Scientific Co-founder Research Articles

PASTE, Don't Cut: Genome Editing Tool Looks Beyond CRISPR and Prime

PASTE, Don't Cut: Genome Editing Tool Looks Beyond CRISPR and Prime

Nested Therapeutics rakes in $90 million series A

Nested Therapeutics has launched with $90 million in series A funding to develop small-molecule cancer medicines by identifying druggable pockets on proteins involved in cancer-relevant pathways. The company’s lead compound, NEST-1, is a molecular glue that targets the proteins involved in the MAPK cellular signaling pathway. One scientific cofounder is Kevan Shokat, a University of California, Berkeley, chemistry professor who first drugged the protein KRas.

3T launches for immunotherapies

3T Biosciences has emerged from stealth with $40 million in series A funding to develop immunotherapies that treat solid tumors and other immune-mediated diseases. The company has a license to technology from Stanford University for discovering therapeutically active T-cell receptor mimetics. K. Christopher Garcia, 3T’s scientific cofounder, is a Stanford structural biologist. The license includes a development-stage therapy.

Conventional Chemotherapy Treatment Induces Unique Patterns of Clonal Evolution in a North American Adult T Cell Leukemia/Lymphoma Cohort

Adult T cell leukemia / lymphoma is a rare neoplasm with dismal cure rates and poor response to chemotherapy. The genetic basis of refractoriness against conventional agents is not known. We follow one of the largest cohorts of ATLL in the US and recently showed that North American ATLL (NA-ATLL) has a distinct mutational profile from Japanese ATLL (J-ATLL) with a higher incidence of epigenetic mutations (Shah et al, Blood, 2018; 132(14):1507-1518). Here, we analyze the mutational landscape of ATLL clones at diagnosis and at relapse in a pilot study of 7 NA-ATLL patients using deep targeted sequencing of 236 recurrently mutated genes. Our goal was to examine clonal evolution patterns that may result as a consequence of the selective pressure of chemotherapy by determining changes in mutational profiles as well as clonal abundance on patients with aggressive ATLL [acute and lymphomatous] (Table 1). All patients were treated with EPOCH (Etoposide, Prednisone, Vincristine, Cyclophosphamide and Doxorubicin) chemotherapy after initial diagnosis except one patient who received interferon therapy. Distinct patterns of clonal evolution were observed in patients with primary refractory disease when compared to those who relapsed after an initial remission. Group 1 has 3 cases (Pts 1 to 3), all of which experienced early relapse and showed mutation patterns suggestive of clonal replacement by a new emerging clone at relapse. Group 2 has 3 patients (Pts 4-6) that are characterized by worsening of primary refractory disease. Mutation patterns in the before-and-after sample pairs suggest linear progression from a previously existing clone during therapy. The clonal evolution status was not resolved in our third group (Pt#7) indicating that mutations outside the 236 genes evaluated might have been present. Frequent epigenetic mutations were found in both diagnostic and refractory/relapsed samples. Surprisingly, 4 epigenetic mutations (KMT2D, EP300, SPEN, SETBP1) identified in 4 cases were confirmed or suspected of germline origin. This suggests that ancestral genomic differences between the Caribbean and Japanese populations could contribute to the mutational and clinical disparities between NA- and J-ATLL. Multiple mutations with similar variant allele frequencies (VAF) at the time of relapse were identified, suggesting simultaneous acquisition of several mutations during clonal evolution. Clonal replacement by an emerging clone (Group 1) such as GATA3 (Pt#3) or KMT2D (Pt#1) (either germline or founding mutation) was observed in patients who achieved an initial response. On the other hand, in primary refractory disease (Group 2), relapse was driven by dominant TBL1XR1 mutations in 2 out of the 3 cases either alone (Pt#6) or in combination with mutated SMARCB1 (Pt#5). The protein encoded by TBL1XR1 is a negative regulator of the nuclear receptor corepressor (NCoR) /histone deacetylase 3 (HDAC3) complex. Therefore, TBL1XR1 inactivation is expected to result in hyper-activity of NCoR/HDAC3, an epigenetic abnormality targetable by HDAC inhibitors. SMARCB1 is also an epigenetic regulator associated with several malignancies and has been targeted with alisertib. A subclonal expansion of EP300 (a histone acetyltransferase) appears to drive relapse in the other case of this group (Pt#4). Therefore, our primary refractory cases show that epigenetic mutations seem to be of utmost importance not only at diagnosis as we have previously shown, but as the driving force behind chemotherapy refractoriness and subsequent relapse. Conclusion: To our knowledge, this is the first mutation-based clonal evolution study aimed to examine dynamic changes induced by chemotherapy among NA-ATLL patients. Early relapse appears to be driven by the emergence of new mutant clones. On the other hand, primary refractory disease appears to have epigenetic drivers that convey chemotherapy refractoriness and evolve from linear progression of a previously existing clone. Serial mutational testing can provide critical information on clonal architecture and identify actionable molecular vulnerabilities in a cohort without effective therapeutic options and a dismal prognosis. Disclosures Sica: Physician's Education Resource (PER): Honoraria. Shah:Physicians' Education Resource: Honoraria. Steidl:Aileron Therapeutics: Consultancy, Research Funding; Stelexis Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Scientific Co-Founder; Pieries Pharmaceuticals: Consultancy; BayerHealthcare: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; GlaxoSmithKline: Research Funding; Celgene: Consultancy. Verma:Stelexis: Equity Ownership, Honoraria; Acceleron: Honoraria; Celgene: Honoraria; BMS: Research Funding; Janssen: Research Funding.

High Burden of Clonal Hematopoiesis in First Responders Exposed to the World Trade Center Disaster

High Burden of Clonal Hematopoiesis in First Responders Exposed to the World Trade Center Disaster

Activating TP53 by Dual Inhibition of MDMX and MDM2

TP53 is often inactivated by mutations or other mechanisms in human cancers. Recent work has demonstrated that its endogenous inhibitor MDMX (or MDM4) is frequently overexpressed in patients with hematologic malignancies including AML, lymphoid malignancies, as well as other cancers. Pharmacological disruption of the interactions of TP53 with both its endogenous inhibitors (MDMX and MDM2) has long been sought after as an attractive strategy to restore p53-dependent tumor suppressor activity. However, selective targeting of this pathway has previously been limited to MDM2-only small-molecule inhibitors, which lack affinity for MDMX. More recently, pharmacological dual targeting of MDMX/MDM2 has become feasible through stapled peptides and is currently being tested in clinical trials. This presentation will discuss such MDMX/MDM2 dual-targeting strategies as well as new insights into MDMX-mediated mechanisms of tumor progression at the stem cell level, which have emerged from recent studies. Disclosures Steidl: Aileron Therapeutics: Consultancy, Research Funding; Stelexis Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Scientific Co-Founder; Pieries Pharmaceuticals: Consultancy; Celgene: Consultancy; BayerHealthcare: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; GlaxoSmithKline: Research Funding.

SF3B1 Mutations Induce Oncogenic IRAK4 Isoforms and Activate Targetable Innate Immune Pathways in MDS and AML

SF3B1 mutations are the most frequently occurring splicing factor mutations in MDS and AML, however the misspliced genes that contribute the malignant state in SF3B mutant MDS or AML remains unclear. We determined that SF3B1 mutant cases of MDS express a longer, active isoform of interleukin 1 receptor associated kinase (IRAK4). IRAK4 is a serine/threonine kinase that is downstream of toll-like receptor (TLR) signaling and leads to activation of oncogenic signaling states, including NF-kB and MAPK. Examination of IRAK4 by RNA sequencing showed that normal cells predominantly express small IRAK4 isoforms resulting from exclusion of the part of exon 6. These isoforms are targeted for proteosomal degradation leading to diminished IRAK4 expression and activation in normal cells. In contrast, a large proportion of MDS/AML samples with SF3B1 mutation show increased expression of an IRAK4 isoform that retains full exon 6, encoding the full-length protein (IRAK4-Long). Consequently, we show that expression of mutant SF3B1-K700E in leukemic cells is associated with increased NF-kB activity, suggesting that mutations in SF3B1 instruct expression of IRAK4 RNA isoforms with maximal functional potential. Furthermore, SF3B1 mutant MDS and AML cells exhibited a block in hematopoietic differentiation in clonogenic assays. This differentiation block was ameliorated with pharmacologic inhibition of IRAK4 with CA-4948, a potent oral clinically useful small-molecule inhibitor of IRAK4. CA-4948 blocked TLR-stimulated cytokine release in various cell models and also led to decreased leukemic burden in mice xenografted with SF3B1 mutant MDS/AML cells. Finally, we determined that SF3B1 mutation induced IRAK4 activation led to TRAF6 mediated K63 ubiquitination of critical cell cycle and regulatory proteins directly implicated in oncogenesis. We had recently shown that U2AF1 mutations can lead to IRAK4 activation via retention of exon 4 (Smith et al, Nat Cell Bio, 2019). Our data now demonstrate that SF3B1 leads to overactivation of IRAK4 via retention of a different exon (exon 6), thus reinforcing that IRAK/TRAF6 activation is a common downstream oncogenic pathway in splicing factor mutated MDS/AML. Taken together, in this study, we find that mutations in SF3B1 induce expression of therapeutically targetable "active" IRAK4 isoforms and provide a genetic link between a spliceosome mutation and oncogenic innate immune signaling in MDS and AML. Disclosures Booher: Curis: Employment. Ramachandra:Aurigene: Employment. Samson:Curis: Employment. Will:Novartis Pharmaceuticals: Research Funding. Steidl:BayerHealthcare: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; GlaxoSmithKline: Research Funding; Celgene: Consultancy; Stelexis Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Scientific Co-Founder; Pieries Pharmaceuticals: Consultancy; Aileron Therapeutics: Consultancy, Research Funding. Starczynowski:Kurome Therapeutics: Consultancy. Verma:Janssen: Research Funding; BMS: Research Funding; Celgene: Honoraria; Stelexis: Equity Ownership, Honoraria; Acceleron: Honoraria.

Clinical ALK5 Inhibitor, Vactosertib, Reverses TGFβ-1 Stimulated Smad-2 Driven Ineffective Hematopoiesis in MDS

Transforming growth factor beta is an important regulator of hematopoiesis and its overactivation has been shown to occur in myelodysplastic syndromes. Even though TGF-beta leads to activation of the transcription factor SMAD2, the exact gene expression program and chromatin changes stimulated in human hematopoiesis are not well studied. We generated primary erythroid progenitors from human CD34+ stem cells and assessed genome occupancy of SMAD2 upon TGF-beta stimulation by Chip-seq analysis. We observed widespread occupancy of important stem cell and differentiation regulators with approximately 7000 binding regions. Parallel RNA-seq identified gene expression changes that are associated with smad2 binding at respective promoters and enhancers. Genes involved in stem cell maintenance as well as erythroid differentiation were prominently altered by TGF-beta and contained prominent SMAD2 peaks in hematopoietic progenitors. Next, to determine the relative contribution of various TGF-beta ligands in bone marrow failure, we analyzed a large cohort of MDS serum samples and found TGF-beta 1 (TGF-b1) to be the predominantly elevated ligand in 30% samples. Exposure to MDS patient serum led to erythroid differentiation blocks in primary hematopoietic stem cells in clonogenic cultures. Finally, we analyzed the effect of constitutive secretion of TGF-b1 in a transgenic mouse containing TGF-b1 gene driven by an albumin promoter. These mice constitutively secrete TGF-b1 and become cytopenic and show marrow histological changes that mimic human MDS. We observed stem cell expansion and block at the stage of common myeloid progenitor (CMP) in these mice; findings that are similar to stem and progenitors alterations seen in human MDS patients. To inhibit TGF-b1 signaling we next used a novel ALK5 (TGF-beta receptor I kinase) inhibitor that has been shown to be safe for human use. Vactosertib is specific small molecule inhibitor of ALK5 with a IC50 between 1 x 10-8 M and 1 x 10-9 M and has been shown to have an acceptable safety profile in phase I trial in solid tumors (NCT02160106). Vactosertib was able to inhibit smad2 phosphorylation and activation in a variety of hematopoietic cell lines. Vactosertib treatment led to reversal of hematopoietic alterations upon exposure to MDS serum in clonogenic assays. Vactosertib dosing in vivo also led to increased blood counts in TGF/alb transgenic mice and resulted in normalization of stem and progenitor changes. In conclusion, we demonstrate increased TGF-b1 levels in subset of MDS samples. We also determined TGF-b1 stimulated smad2 binding in important regulatory regions of the genome in primary human erythroid progenitors. We also demonstrate the preclinical efficacy of novel TGF-beta receptor 1 kinase inhibitor in vitro and in vivo. These studies provide preclinical rationale for using this ALK5 inhibitor in trials. Disclosures Huh: MedPacto Inc: Employment. Hwang:MedPacto Inc: Employment. Seong-Jin:MedPacto Inc: Employment. Will:Novartis Pharmaceuticals: Research Funding. Steidl:BayerHealthcare: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; GlaxoSmithKline: Research Funding; Celgene: Consultancy; Aileron Therapeutics: Consultancy, Research Funding; Stelexis Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Scientific Co-Founder; Pieries Pharmaceuticals: Consultancy. Verma:Stelexis: Equity Ownership, Honoraria; Acceleron: Honoraria; Celgene: Honoraria; BMS: Research Funding; Janssen: Research Funding.

Keeping the Heart Fitm2 during Chemotherapy.

Keeping the Heart Fitm2 during Chemotherapy.

Antiaging firm Elevian launches

Elevian has launched with $5.5 million in seed funding to pursue regenerative medicines for aging and age-related diseases. Based on work done in the labs of its scientific cofounder, Harvard University professor Amy Wagers, the biotech is developing therapeutics that increase the activity of growth-differentiating factor 11, a circulating factor that when given to mice elicits the regenerative benefits of young blood. Enhancing GDF11 levels could treat diseases such as Alzheimer’s, type 2 diabetes, and age-related muscle disorders, Elevian says.

Cancer drug start-up Navire sets sail

BridgeBio Pharma and the University of Texas MD Anderson Cancer Center have launched a new company called Navire Pharma. Backed by $30 million in financing from BridgeBio, Navire will develop small-molecule inhibitors of an enzyme called SHP2, implicated in many cancers. “I’ve been working on this protein for longer than I want to admit, so this is exciting,” says Benjamin G. Neel, director of New York University’s Perlmutter Cancer Center. Neel, who has studied the proteins since 1992, is a scientific cofounder of Navire and chair of its scientific advisory board. SHP2 belongs to the tyrosine phosphatase family of proteins, which help relay chemical messages controlling cell growth and division. MD Anderson is contributing its SHP2 inhibitors to the collaboration and will continue to spearhead studies until a compound is ready for human trials. Researchers think that blocking SHP2 could halt the growth of tumors spurred by malfunctions in the

Exosomal MicroRNA Clusters Are Important for the Therapeutic Effect of Cardiac Progenitor Cells

Although current evidence indicates that cardiac progenitor cells (CPCs) may improve cardiac repair after ischemic injury, the exact mechanism for this cardioprotective effect to date remains undefined. In this issue of Circulation Research , Gray et al1 report on the use of systems biology to link the change in exosomal microRNAs (miRNA) to the beneficial effects of CPCs during ischemic injury in mice. Expression analysis showed that exosomes from hypoxic CPCs contain a higher level of a subset of miRNAs than exosomes coming from normoxic CPCs. To connect the relevance of the change in miRNA levels to the biological effect of exosomes, the authors performed a principle component analysis of the miRNA expression profiles in exosomes, which identified 4 unique miRNA clusters, which after further mathematical modeling seemed to correlate to a biological effect related to the therapeutic benefit. Instead of studying the biological function of a single miRNA, computational modeling can be applied to integrate multiple variants to gain more insight into the overall relationship and interaction between the different datasets. Gray et al1 show that especially for miRNA biology, where a miRNA can target up to hundreds of genes in a parallel, systems biology could turn out to be a suitable approach for predicting a biological outcome or therapeutic benefit. Article, see p 255 Cells communicate with each other via direct cell–cell contact and secretion of soluble factors. Soluble factors that are gaining an increasing amount of attention are extracellular membrane vesicles. The ability of vesicles to transport different molecules, such as proteins, peptides, mRNA, and microRNAs, from one cell to the other and their general body-wide distribution make them attractive candidates for horizontal information transfer between cells.2 Although the release of vesicles to influence other cells seems to be straightforward, multiple mechanisms, size distributions, …

Chimeric Antigen Receptor (CAR) T Cells Targeting the CD19 Antigen for the Treatment of Pediatric Relapsed B Cell ALL

T cells can be genetically modified to target tumor antigens through the expression of a chimeric antigen receptor (CAR). CAR T cells targeting the CD19 antigen is a novel therapeutic approach for patients with relapsed B cell acute lymphoblastic leukemia (B-ALL). We have previously demonstrated that CAR T cells have a significant clinical benefit in adult patients with relapsed B-ALL. The primary objective of this study (NCT01860937) is to extend the use and test the safety of CD19 specific CAR T cells in children with relapsed CD19+ B-ALL. To date, 11 pediatric patients with very high risk (VHR) or relapsed B-ALL have been enrolled on protocol with a median age of 13 years (range 2-23 years) at time of T cell collection. We have treated 4 pediatric patients with relapsed B-ALL (ages 13, 14, 19, and 22 years) using patient derived T cells expressing a CD19 specific CAR (19-28z). Complete response (complete remission or complete remission with incomplete count recovery) occurred in 2/4 (50%) patients. Severe cytokine release syndrome (sCRS) defined by fever for ≥3 consecutive days, elevated serum cytokine levels, and one clinical sign of toxicity (hypotension, hypoxia, neurologic disorder including altered mental status, obtundation, and/or seizure) occurred in both patients who responded to CAR T cells. Morphologic disease (≥5% bone marrow blasts) at time of treatment was present in three patients including both patients with sCRS. Systemic immunosuppressants (corticosteroids or anti-IL6 receptor antibody tocilizumab) abrogated clinical symptoms of sCRS. Elevated serum cytokines of IFN-g (>20 fold), fractalkine (>20 fold), Flt-3L (>55 fold), IL-5 (>15 fold), IL-6 (>100 fold), and IL-10 (>15 fold) were demonstrated in patients with sCRS. Monitoring of bone marrow demonstrated peak 19-28z CAR T-cell detection within 1-2 weeks following infusion with gradual contracture over 2-3 months. These early results demonstrate the feasibility and significant clinical impact of this approach in patients with relapsed B-ALL. To more rapidly generate statistically relevant data, demonstrate the “exportability” of this technology between academic institutions, and offer this therapeutic option to a broader number of pediatric patients with chemo-refractory B-ALL our trial will expand into a phase I multicenter clinical trial with a collaborating institution. Subsequent cohorts of patients will receive 19-28z CAR T cells and will be evaluated for toxicity, persistence of CAR T cells, and for anti-leukemic efficacy. DisclosuresOff Label Use: CAR T cells for relapsed B-ALL. Riviere:Juno Therapeutics: Consultancy, Scientific co-founder and Stock holder Other. Boulad:Genzyme Sanofi: Trials partially funded by Genzyme Sanofi Other. Sadelain:Juno Therapeutics: Consultancy, Scientific co-founder and Stock holder Other. Brentjens:Juno Therapeutics: Consultancy, Scientific co-founder and Stock holder Other.

CD19-Targeted 19-28z CAR Modified Autologous T Cells Induce High Rates of Complete Remission and Durable Responses in Adult Patients with Relapsed, Refractory B-Cell ALL

Background: Relapsed adult acute lymphoblastic leukemia (ALL) is associated with high reinduction mortality, chemotherapy resistance, and dismal prognosis with a median overall survival (OS) < 6 months and 5-year OS ≤10%. We have previously reported a high anti-tumor activity of autologous T cells genetically modified to express 19-28z chimeric antigen receptor (19-28z CAR) targeting CD19 in adult patients with CLL and ALL (Brentjens R et al. Blood 2011; Davila M et al. Sci Transl Med2014). Herein, for the first time, we further report the long-term outcome of our phase I clinical trial in adults with relapsed/refractory (R/R) ALL (NCT01044069) with analysis on potential predictive markers of response and neurological toxicities.Patients and Methods: Adult patients with R/R B-ALL were enrolled. Eligible patients underwent leukapheresis, and T cells were transduced with a retrovirus encoding a CAR construct composed of anti-CD19 scFV linked to CD28 and CD3ζ signaling domains (19-28z). All patients received lymphodepleting chemotherapy followed 2 days later by 1x106 – 3x10619-28z CAR T cells/kg. The primary objective of the study was to evaluate the safety and anti-tumor activity of 19-28z CAR T cells in ALL. Post-treatment minimal residual disease (MRD) was assessed at day 14-28 by multiparameter flow cytometry and deep sequencing in the bone marrow (BM) samples (Adaptive Biotech Corp.)Results: 24 patients have been treated. The median age was 56 years (range, 23-74). 6 patients (25%) had Ph+ B-ALL (T315I mutation in 2 patients), 6 patients (25%) had prior allogeneic hematopoietic stem cell transplant (allo-HSCT), and 11 patients (46%) had 3 or more prior lines of ALL therapy before receiving the 19-28z CAR T cell therapy.Of the 24 patients, 22 patients were evaluable for response. At the time of 19-28z CAR T cell infusion, 12 of 22 patients had morphologic disease (6 to 97% blasts in the BM) and the remaining 10 patients had MRD. Twenty out of 22 patients (91%) were in complete remission (CR) after 19-28z CAR T-cell infusion, and 18 of these 20 patients (90%) achieved an MRD-negative CR. Ten of the 13 transplant eligible patients (77%) successfully underwent allo-HSCT following the 19-28z CAR T cell therapy. As of July 1, 2014, the median follow-up was 7.4 months (range 1-34), with 13 patients having at least 6 months of follow-up. Responses appear durable with 6 patients remaining disease-free beyond 1 year (range 12.6 – 34 months). Median overall survial (OS) is 9 months. 5 patients relapsed during the follow-up, including 1 patient with CD19 negative relapse. Three of the relapsed patients were treated again with the 19-28z CAR T cells, and two patients achieved a second CR.Comparing responders to non-responders, no association was observed between response and age (<60 vs. ≥60), prior allo-HSCT, number of prior therapies, or pre-treatment blast percentage.While none of the 10 patients with MRD at the time of T cell infusion developed cytokine release syndrome (CRS), 9 of 13 patients with morphologic disease at the time of the T cell infusion developed CRS with or without neurological symptoms that required intervention with an IL-6R antagonist or corticosteroid. A detailed analysis of serum cytokines demonstrated a consistent peak of IL-6 (22.2 to 553-fold increase) immediately prior to the development of neurological toxicities. Based on these data, we have developed a multi-disciplinary CRS management algorithm for patients at high risk in order to reduce the severity of CRS and improve safety of the 19-28z CAR T cell therapy.Conclusions: While longer follow-up is needed to confirm the durability of the observed responses, the potent induction of MRD-negative responses and successful long-term outcomes, including subsequent allo-HSCT without apparent additional post-transplant toxicities, strongly support the use of 19-28z CAR T cells in adult patients with B-ALL. A temporal relationship between serum IL-6 levels and neurological toxicities indicates that early intervention with IL-6 directed therapy may be more effective in ameliorating neurological toxicities in patients with morphologic disease at the time of T-cell infusion. These findings will need to be evaluated systematically and confirmed in a larger phase 2 trial. DisclosuresPark:Juno Therapeutics: Research Funding. Riviere:Juno Therapeutics: Consultancy, scientific co-founders Other. Sadelain:Juno Therapeutics: Consultancy, Scientific co-founder and Stock holder Other. Brentjens:Juno Therapeutics: Consultancy, Scientific co-founder and Stock holder Other.

Interim Safety Analysis: a Phase I Trial of High Dose Therapy and Autologous Stem Cell Transplantation Followed By Infusion of Chimeric Antigen Receptor Modified T-Cells (19-28z CAR-T) Directed Against CD19+ B-Cells for Relapsed and Refractory Aggressive B Cell Non-Hodgkin Lymphoma (B-NHL)

Background: Diffuse large B-cell lymphoma (DLBCL) is the most common aggressive B-NHL. While 50-80% of patients with DLBCL are cured with standard induction therapy, a large fraction of patients either relapse or have primary refractory (rel/ref) disease. High-dose therapy followed by autologous stem cell transplantation (HDT-ASCT) is the established standard of care for these patients. Despite this, approximately half of all rel/ref patients that are chemosensitive to salvage therapy are cured with this approach. Relapse is the major cause of treatment failure, and ultimately death, in these patients. Our group has demonstrated encouraging activity with 19-28z chimeric antigen receptor modified T cells (19-28z CAR-T) directed against CD19 in rel/ref ALL. We hypothesize that biologic optimization of cellular therapy with 19-28z CAR-T can be met immediately post-HDT-ASCT given: lymphoproliferative cytokine availability through lymphodepletion post-HDT, depletion of prohibitive regulatory cellular elements post-HDT and achievement of minimal residual disease prior to 19-28z CAR-T consolidation. Herein, we report safety data on the first 6 patients of the phase I MSKCC #12-117: 19-28z CAR-T post HDT-ASCT for poor-risk rel/ref aggressive B-NHL.Methods: Eligibility for this study includes rel/ref aggressive B-NHL appropriate for HDT-ASCT as defined by chemosensitivity to salvage therapy and poor risk features including: 1) FDG-PET positivity following 2 cycles of salvage therapy or 2) bone marrow involvement of B-NHL at the time of rel/ref clinical restaging. Patients underwent separate apheresis for CD34+ progenitor cells and CD3+ T cells. T cells were transduced with a retrovirus encoding a CAR construct composed of anti-CD19 scFV linked to CD28 and CD3ζ signaling domains (19-28z). Patients were admitted for BEAM conditioned HDT and ASCT occurred day 0. Pegfilgrastim was administered on day+1 and 19-28z CAR-T dose per phase I study was split on days +2 and +3.Results: This analysis includes the first six patients, all male, on the phase I study. The median age is 61 (range 34-68) years at the time of HDT-ASCT. Diagnoses included: n=2 relapsed and transformed follicular lymphoma (one with double-hit biology), n=3 relapsed DLBCL (one CD5+) and one subject with relapsed and transformed marginal zone lymphoma involving the bone marrow. Five patients were treated at dose level #1 (5 x106 19-28z CAR-T/kg) with no dose-limiting toxicity (DLT) observed. Four of the five patients at dose level #1 experienced grade 3 febrile neutropenia and one patient met-criterion for non-severe cytokine-release syndrome (nCRS) effectively treated with tocilizumab 4 mg/kg x1. One patient was treated at dose-level #2, 1 x107 CAR-T/kg, and experienced a DLT of grade 4 severe CRS (sCRS) manifested with acute kidney injury, hypotension and mental status changes effectively treated and fully recovered with tocilizumab in combination with dexamethasone. Peak CRP in all patients was observed at a median of 3.5 days (range 3-4 days) post-19-28z CAR-T infusion (median peak CRP= 17 mg/dL, range: 5-43.1 mg/dL), with CRP >20 mg/dL identified in the two patients that experienced CRS (nCRS=27 mg/dL, sCRS=43 mg/dL). Previously associated serum cytokine elevations (Davila et al Sci Trans Med, 2014) were observed in the two patients that experienced CRS (Figure). All patients engrafted neutrophils at a median of 10 days (range: 9-10 days) post-ASCT, and achieved a complete remission at first post-HDT-ASCT restaging. No sequelae of autoimmune phenomenon were observed. At a median follow-up of six months, with 2 patients >one year post HDT-ASCT, all patients remain alive and in remission.Conclusions: This is the first report of 19-28z CAR-T cells in conjunction with consolidative HDT-ASCT for poor-risk rel/ref aggressive B-NHL. No DLTs have been observed at dose level #1, 5 x106 19-28z CAR-T/kg, while sCRS was observed in n=1 at 1 x107 CAR-T/kg resulting in a DLT. CRS was associated with pro-inflammatory serum cytokine elevation. CRS was aborted with tocilizumab with (sCRS=1) or without (nCRS=1) dexamethasone. All patients engrafted neutrophils at the expected time point. The use of 19-28z CAR T cells is a promising approach in this small group of poor-risk PET+ NHL patients undergoing autologous transplant. This is an ongoing trial and updated data will be presented. [Display omitted] DisclosuresRiviere:Juno Therapeutics: Consultancy, scientific co-founders Other. Sadelain:Juno Therapeutics: Consultancy, Scientific co-founder and Stock holder Other. Brentjens:Juno Therapeutics: Consultancy, scientific co-founder Other.

A Systemic Xenograft Model of Waldenström’s Macroglobulinemia Demonstrates the Potent Anti-Tumor Effect of Second Generation CD19 Directed Chimeric Antigen Receptor Modified T Cells in This Disease

Chimeric antigen receptor (CAR) modified T-cell therapy consists of ex-vivo genetic manipulation of autologous lymphocytes in order to establish robust T-cell mediated anti-tumor immunity. Our group was the first to design and evaluate a CAR targeted toward the B cell antigen CD19 in mice. Currently we utilize a second generation CAR comprised of a single-chain variable fragment (scFv) derived from an antibody against CD19 fused to the CD3 ζ chain and the CD28 intracellular signaling domain (19-28z) to provide the necessary signal 1 and signal 2 for enhanced T-cell activation and persistence. We have gone on to test the safety and efficacy of 19-28z CAR T-cells in patients with chronic lymphocytic leukemia (CLL) and acute lymphoblastic leukemia (ALL). We observed rapid complete molecular remissions in the first 14/16 patients treated with relapsed/refractory ALL. We hypothesize that contributing to the enhanced efficacy seen in ALL, when compared to solid tumors or extra-medullary CLL, is the fact that ALL is a bone marrow predominant disease, which may provide a microenvironment more amenable to T-cell therapy.Waldenström’s Macroglobulinemia (WM) is an ideal disease to test 19-28z CAR-modified T-cell therapy, as it is often bone marrow predominant, and WM cells from patient samples typically uniformly express high levels of CD19. Furthermore, despite recent progress made with novel BCR-directed therapy, complete eradication of the WM clone from the bone marrow niche remains elusive, therefore providing an ideal clinical scenario for treatment consolidation via alternative cytotoxic methods such as cellular immunotherapy.Using the human WM cell line, BCWM.1, we evaluated the in vitro efficacy of 19-28z CAR-modified T-cells when compared to mock transduced T cells or T cells transduced with an irrelevant second generation CAR directed towards the ovarian antigen MUC16. In a 4 hour co-culture assay, we observed significant cytotoxicity, even at low effector:target ratios (52% lysis at 1:1; 92% lysis at 10:1; p<0.01). This corresponded to increased secretion of INFγ and IL-2, markers of T-cell activation (p<0.01).We then conducted in vivo studies using sublethally irradiated SCID/beige mice to generate a systemic model of WM via tail vein injection of 1x106 luciferase transduced BCWM.1 cells. This model is characterized by tumor growth in the bone marrow followed by rapid spread to the liver, lungs, kidney, and CNS. Mice were monitored by weekly bioluminescent imaging (BLI) and ultimately were sacrificed when they developed hind leg paralysis. 19-28z CAR modified T-cells administered at day 7, after tumor establishment, when compared to non-treated and irrelevant CAR-modified T cell controls, delayed the progression of disease and doubled the median survival time of the mice after treatment (p=0.001).Taken together, the pre-clinical efficacy demonstrated in this abstract and the clinical features of WM, listed above, provide the rational for testing 19-28z CAR modified T cells clinically for WM. We have now opened a clinical trial for patients with relapsed or refractory WM, in which chemotherapy preconditioning is followed by a single dose of 19-28z CAR modified autologous T-cells (NCT00466531). DisclosuresBrentjens:Juno Therapeutics: Consultancy, Scientific co-founder and Stock holder Other.

Engineered T Cell Receptor-Mimic Antibody, (TCRm) Chimeric Antigen Receptor (CAR) T Cells Against the Intracellular Protein Wilms Tumor-1 (WT1) for Treatment of Hematologic and Solid Cancers

Adoptive transfer therapy of T cells expressing chimeric antigen receptors (CAR) against tumor-associated antigens has been shown to be clinically successful in a limited set of leukemia. However, novel antigen targets for both hematological and solid malignancies are required. Most CARs described thus far are targeted against external antigens on particular cell types. We have designed and engineered the first CAR T cell against a human intracellular protein, WT1. WT1 is overexpressed in many cancers, including acute and chronic leukemias and numerous solid tumors. Our TCRm CAR, derived from the ESK1 TCRm mAb, termed WT1 28z, is reactive with the RMFPNAPYL peptide of the WT1 protein that is processed and presented on the surface of cells in the context of HLA-A*02:01. WT1 28z expressing T cells have high expression of the CAR on their surface. They are cytotoxic in standard 51Cr assays against a range of cancer cell lines, including the megakaryoblastic cell line SET2, the acute myeloid leukemia (AML) cell line AML14, the multiple myeloma cell line KARPAS, and the ovarian cancer line, OVCAR3, as compared to CAR T cells against an irrelevant antigen. The WT1 28z CAR T cells are also cytotoxic against primary AML bone marrow blasts in this assay. When co-cultured with these primary cells or cancer cell lines, the WT1 28z CAR T cells have enhanced production of proinflammatory cytokines such as IFN-g, IL-2, and GM-CSF, as compared to irrelevant CAR T cells. Importantly, WT1 28z T cells are specific for the WT1-HLA-A*02:01 complex. The cells do not show cytotoxicity against cell lines or primary cells that are not both HLA-A*02:01- positive and WT1 positive. WT1 28z T cells are currently being tested alongside irrelevant antigen CAR T cells in AML and ovarian cancer murine models in vivo to assess efficacy, with the ultimate goal of translating this novel approach into the clinical setting for both hematological and solid cancers. The data provide the proof-of-concept that CAR T cells also may be directed at intracellular antigens. DisclosuresDao:Novartis: Patents & Royalties. Liu:Eureka: Employment, Inventor Other. Scheinberg:Novartis: Patents & Royalties. Brentjens:Juno Therapeutics: Consultancy, Scientific co-founder and Stock holder Other.

Five-grass pollen immunotherapy tablet: an update on the latest findings from clinical trials: an interview with Olivier de Beaumont.

Interview by Jonathan Wilkinson (Managing Commissioning Editor, Future Science Group). Olivier de Beaumont became Doctor of Medicine at the University of Paris Descartes in 1993. In the same year, he also took a Master of Health Economics degree at Paris Dauphine University. He is also a Master of Business Administration, ESCP Paris, 1999. At Stallergenes, he has been serving as Vice President/Head of Corporate Clinical Development since 2005, responsible for the clinical development plan, clinical operations, biometry and pharmacovigilance. In 2011, he took the responsibility of Senior Vice President Global Medical Affairs responsible for medical information and education, medical communication and nonregistration clinical studies. In 2014 he became Senior Vice President Global Scientific and Medical Affairs. From 2002 to 2005 he led the European business development of the world's leading clinical research organization Quintiles, developing Phase I-IV clinical trial programs for pharmaceutical companies. Previous roles included: Chief Scientific Officer and cofounder of Direct Medica (2000-2002), product champion and lifecycle management at Aventis (1998-2000), medical affairs manager in oncology and respiratory diseases at corporate Rhone-Poulenc Rorer (1993-1998).

MiR-25 in heart failure.

Recent work by Wahlquist et al 1 showed miR-25 to reduce levels of rco/endoplasmic reticulum calcium-ATPase (SERCA) 2a during heart failure, whereas therapeutic inhibition of this microRNA (miRNA) was able to reverse pre-established heart failure in a mouse model of pressure overload. Although these data suggest that miR-25 might be a drug target for the treatment of heart failure, further in vivo analysis is required before we can consider miR-25 inhibition for clinical application. Recently, Wahlquist et al1 reported on the pathological upregulation of miR-25 during heart failure and showed that inhibition was able to block and reverse the disease in mice. Although an increase in cardiac miR-25 levels caused a decline in cardiac function, anti–miR-based inhibition of miR-25 halted established heart failure at least in part by increasing the mRNA of SERCA2a. This is intriguing because phase II clinical data from the Calcium Upregulation by Percutaneous Administration of Gene Therapy in Cardiac Disease (CUPID) trial has demonstrated that gene therapy to increase SERCA2a levels in humans delays the progression of chronic heart failure. This could imply that pharmacological regulation of miR-25 may regulate at least 1 clinically validated target, which may be more straightforward than current gene therapy approaches. However, miRNA biology seems to sketch a somewhat more complex picture than a miRNA functioning through 1 single-gene target. In addition, conflicting data on the cardiac functions of miR-25 highlight the need for further investigation before advancing miR-25 as a clinical target for heart failure. miRNAs are short single-stranded noncoding RNAs that modulate the expression of target proteins by annealing to complementary sequences in their target mRNAs.2 It is thought that a single vertebrate miRNA on average targets a few hundred transcripts.3,4 Because individual miRNAs often target numerous related mRNAs that encode multiple components of …

Ancora Pharmaceuticals: Counting Carbs Synthetically

With many biotech companies reaching for commercial success, it helps when the founding science comes from an unquestioned leader in the field. Such is the case with Ancora Pharmaceuticals (http://www.ancorapharma.com), founded in 2003 to develop a synthetic carbohydrate platform based on work licensed from MIT. In 2002, the company's scientific cofounder, Dr. Peter Seeberger, a world-class carbohydrate chemist then based at MIT, helped establish the company after winning an MIT-based entrepreneurial competition, setting in motion the foundation for the now 20 person company based in Medford, Massachusetts.