ALBERT

Description: Bortezomib (Velcade) is the first proteasome inhibitor that has been introduced into the clinic for the treatment of multiple myeloma. Bortezomib, by inhibiting the NFkB function, results in increased tumor cell sensitivity to chemotherapy and induces apoptosis of alloreactive T-cells. Since NF-kB activation results also in transcription of pro-inflammatory molecules, it is speculated that NF-kB inhibition may also ameliorate inflammatory and autoimmune conditions. Rheumatoid arthritis may be a possible target disease for proteasome inhibitors because the most important pro-inflammatory mediators (TNF-a, IL-1, IL-6, V-CAM-1) are regulated by NF-kB. We investigated the effect of Bortezomib in a rat model of Adjuvant Arthritis (AA) which closely resembles human rheumatoid arthritis. The presence of 5,10,100 nM bortezomib for 72h or the presence of 10 nM bortezomib for 48 and 72 h in culture with ConA–activated T-cells from the spleen of rats after AA induction, significanlty inhibited their proliferation as measured by radioactive thymidine incorporation (p

Description: Viral infections remain a significant cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HSCT). Adoptive immunotherapy with donor-derived virus-specific T cells (VSTs) has proven safe and effective for the prophylaxis and treatment of EBV, CMV, AdV, BKV and HHV-6 infections post-HSCT. However, broader application is restricted by the time taken to prepare patient-specific products and the lack of virus-specific T cell precursors in cord blood and seronegative donors. Thus, to assess whether 3rd party multivirus-directed VSTs could produce clinical benefit when administered as an "off the shelf" product to allogeneic HSCT recipients with refractory BKV, EBV, CMV, AdV and/or HHV-6 infections we initiated a Phase II clinical trial and now report interim results for 22 patients infused to date. We prepared a bank of 58 VST lines from individuals with common HLA polymorphisms by exposing donor PBMCs (3x107) to overlapping peptide libraries spanning AdV (Hexon, Penton), EBV (LMP2, EBNA1, BZLF1), CMV (pp65, IE1), BKV (Large T, VP1) and HHV-6 (U11, U14, U90) antigens followed by a 9-11 day expansion phase in a G-Rex device in the presence of IL4 and IL7. This produced a mean of 4.2±1x108 VSTs that were polyclonal, comprising both CD4+ (61±2.4%) and CD8+ (34±2.1%) cells that expressed central (CD45RO+/CD62L+/CCR7+ - 43±3.9%) and effector memory markers (CD45RO+/CD62L- - 10±1%). 56/58 lines had activity against AdV, 50/58 against EBV, 35/58 against BKV, 34/58 against HHV-6, and 33/58 against CMV. To date, 62 HSCT recipients have been screened for study participation and a potential line, based on target virus specificity through a shared allele and overall HLA match, was identified for 58. Of these, 22 patients with infections that were unresponsive to at least 2 lines of antiviral treatment have been infused (fixed dose level - 2x107 VSTs/m2) with VST lines matched at 1 to 6 HLA antigens. Seventeen received just 1 infusion, while 5 patients required 2 or more infusions for sustained benefit. Eight patients received VSTs for CMV infections, including 3 cases of CMV colitis, 8 for BKV (6 for cystitis, 2 for nephritis), 1 for HHV-6, 1 for EBV-PTLD, 1 for AdV, 1 for BKV and EBV, 1 for CMV and AdV and 1 for CMV and BKV infections. There were no immediate adverse effects related to infusion. Based on viral load measurements by quantitative PCR a single VST infusion successfully controlled active infections in 19/21 evaluable patients: CMV (4 CR, 5 PR, 1NR); EBV (2 CR); AdV (1 CR, 1 NR); BKV (1 CR, 7 PR); and HHV-6 (1 PR). Of note, all 6 patients with BK hemorrhagic cystitis and 2/3 patients with CMV colitis had marked improvement/resolution of symptoms following VST treatment. In 8 subjects who responded to VST therapy we saw an increase in virus-specific T cells post-infusion. These expanded cells were confirmed to be of 3rd party VST origin in 3 patients and persisted for up to 6 weeks post-infusion. Finally, despite the HLA disparity of VSTs and recipients, de novo GvHD occurred in only one subject, who developed Grade I skin GVHD 1 week post-infusion, which resolved with the administration of topical steroids. One additional patient had a flare of chronic skin GVHD coincident with tapering of immunosuppression and 1 patient developed a transient fever 5 hours post-infusion, which spontaneously resolved. These results demonstrate the feasibility and safety of 3rd party multivirus-directed VSTs, generated by direct stimulation of PBMCs with synthetic peptides and administered as an "off the shelf" product. The infused cells were capable of in vivo expansion in allogeneic HSCT recipients and proved clinically effective against refractory EBV, CMV and AdV infections and also controlled HHV-6 reactivation and BKV-associated hemorrhagic cystitis. Disclosures Off Label Use: Adoptively transferred T cells administered under an IND. Brenner:Bluebird Bio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Celgene: Other: Collaborative Research Agreement; Cell Medica: Other: Licensing Agreement. Rooney:Celgene: Other: Collaborative research agreement; Cell Medica: Other: Licensing Agreement. Heslop:Celgene: Other: Collaborative research agreement; Cell Medica: Other: Licensing Agreement.

Description: Epstein-Barr virus (EBV) reactivation post allogeneic hematopoietic stem cell transplantation (HSCT) can lead to the outgrowth of EBV-infected B cells and the development of post-transplant lymphoproliferative disease (EBV-PTLD). Since 1993 our group has used adoptive transfer of in vitro expanded EBV-specific T cells as a means to prevent or treat these EBV-driven lymphomas. In a series of Phase I and II clinical trials we have assessed the safety and clinical benefit associated with these transferred cells in allogeneic HSCT recipients. The T cell products infused were generated using 3 different manufacturing methodologies - [(i) EBV-transformed lymphoblastoid cell lines (EBV-LCLs) (~12 weeks manufacturing time), (ii) plasmid-nucloefected dendritic cells (DCs) (17 day manufacturing) and (iii) direct stimulation of PBMCs using overlapping peptide libraries (10 day manufacturing)] and were administered to either prevent (n=162) or treat (n=47) EBV reactivation/disease in a total of 209 allogeneic HSCT recipients ranging in age from 6 months to 63 years. 198/209 patients were infused with donor-derived virus-specific T cells (VSTs) and administered at doses ranging from 5x106 to 1.2x108 cells/m2. Infusions were well tolerated, with acute graft versus host disease (aGvHD) grade I-II documented in just 10 patients, of which 2 cases were de novo. Chronic GvHD occurred in 13 patients and was extensive in 2. Other toxicities were seen only in patients with active disease and included localized swelling at the tumor site due to EBV-specific T cell infiltration (n=4), which was severe in 2 causing transient airway obstruction. An additional patient with bulky, Rituximab-resistant EBV-PTLD developed fever and sepsis-like signs post-infusion, coincident with a decrease in viral load, an increase in the frequency of circulating EBV-specific T cells and an elevation in plasma cytokines consistent with a cytokine release syndrome. However, the symptoms resolved within hours of administering steroids and an anti-TNF antibody and did not recur. Of 162 patients infused prophylactically only 1 (0.6%) developed EBV-PTLD, which occurred following the administration of steroids 3 weeks-post VST infusion. However, this patient responded to a second VST infusion after the steroids. 31 of 36 (86%) patients with elevated viral load (n=21) or biopsy-proven/probable LPD (n=15) achieved durable complete remissions and the infused cells persisted long term as demonstrated in 26 patients who received gene-marked VSTs that were detectable for up to 9 years post-infusion. Based on the safety and efficacy profile of donor-derived EBV-specific VSTs we extended our approach to provide an "off the shelf" product to third party recipients, generating and banking T cell lines from 88 consenting normal donors. To date we have treated 11 patients, all of whom had drug-refractory EBV infections. Two patients were treated for elevated viral loads while 9 had established PTLD. Patients received 1-5 doses (fixed dose - 2x107 cells/m2) with 3rd party VSTs matching at one to three of six HLA alleles. Despite the HLA disparity the cells have proven safe with a single case of grade I GvHD reported. Of the 11 patients infused, 8 responded to therapy with 5 complete and 3 partial responses for an overall response rate of 73%. Overall our experience with adoptively-transferred EBV-specific T cells over a 22-year period demonstrates the safety and efficacy of this approach for the prevention and treatment of post-transplant EBV disease. Disclosures Off Label Use: Adoptively transfered T cells administered under an IND. Rooney:Celgene: Other: Collaborative research agreement; Cell Medica: Other: Licensing Agreement. Heslop:Cell Medica: Other: Licensing Agreement; Celgene: Other: Collaborative research agreement.

Description: Viral infections remain a significant cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HSCT). Adoptive immunotherapy with donor-derived virus-specific T cells (VSTs) has proven safe and effective for the prophylaxis and treatment of EBV, CMV, AdV, BKV and HHV-6 infections post-HSCT. However, broader application is restricted by the time taken to prepare patient-specific products and the lack of virus-specific T cell precursors in cord blood and seronegative donors. Thus, to assess whether 3rd party multivirus-directed VSTs could produce clinical benefit when administered as an "off the shelf" product to partially HLA-matched allogeneic HSCT recipients with drug-refractory BKV, EBV, CMV, AdV and/or HHV-6 infections we initiated a Phase II clinical trial (fixed cell dose of 2x107 VSTs/m2) using a bank of 59 VST lines generated from healthy, eligible donors. To date, 32 patients with one (n=28) or two (n=4) drug-refractory infections have been infused with lines matched at 1 to 6 HLA antigens. Fifteen patients received VSTs to treat CMV (including 3 cases of CMV colitis), 13 for BKV (11 for hemorrhagic cystitis, 2 for nephritis), 4 for AdV (including 1 case of pneumonia), 2 for HHV-6 (including 1 case of encephalitis) and 2 for EBV (including 1 case of EBV-PTLD). Twenty-one patients received just 1 infusion of cells, while 11 required 2 or 3 infusions for sustained benefit. Despite the HLA disparity between the VST lines and the recipients, de novo graft versus host disease (GVHD) occurred in only 1 subject, who developed Grade I skin GVHD that resolved with topical steroids. One additional patient had a flare of chronic skin GVHD coincident with tapering of immunosuppression and 2 patients developed transient fevers a few hours post-infusion, which spontaneously resolved within a couple of hours. Based on viral load, as measured by quantitative PCR, 3rd party VSTs successfully controlled active infections in 29 of 31 evaluable patients: CMV (9 CR, 5 PR, 1 NR); EBV (2 CR); AdV (2 CR, 1 PR, 1 NR); BKV (4 CR, 9 PR); and HHV-6 (1 PR). Of note, all 11 patients with BK hemorrhagic cystitis, 2 of 3 patients with CMV colitis, and the patients with AdV pneumonia and HHV-6 encephalitis, all had marked improvement or resolution of symptoms within 6 weeks following VST treatment. In more than 50% of responders (n=16) we detected an increase in the circulating frequency of VSTs post-infusion, which were confirmed to be 3rd party VST origin in 8 and remained detectable for up to 12 weeks post-infusion. The eventual decrease in 3rd party VSTs coincided with endogenous immune reconstitution in most patients. These results confirm the feasibility and safety of 3rd party multivirus-directed VSTs, administered as an "off the shelf" product to treat drug-refractory infections/disease. The infused cells were capable of expanding in vivo and proved clinically effective against refractory EBV, CMV, AdV, BKV and HHV-6 reactivation and virus associated disease. Disclosures Tzannou: ViraCyte LLC: Consultancy. Brenner:Viracyte: Equity Ownership; Cell Medica: Patents & Royalties. Rooney:Cell Medica: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Viracyte: Equity Ownership. Heslop:Cell Medica: Patents & Royalties: Licensing agreement EBV-specific T cells; Viracyte: Equity Ownership; Chimerix: Other: Endpoint adjudication committee; Celgene: Patents & Royalties, Research Funding. Leen:ViraCyte LLC: Equity Ownership, Patents & Royalties.

Description: Drinking water distribution networks are among the most resilient infrastructure systems to disasters, specifically hazards such as accidental pollution, floods, droughts, earthquakes, and pandemics. Water operators experiencing these kinds of hazards should focus on the establishment of more effective response systems. The paper presents the outputs and results of improving response time and effectiveness of the capacity developed by national, bilateral, and EU Civil Protection mechanisms. The methodology used for the hazard risk assessment procedures and the analysis of the Water Safety Plans (WSPs) lead to improved preparedness mechanisms. The results showed that water use efficiency is a key component in resiliency.

Description: Plant growth promoting rhizobacteria (PGPR) are able to provide cross-protection against multiple stress factors and facilitate growth of their plant symbionts in many ways. The aim of this study was to isolate and characterize rhizobacterial strains under natural conditions, associated with naturally occurring representatives of wild plant species and a local tomato cultivar, growing in differently stressed Mediterranean ecosystems. A total of 85 morphologically different rhizospheric strains were isolated; twenty-five exhibited multiple in vitro PGP-associated traits, including phosphate solubilization, indole-3-acetic acid production, and 1-aminocyclopropane-1-carboxylate deaminase activity. Whole genome analysis was applied to eight selected strains for their PGP potential and assigned seven strains to Gammaproteobacteria, and one to Bacteroidetes. The genomes harboured numerous genes involved in plant growth promotion and stress regulation. They also support the notion that the presence of gene clusters with potential PGP functions is affirmative but not necessary for a strain to promote plant growth under abiotic stress conditions. The selected strains were further tested for their ability to stimulate growth under stress. This initial screening led to the identification of some strains as potential PGPR for increasing crop production in a sustainable manner.

Description: Background: Dendritic cells (DCs) have received much attention as a therapeutic tool for infections and immunotherapy of advanced malignancies, due to their unique ability of antigen-presentation and initiation of the T-cell-dependent immune response. However, the broader application of vaccine- or antitumor specific T cell–based immunotherapy is limited by DC’s scarcity in the peripheral blood and the complex and costly methodology required for their production. To date, peripheral blood monocytes are the most common source for DCs generation and recently hematopoietic stem cell (CD34+)-derived DCs have also been produced. However, the accessibility to stem cell sources is limited and the number of generated DCs are barely enough for wide clinical use. Aims: The non-transplantable umbilical cord blood (UCB) units could be a readily available and promising source for large scale DC production, by offering adequate number of CD34+ cells. In the present study, we developed a new culture method for the generation of high numbers of myeloid DCs from non-transplantable UCB units by using new generation bioreactors (G-rex) and estimated the minimum required volume of a UCB unit to produce adequate number of DCs for clinical application. Methods: CD34+cells from non-transplantable UCBs were purified by immunomagnetic separation, cultured in the presence of a cytokine cocktail [stem cell factor (SCF), granulocyte-macrophage colony-stimulating factor (GM-SCF) and interleukin-4 (IL-4)) for 10 days in tissue culture plates and subsequently expanded in G-rex bioreactors for 25 additional days. Control cultures were completed in conventional plates. DCs were matured either with a combination of Toll-like receptor ligands [(TLR-Ls), Poly I:C and R848] or with commonly used vaccines containing TLR-Ls [Act-HIV (bacterial form of influenza), Influvac (viral form of influenza), Typhim Vi, HBV and BCG ]. DCs phenotype was analyzed by flow cytometry (CD40, HLA-DR, CD83 and CD86) and the cytokine secretion values were measured by ELISA. The endophagocytic activity of DCs was assessed by a dead yeast engulfment assay. Results: DCs derived from UCB CD34+ cells with more than 85% purity, were cultured in bioreactors or in conventional plates. DCs cultured in Grexes reached a median fold expansion 20.875 and a median absolute number of 2 billions, in sharp contrast to a median 100-fold increase of conventionally cultured DCs. The expanded DCs had the typical morphology and surface markers of myeloid DCs (CD33+/CD11+: 70.47%).To address whether those highly expanded DCs are functional and have the ability, upon maturation, to induce Th1 response, the cells were matured with either a cocktail of TLR-Ls or commonly used vaccines, and tested for the expression of molecules needed for antigen presentation, endophagocytic activity and cytokine production. Similar to conventional DCs, the highly-expanded and matured with vaccines or TLR-Ls DCs, showed dendrites and endocytotic capacity. The expanded and matured with either way DCs, expressed also HLA-DR and co-stimulatory molecules (CD40, CD86 and CD83) whereas they produced increased levels of IL-12p70, TNF-α and IL-6 but undetectable IL-10, thus suggesting a strong potential for Th1 response. We tested different volumes and UCB units for CD34+cell-derived DCs and showed that the minimum volume of a UCB unit that could be used for large scale DCs generation with our optimized culture system, is 9ml, corresponding to approximately 10% of the volume of a transplantable UCB unit. Conclusion: We established an optimized and simple culture system to expand at clinically relevant numbers, the production of human, myeloid DCs from UCB-derived CD34+ cells. These DCs have the functional properties that are necessary to obtain therapeutic gains against malignancies and viral infections. Billions of DCs were derived from a minimum of 10% of the volume of an average transplantable UCB unit. This new method could be one step closer to a broader clinical application of DC- and T-cell based immunotherapy for various malignancies. Disclosures No relevant conflicts of interest to declare.

Description: Viral infections, mainly by cytomegalovirus (CMV), Epstein Barr virus (EBV) and polyomavirus type I (BKV), are major causes of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (allo-HSCT). As effective immune responses against human viruses rely on an armamentarium of T-cell receptor (TR) repertoire capable of recognizing a broad range of antigenic peptides of those pathogens, reconstitution of antiviral immunity, either by spontaneous generation of endogenous virus-specific T cells (VSTs) or by adoptive immunotherapy with VSTs, plays a critical role to fight infections. We here evaluated the diversity and clonality of TR repertoire of functional tri-virus-specific T cell products generated from immunocompetent donors (n=10) and compared their TR gene repertoire to that of peripheral blood mononuclear cells (PBMCs) from patients who had undergone allo-HSCT (n=5). To generate tri-VSTs, PBMCs derived from 15-20ml of peripheral blood of normal donors, were exposed to EBV, CMV and BKV overlapping peptides and cultured in the presence of interleukin 4 (IL-4) and IL-7 for 10 days in G-rex bioreactors. Specificity of donor-derived VSTs and patient-derived PBMCs was measured by IFN-γElispot. TR diversity was investigated by next-generation sequencing on a MiSeq Sequencer, after amplification of TR beta chain gene rearrangements by RT-PCR with the BIOMED-2 protocol. Raw NGS reads were filtered based on their length and quality and the filtered-in sequences were submitted to IMGT/HighVQUEST. Metadata analysis and clonotype computation were performed using a validated in-house bioinformatics platform. As clonotype we defined sequences carrying the same TRBV gene and identical CDR3 amino acid sequence. Tri-VSTs provided 947,298 productive TRBV-TRBD-TRBJ rearrangements and a polyclonal and highly diverse TR gene repertoire, consisting of a total of 169,502 unique clonotypes (average: 16,950/sample, range 4,057-45,602), 64,971 (38.3%) of which were expanded (corresponding to more than one sequence). In terms of clonality, the mean relative frequency of the major clonotype in all tri-VSTs was 12.6% (range 3.3-29.2%). Interestingly, among tri-VST cell lines, 637 clonotypes were shared (present in 〉2/10 samples), 80 were highly shared (present in 〉3/10 samples) while 7 were present in 6-8 different VST lines and largely expanded, accounting for up to 29.2% of all sequences. Importantly, there were 65 of 96 major VST clonotypes shared, thus suggesting that they were potentially associated with recognition of the targeted viruses. Given that 4/10 VSTs cell lines were not specific for CMV, while being EBV-and BKV-specific, dominant TRs in those 4 cell lines can potentially be associated with EBV- or BKV-activity. By searching a public database of TR clonotypes with known reactivity against EBV and/or CMV (ShugayM, Nucleic Acids Research, 2018), we found 8 shared EBV-specific and 4 shared CMV-specific clonotypes among our VSTs and the 499 public clonotypes. When we compared the produced VSTs with PBMCs from 3 allo-grafted patients with circulating CMV-, BKV- and EBV-specific T cells and previous viral reactivation, we detected 163 shared clonotypes. Likewise, we observed 21 and 23 shared clonotypes in similar frequencies, between VSTs and PBMCs from 2 patients with CMV- or BKV-specific T cell immunity. These data identify clones that potentially expand in vivo and protect patients from viral infections. Overall, our findings reveal high levels of TR clonality in cell lines enriched for T cells reactive against EBV and/or CMV and/or BKV and provide insights into the TR repertoire of ex vivo- or endogenously-generated VSTs. Our approach may help to identify optimal TRs for immunotherapy as well as TRs which can be used as a tool for risk stratification of viral infections. Disclosures Agathangelidis: Gilead: Research Funding. Gemenetzi:Gilead: Research Funding. Stamatopoulos:Abbvie: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Gilead: Honoraria, Research Funding. Hadzidimitriou:Gilead: Research Funding; Abbvie: Research Funding; Janssen: Honoraria, Research Funding.