ALBERT

Description: Significant resources have been invested in sequencing studies to investigate the role of rare variants in complex disease etiology. However, the diagnostic interpretation of individual rare variants remains a major challenge, and may require accurate variant functional classification and the collection of large numbers of variant carriers. Utilizing sequence data from 458 individuals with hypertriglyceridemia and 333 controls with normal plasma triglyceride levels, we investigated these issues using GCKR , encoding glucokinase regulatory protein. Eighteen rare non-synonymous GCKR variants identified in these 791 individuals were comprehensively characterized by a range of biochemical and cell biological assays, including a novel high-throughput-screening-based approach capable of measuring all variant proteins simultaneously. Functionally deleterious variants were collectively associated with hypertriglyceridemia, but a range of in silico prediction algorithms showed little consistency between algorithms and poor agreement with functional data. We extended our study by obtaining sequence data on family members; however, functional variants did not co-segregate with triglyceride levels. Therefore, despite evidence for their collective functional and clinical relevance, our results emphasize the low predictive value of rare GCKR variants in individuals and the complex heritability of lipid traits.

Description: Mutations in glucokinase ( GCK ) cause a spectrum of glycemic disorders. Heterozygous loss-of-function mutations cause mild fasting hyperglycemia irrespective of mutation severity due to compensation from the unaffected allele. Conversely, homozygous loss-of-function mutations cause permanent neonatal diabetes requiring lifelong insulin treatment. This study aimed to determine the relationship between in vitro mutation severity and clinical phenotype in a large international case series of patients with homozygous GCK mutations. Clinical characteristics for 30 patients with diabetes due to homozygous GCK mutations (19 unique mutations, including 16 missense) were compiled and assigned a clinical severity grade (CSG) based on birth weight and age at diagnosis. The majority (28 of 30) of subjects were diagnosed before 9 months, with the remaining two at 9 and 15 years. These are the first two cases of a homozygous GCK mutation diagnosed outside infancy. Recombinant mutant GCK proteins were analyzed for kinetic and thermostability characteristics and assigned a relative activity index (RAI) or relative stability index (RSI) value. Six of 16 missense mutations exhibited severe kinetic defects (RAI ≤ 0.01). There was no correlation between CSG and RAI ( r 2 = 0.05, P = 0.39), indicating that kinetics alone did not explain the phenotype. Eighty percent of the remaining mutations showed reduced thermostability, the exceptions being the two later-onset mutations which exhibited increased thermostability. Comparison of CSG with RSI detected a highly significant correlation ( r 2 = 0.74, P = 0.002). We report the largest case series of homozygous GCK mutations to date and demonstrate that they can cause childhood-onset diabetes, with protein instability being the major determinant of mutation severity.

Description: Author(s): C. Mok, B. Barrett, A. Carew, R. Berthiaume, S. Beattie, and A. Kumarakrishnan We have developed two configurations of an echo interferometer that rely on standing-wave excitation of a laser-cooled sample of rubidium atoms. Both configurations can be used to measure acceleration a along the axis of excitation. For a two-pulse configuration, the signal from the interferometer i... [Phys. Rev. A 88, 023614] Published Tue Aug 20, 2013

Description: Author(s): B. Barrett, A. Carew, S. Beattie, and A. Kumarakrishnan We describe progress toward a precise measurement of the recoil energy of an atom measured using a modified grating-echo atom interferometer (AI) that involves three standing-wave (sw) pulses. With this technique, an additional sw pulse is used to shift the phase of excited momentum states, which pr... [Phys. Rev. A 87, 033626] Published Mon Mar 25, 2013

Description: Peptidases, their substrates and inhibitors are of great relevance to biology, medicine and biotechnology. The MEROPS database ( http://merops.sanger.ac.uk ) aims to fulfill the need for an integrated source of information about these. The database has hierarchical classifications in which homologous sets of peptidases and protein inhibitors are grouped into protein species, which are grouped into families, which are in turn grouped into clans. Recent developments include the following. A community annotation project has been instigated in which acknowledged experts are invited to contribute summaries for peptidases. Software has been written to provide an Internet-based data entry form. Contributors are acknowledged on the relevant web page. A new display showing the intron/exon structures of eukaryote peptidase genes and the phasing of the junctions has been implemented. It is now possible to filter the list of peptidases from a completely sequenced bacterial genome for a particular strain of the organism. The MEROPS filing pipeline has been altered to circumvent the restrictions imposed on non-interactive blastp searches, and a HMMER search using specially generated alignments to maximize the distribution of organisms returned in the search results has been added.

Description: Abstract 3283 Donor-derived regulatory T cells (Treg) and natural killer (NK) cells can respectively improve stem cell transplant (SCT) outcome by reducing graft versus host disease (GVHD) severity and exerting a graft-versus-leukemia effect. High frequencies of donor Treg are associated with less GVHD, and low doses of interleukin-2 (IL-2) can expand both NK and Treg after allogeneic SCT. To explore the feasibility of improving the quality of peripheral blood SCT donations, we evaluated the safety and the tolerability of ultra-low dose IL-2 administration to volunteers with the aim of preferentially expanding Treg and NK cells. Twelve healthy volunteers (mean age 34 years; range 22–57) received 0.1 or 0.2 million U/m2/day IL-2 subcutaneously for 5 days (NIH protocol 11-H-0268). Blood samples were collected before and 1, 2, 3, 4, 7 and 28 days after IL-2 injection. Samples were analyzed by multiplex techniques including whole transcriptome gene expression with HumanGene 1.0ST microarrays; serum levels of 69 cytokines and chemokines by Luminex assay; and lymphocyte phenotyping by flow cytometry, to comprehensively characterize the cellular and molecular immune response to IL-2 (“IL-2 immunome”). Treg subsets were determined within the CD4+ T cell population using FoxP3, Helios, CD45RA and CD31 to identify thymus-derived natural Treg (nTreg), induced Tregs (iTreg) and their recent thymic emigrants (RTE). NK cell subsets were determined within CD56+CD3- population using NKG2A, KIR2DL1, KIR2DL2/3, KIR3DL1 and CD57 to identify CD56bright, CD56dim NKG2A+KIR-, and CD56dim KIR+CD57+ cells. All subjects tolerated ultra-low dose IL-2 with minimal adverse events (mainly grade 1–2 injection site reactions). The fraction of FoxP3+Treg in CD4 rose significantly above baseline peaking at 4 days (3.7% vs 5.8%; p=0.0004) after the first dose of IL-2. Treg subset analysis demonstrated that the fraction of nTreg and RTE nTreg in CD4 expanded significantly in the lower dose cohort compared to the higher dose cohort (p=0.004 and p=0.005 respectively). %CD56bright NK significantly increased at 7 days (p=0.008), whereas CD56dimNKG2A+KIR-, and CD56dimKIR+CD57+ NK cells remained at baseline. The Ki67 proliferation marker further verified a significant in vivo expansion of CD56bright NK cells with ultra-low dose IL-2. Cytokine and chemokine profiling demonstrated significant increase circulating level of IP-10 (P=0.0018) through day 2 to 4 after IL-2 injections. In contrast, circulating levels of IL-2, IL-6, IL-10, IL-15 and IL-17 remained unchanged after IL-2 injection. Gene expression microarray studies revealed significant changes in 24 genes (P value 〈 0.1 corrected by false discovery rate (FDR) for multiple testing), including up-regulation of IL-2RA and FOXP3 as early as 2 days after IL-2 injections. Gene Set Analysis (GSA) revealed significant changes (P value 〈 0.1 after FDR) in innate immune response pathways, including Toll-like receptor signaling and interferon signaling. This is the first study to show that ultra-low dose IL-2 could be safely administrated to healthy volunteers to expand thymic-derived natural Treg and CD56bright NK cells. These results raise the possibility of using ultra-low dose IL-2 to boost Treg and NK cells in stem cell donors. Disclosures: No relevant conflicts of interest to declare.

Description: Donor lymphocyte infusions (DLI) following hematopoietic stem cell transplantation may reduce or control opportunistic infections and leukemia/lymphoma relapse, but the associated graft versus host disease (GvHD) limits the clinical success of this procedure. Since T cell immunotherapy may be a safer alternative to DLI we have now used a single T cell platform that mediates both antileukemic and antiviral activity. Autologous T cells modified to express CD19-specific chimeric antigen receptors (CD19.CAR) have had clinical activity against CD19-expressing malignancies, but it is unknown if similarly modified allogeneic T cells will be equally effective. Allogeneic virus specific T cells (VSTs) directed to cytomegalovirus (CMV), adenovirus (Adv), and Epstein Barr virus (EBV) have been shown to be safe and effective in preventing and treating life-threatening viral infections post HSCT. Therefore, we sought to determine whether allogeneic VSTs could be engineered to express CD19.CAR and would retain the safety and effectiveness of unmodified VSTs whilst gaining anti-tumor activity. VSTs were expanded ex vivo using antigen presenting cells engineered to express adenovirus and cytomegalovirus (using an Ad5f35 adenoviral vector expressing the CMV pp65 gene), and Epstein Barr virus (using EBV-infected lymphoblastoid cell lines) antigens. After 3 stimulations, the VST’s were modified to express CD19.CAR.28ζ using a retroviral vector encoding the CAR-CD19 receptor coupled to the CD28 co-stimulatory molecule and the T cell receptor zeta (ζ) chain. Nine CD19.CAR-modified virus specific T cell (CD19.CAR-VSTs) products were generated for infusion. All VST lines recognized at least one viral antigen as determined by Elispot or chromium release assays and 20% to 48% of cells expressed the CD19.CAR. All lines killed CD19-expressing cells in vitro. We treated nine patients with these CD19.CAR-VSTs, 3 months to 13 years after HSCT. Six patients received CD19.CAR-VSTs for relapsed disease and 3 patients received the T cells as adjuvant therapy to prevent viral infection and relapse after HSCT. Safety. There were no infusion-related toxicities. One patient presented with gastrointestinal symptoms following infusion subsequently determined to be unrelated to the T cells. Persistence. VSTs persisted a median of 8 weeks in the peripheral blood and up to 9 weeks at disease sites. In three patients (#1, #3 and #5), CD19.CAR signals were detectable in the bone marrow or the lymph nodes (44.8, 25.85, and 32 copies/1000 ng DNA) even when no signal was measurable in peripheral blood, indicating preferential accumulation of the infused T cells at the disease site. Anti-Tumor Activity. During the period of CD19.CAR-VST persistence, objective anti-tumor activity was evident in 2/6 patients with relapsed disease (patient # 1 had detectable blasts in the peripheral blood which disappeared within 1-2 weeks following infusion, patient # 2 had 16% circulating CLL cells which decreased within 2 weeks of T cell infusion) but disease recurred after 3 and 2 months, respectively. The two patients who received cells while in remission remain disease-free 〉3 and 〉9 months later. Anti-Viral Activity. In two patients with EBV reactivation, donor CD19.CAR-VSTs expanded concomitant with an increase in virus-specific T cell responses, and decreased viral load. A third patient had a rise in adenovirus specific VSTs during an episode of adenovirus associated diarrhea. Although the infection was controlled, there was no concomitant rise in CD19-CAR expressing T cells in this patient. No other patient had viral disease. In conclusion, allogeneic CD19.CAR-VSTs administered after allogeneic HSCT are safe and can exert both anti-tumor and anti-viral activity in the absence of GvHD. Earlier administration of CD19.CAR-VSTs after HSCT, when the host is lymphodepleted and the incidence of viral infection is higher, may allow these cells to better capture the potential advantages of native TCR stimulation (and associated co-stimulation) for expansion and persistence, and thereby produce a higher frequency of sustained tumor responses. Alternatively, intentional stimulation of the native TCRs by viral vaccines may produce equal benefit, with greater predictability. Disclosures: Savoldo: Celgene: Patents & Royalties, Research Funding. Heslop:Celgene: Patents & Royalties, Research Funding; Cell Medica: Patents & Royalties. Rooney:Cell Medica: Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties, Research Funding; Celgene: Patents & Royalties, Research Funding. Brenner:Celgene: Patents & Royalties, Research Funding. Dotti:Celgene: Patents & Royalties, Research Funding.

Description: Abstract 3002 Adoptive transfer of CMV-specific T cells derived from adult CMV-seropositive (CMVpos) donors can effectively restore antiviral immunity after stem cell transplantation. However due to the absence of CMV antigen-specific memory T cells in cord blood (CB) and adult CMV-seronegative (CMVneg) donors, different culture systems are required to generate virus-specific T cells for adoptive transfer. With a novel protocol we have generated CMVpp65-specific T cells from CB and found that 15/15 CB T cell lines recognized atypical epitopes of pp65. We then explored the generation of CMV-specific CTL from CMVneg donors using a GMP-compliant methodology and studied the epitopes recognized. CD45RA+ naive T cells were selected from the peripheral blood of CMVneg donors and stimulated with pp65-Pepmix-pulsed dendritic cells with supplemented with IL-7, IL-12, and IL-15. For subsequent stimulations T cells were stimulated with pp65-Pepmix-pulsed EBV-LCL and IL-15 or IL-2. CMVpp65-specific T cells (CMV-CTL) expanded from 8 of 11 CMVneg donors were primarily CD8+ T cells (mean 71%). Naïve donor CMV-CTL secreted IFN- γ in response to pp65 peptides (mean 224; range: 38–611 SFC/1×105 cells) compared to irrelevant peptides (mean 12;Range 3–37) as measured in Elispot assays and lysed pp65-pulsed target cells (mean :48; range :15–70%) but not negative controls (mean 22; range 4–40%). These CMV-CTL derived from naive (but not memory) T cells recognized only novel and atypical pp65 epitopes (such as the HLA-A2-restricted epitopes LQTGIHVRV and MLNIPSINV) but not the typical HLA-A2-restricted epitope NLVPMVATV as confirmed by ELISPOT and multimer analysis. These results are similar to CB-derived CTL. Analysis of the avidity of naïve donor CTL specific for the atypical CMV epitopes revealed that the 1/2 maximum effective concentration was similar (mean: 600 pM) to CMVpos CTL recognizing typical epitopes (mean: 300 pM), and more avid than CMVpos CTL recognizing atypical epitopes (mean: 4 nM), highlighting the difference between naïve-derived and memory-derived CTL. TCR sequencing performed on T cells specific for typical (CMVpos) and atypical (CMVpos, CMVneg, and CB) epitopes revealed that CMVpos donor CMV-CTL recognizing typical epitopes were markedly more oligoclonal than CTL recognizing the atypical epitopes derived from CB, CMVpos, or CMVneg donors. To address the concern that atypical epitopes might not be naturally presented by CMV-infected cells and therefore not recognized by in vitro generated CTL, we tested whether CMV CTL (from CB, CMVpos, CMVneg) generated using CMV AD169-infected fibroblasts or CMV VR1814-infected DCs would recognize the same epitopes. As before, CMVpos CMV CTL recognized typical epitopes of pp65 while CB and CMVneg CMV CTL recognized only atypical epitopes, suggesting that the epitopes are naturally processed and presented by APCs, and that the atypical epitopes observed are not an artifact of using exogenous antigens like the pp65 Pepmix. Thus, despite their unusual repertoire, T cells derived from CB or CMVneg donors are likely to control CMV infection. These results reveal major differences in the naïve and memory CMV specific T cell repertoire that merits further exploration. Nevertheless, we demonstrated that atypical epitopes are naturally presented by CMV infected cells and we are now evaluating the clinical efficacy of these CTL in recipients of CBT. These studies should determine if naive T cells primed in vitro are able to persist and establish memory and virus protection in vivo. Disclosures: No relevant conflicts of interest to declare.