDEB subjects enrolled in this trial carried various heterozygous COL7A1 mutations resulting in expression of only truncated C7 protein undetectable by NC2 antibodies and displayed absent, or sparse rudimentary anchoring fibrils (AF) by EM. Autologous RDEB KC isolated from skin biopsies were transduced with GMP grade retrovirus carrying the full-length COL7A1. 6 ∼35cm2 autologous epidermal sheet grafts were manufactured and grafted onto prepared wound beds of each subject (N=24 grafts). The primary endpoints included expression of full-length C7 and restoration of AF at 3 and 6 months, as well as absence of RCR at 3, 6, 12 months post grafting. All 24 grafts were well tolerated by all subjects, and no serious adverse events were reported. At 3 months, 87% (21/24 grafts) and at 6 months, 67% (16/24 grafts) showed significant wound healing, as of 75% healing compared with baseline. Molecular analysis of the skin biopsies obtained from the grafted sites revealed robust C7 expression detected by both NC1 and NC2 C7 antibodies, with morphologically normal appearance and frequency of NC2 reactive AF identified at the basement membrane zone using IEM. These data indicate that COL7A1 ex-vivo gene transfer has a favorable safety profile and capable of cutaneous type VII collagen delivery highlighting the potential of durable cell based RDEB therapy in humans.

18F-nanobody for PET imaging of HER2 overexpressing tumors

IntroductionRadiolabeled nanobodies are exciting new probes for molecular imaging due to high affinity, high specificity and fast washout from the blood. Here we present the labeling of an anti-HER2 nanobody with 18F and its validation for in vivo assessment of HER2 overexpression. MethodsThe GMP grade anti-HER2 nanobody was labeled with the prosthetic group, N-succinimidyl-4-[18F]fluorobenzoate ([18F]-SFB), and its biodistribution, tumor targeting and specificity were evaluated in mouse and rat tumor models. Results[18F]FB-anti-HER2 nanobody was prepared with a 5–15% global yield (decay corrected) and a specific activity of 24.7±8.2MBq/nmol. In vivo studies demonstrated a high specific uptake for HER2 positive xenografts (5.94±1.17 and 3.74±0.52%IA/g, 1 and 3h p.i.) with high tumor-to-blood and tumor-to-muscle ratios generating high contrast PET imaging. The probe presented fast clearance through the kidneys (4%IA/g at 3h p.i.). [18F]FB-anti-HER2 nanobody is able to image HER2 expressing tumors when co-administered with the anti-HER2 therapeutic antibody trastuzumab (Herceptin), indicating the possibility of using the tracer in patients undergoing Herceptin therapy. ConclusionsThe GMP grade anti-HER2 nanobody was labeled with 18F. This new PET probe for imaging HER2 overexpression in tumors has ample potential for clinical translation.